Rebeca Rodríguez saw that a place was available a few metres from Karmela Restaurant where she was cooking under the guidance of Daniel Peregrina made Rebeca a proposal to become partners and she accepted Today she is in charge of the kitchen of this new business that was launched just five months ago under the name of Vendaval in Calle Málaga in Alhaurín de la Torre "The idea was to set up a seafood restaurant something informal and with a lot of emphasis on fried fish because there are not so many places to eat it here but little by little people have been asking us for the more innovative cuisine that we do at Karmela and we have been incorporating other dishes such as the prawn pil pil rolls." explains Daniel Peregrina about a constantly evolving menu and an affordable average price (around 15-20 euros) gildas (a salty tapa) and fried fish such as lemon anchovies scrambled eggs with pork cheeks and flambéed kimchi aioli among other proposals with which they aim to offer something different to the town's gastronomic offer This is how Rebeca Rodríguez sees it: "Many people knew us from Karmela and are used to the creative cuisine that is done there while at the same time offering the essence of a seafood restaurant" Although some very peculiar drawings also hang on the walls: the ones left on the paper tablecloths by the youngest members of the family in addition to the dishes 'For the little ones' and the location in a pedestrian street says Peregrina about a space that can seat around twenty diners A team of four people are in charge of running this new venture which aspires to establish itself as a casual alternative to Alhaurinos' lunch and dinner And another date for wine lovers: Barbadillo wine tasting with four courses on 14 May at 9pm (26 euros) Comentar es una ventaja exclusiva para registrados Sign up for our daily Newsletter and stay up to date with all the latest news You are receiving this pop-up because this is the first time you are visiting our site You are using software which is blocking our advertisements (adblocker) we are relying on revenues from our banners So please disable your adblocker and reload the page to continue using this site.Thanks EV Produce International announced strategic expansion plans to bolster its import and shipping operations The company will diversify its product portfolio and extend its distribution network to encompass the entire continental United States Building on eight years of market presence the company supplies premium limes from its Mexican partner which maintains operations in Martínez de la Torre While limes and chayote have been core products the company has recently integrated new offerings it has commenced production and export of chiles (Jalapeño The company has started its production of Serrano chiles The company initiated the import and distribution of these new products in December 2024 on the West Coast "We are now expanding our reach to serve the entire U.S market through our distribution centers in California and McAllen vice president of EV Produce International the company primarily targeted the Latino market in the U.S Carranza noted a significant increase in consumption across diverse demographics driven by the growing popularity of Mexican cuisine "While chiles have traditionally been a staple within the Latino community the widespread appreciation of Mexican food has fueled increased consumption nationwide," he says Tomatillos have also been added to the product line EV Produce International has witnessed a surge in demand for limes as consumption extends beyond the Latino demographic to encompass the broader U.S lime consumption was largely concentrated within the Latino market which prompted us to establish a distribution center in California," Carranza says we have observed a consistent increase in consumption attributed to the prevalence of limes in cocktails and various Mexican culinary preparations." EV Produce International's operations extend beyond the U.S with current lime exports to South Korea and Japan For more information:Erick Carranza EV Produce Tel: +1 (619) 883-3865 http://evproduce.com/ FreshPublishers © 2005-2025 HortiDaily.com Volume 15 - 2021 | https://doi.org/10.3389/fnana.2021.785840 This article is part of the Research TopicIn the Footsteps of the Prosomeric ModelView all 15 articles The trigeminal column is a hindbrain structure formed by second order sensory neurons that receive afferences from trigeminal primary (ganglionic) nerve fibers Classical studies subdivide it into the principal sensory trigeminal nucleus located next to the pontine nerve root and the spinal trigeminal nucleus which in turn consists of oral this column would be subdivided into segmental units derived from respective rhombomeres Experimental studies have mapped the principal sensory trigeminal nucleus to pontine rhombomeres (r) r2-r3 in the mouse The spinal trigeminal nucleus emerges as a plurisegmental formation covering several rhombomeres (r4 to r11 in mice) across pontine retropontine and medullary hindbrain regions In the present work we reexamined the issue of rhombomeric vs we analyzed its subdivisions in an AZIN2-lacZ transgenic mouse known as a reference model for hindbrain topography together with transgenic reporter lines for trigeminal fibers We screened as well for genes differentially expressed along the axial dimension of this structure in the adult and juvenile mouse brain This analysis yielded genes from multiple functional families that display transverse domains fitting the mentioned rhombomeric map The spinal trigeminal nucleus thus represents a plurisegmental structure with a series of distinct neuromeric units having unique combinatorial molecular profiles The rostrocaudal organization of successive Pr5 and Sp5C units raises the question of the possible correspondence of their mutual boundaries with interrhombomeric limits the hypothesis may be considered that the underlying rhombomeric scaffold may actually establish a finer segment-by-segment organization of the trigeminal column it is possible that the segmental units of the trigeminal column develop characteristic molecular and cellular identities with probable neurochemical and/or functional implications On the whole we propose a novel segmental map of the mouse trigeminal column according to gene expression characterizing it as a plurineuromeric modular complex in relation to a series of rhombomere-derived domains and care of laboratory animals were conducted in compliance with the current normative standards of the European Union (Directive 2010/63/EU) the Spanish Government (Royal Decree 1201/2005 and 53/2013; Law 32/107) and had the approval of the University of Murcia Committee for Animal Experimental Ethics We used adult brains of a heterozygotic mice line developed at the Department of Biochemistry, School of Medicine, University of Murcia (López-Garcia et al., 2013) These mice express recombinant beta-galactosidase protein under control of the Azin2 promoter vibratome 120 μm thick serial sections were obtained Serial sections were obtained in either sagittal or horizontal planes Floating sections were then reacted for beta-galactosidase and were finally washed Digital microphotographs were acquired using Aperio CS2 technology (Leica Microsystems GmbH This database provides images from transgenic mice lines with EGFP as reporter for the expression of diverse genes According to the recorded GENSAT procedures the brain sections were processed for immunohistochemistry against EGFP which normally leads to full labeling of the positive neurons including their soma and fibers We searched for genes expressed in the trigeminal ganglion whose axons reach the brain and form the ascending and descending trigeminal tracts delimiting in this way the extent of the trigeminal sensory column and cropped them to show our region of interest We searched the AMBA and ADMBA for genes whose respective in situ hybridization (ISH) experiments included both sagittal and coronal section series at P561 as well as sagittal section series from stages P4, P14, and/or P28.2 We screened these image series visually selecting the genes with significant expression within subregions of Sp5 This analysis was initially carried out by two of the authors independently whose preliminary results were discussed to reach a consensus Our criteria to select genes was firstly that they displayed discrete expression patterns with positive and negative regions visible along the longitudinal axis of Sp5; secondly we checked that these patterns were coherently reproduced in both sagittal and coronal P56 stage series part of the image series from these juvenile stages displayed generalized low ISH signal or high background so that these image series were discarded from analysis As a result, we identified 12 genes (Table 1) that were differentially expressed in the trigeminal column including the brightfield microphotographs or scanned images as well as their respective counterpart with color-coding of the expression level both of them available for each brain section the positive cells appear with a blue precipitate accordingly to standard ISH protocols The sections corresponding to juvenile stages (P4 and P28) are counterstained with HP Yellow In the color-coded images from adult and juvenile stages the expression intensity ranges from blue (low expression intensity) through green (medium intensity) to red (high intensity) Summary of the expression of each gene in the rhombomeric portions of the trigeminal column We downloaded the images that included the whole or part of the trigeminal column cropping them to show the region of interest the images from parasagittal sections are oriented with the rostral end to the left while those from coronal sections are details of the right side of the original image The list of the selected genes, together with the references of their respective experiments and downloaded images from the AMBA and ADMBA databases, is indicated in Supplementary Table 1 AZIN2-LacZ expression in the adult hindbrain (A,B) Respective details of parasagittal and horizontal sections In each of them the rostral end is to the left The black or white dotted lines indicate interrhombomeric boundaries The white dashed lines delimit the principal sensory (Pr5) and the spinal trigeminal nucleus (Sp5) The dot-dash line in B indicates the midline The rectangles delimit the respective positions of A and B images The dashed lines indicate the respective approximate positions of their sectioning planes mandibular fibers of the trigeminal tract; sp5ot ophthalmic fibers of the trigeminal tract; VC Labeling of trigeminal primary afferent fibers in adult transgenic mice from the GENSAT database processed for immunohistochemical detection of EGFP (A,C) Respective parasagittal sections of adult brains from Calca and Avil mouse lines The principal sensory (Pr5) and the spinal trigeminal nucleus (Sp5) the dorsal cochlear nuclei (DC) and the medial cerebellar peduncle (mcp) are there is expression in the ventral nucleus of the lateral lemniscus (VLL) and in the Kölliker-Fuse nucleus (KF) in the section from the Calca line (B,D) Respective details of the former sections showing labeling in the trigeminal nerve root (5n) and labeled ascending trigeminal fibers (black and white arrows) within Pr5 Schematic drawing showing a sagittal view of the hindbrain plus the first myelomere with the morphological landmarks used to delimit the rhombomeric domains in this work CBN) and the ventral nucleus of the lateral lemniscus (VLL) are located in r1 The medial cerebellar peduncle (mcp) crosses superficially r2 and r1 The dorsal and ventral cochlear nuclei (DC The trigeminal motor nucleus (5N) and the root of the trigeminal nerve (5n) are located across r2 and r3 The pontine nuclei (Pn) are located superficial and medially in r3 plus r4 The bundles of fibers of the vestibulocochlear nerve (8n) and the descending fibers of the facial nerve (7n) are in r4 The abducens motor nucleus (6N) is medially in r5 The superior olive (SO) and the trapezoid body (tz) are located principally in r5 The facial motor nucleus (7N) is located in r6 The inferior olive (IO) extends from r8 to r11 The complex formed by the hypoglossal and vagal motor nuclei (12N The ambiguous motor nucleus (Amb) extends from r7 to r10 The external cuneatus nucleus (ECu) appears in r9 at lateral section levels The area postrema (AP) is located at the midline in r10 The pyramidal decussation (pyx) is located in the first myelomere (my1) the principal sensory nucleus (Pr5) is located in r2 and r3 while the spinal trigeminal nucleus (Sp5) extends from r4 caudalwards the limit between interpolar and caudal subnuclei is located at the r9/r10 limit We have drawn Sp5 including the dorsal horn of the first myelomere considering their similarity in morphology and gene expression (B) Coronal section from adult brain at the level of the trigeminal motor nucleus (5N) crossing through r1 There is Baiap3 expression in KF and other parabrachial nuclei (PB) within r1 while Pr5 (encircled by dashed lines) remains negative in this section (C) Coronal section from adult brain at the level of r10 showing Baiap3 expression in the gelatinous layer of Sp5 (encircled by dashed lines) as well as in the lateral reticular nucleus (LRt) the trigeminal-solitary transition zone (5Sol) and the matrix region of the medulla (Mx) (D,E) Respective brightfield and color-coded images of a parasagittal section from a P28 brain processed for detection of Camk2a expression there is expression in the r10 and r11 portions of Sp5 with a laminar pattern showing higher intensity in its gelatinous layer The r9 portion of Sp5 express this gene homogeneously with a gradient expression extending into r8 there is expression in some scattered cells (F,G) Respective coronal sections from adult brain at r9 and r10 levels of an adult brain displaying their aforementioned expression pattern in the respective portions of Sp5 Positive zones for Camk2a expression close to Sp5 include Sol as well as other structures indicated below (A–D) Details of the expression pattern from an adult brain in Pr5 (A,B) Brightfield and color-coded images from a parasagittal section at lateral level (C,D) Brightfield and color-coded images from a coronal section R2 and r3 are delimited according to the position of 5N across both rhombomeres 7n and 8n bundles determine the position of r4 (E,F) Brightfield and color-coded images from a parasagittal section There is mild expression in the r2 and r3 trigeminal portions (G,H) Brightfield and color-coded images from a parasagittal section of a P14 brain The pattern is similar to the one shown for the adult in the former figures but with higher intensity and a greater number of labeled cells in the positive zones (r2 (A,B) Brightfield and color-coded images from a parasagittal section from an adult brain There is homogenous expression within the r2 and r3 portions of Pr5 From r9 the expression extends gradientally into the r8 and r7 portions of Sp5 There are also some disperse labeled cells within the r4 in the brainstem this gene is expressed in KF (C,D) Brightfield and color-coded images of a coronal section from an adult brain at the level of 5N There is dense and homogenous expression within the r2 and r3 portions of Pr5 (E,F) Brightfield and color-coded images of a coronal section from an adult brain at the level of 7N The limits of r4 are tentatively drawn enclosing the positions of the intraencephalic portion of 7n and the entry root of 8n The r4 portion of Sp5 displays some labeled cells This gene shows also expression in 5N and 7N as well as in VC (G,H) Brightfield images of parasagittal sections although in the r2 portion of this nucleus the labeled cells are located only in its dorsal region Within Sp5 the expression pattern from r4 to r9 is similar to that described for the adult brain (A,B) Brightfield and color-coded images of a parasagittal section of an adult brain There are sparse labeled cells along all the trigeminal column from the r2 to the r11 trigeminal portions The expression is strongest in the r5 and r6 portions of Sp5 showing also moderate labeling in r4 and in r7 plus r8 within Sp5 (C) Brightfield image of a parasagittal section from a P4 brain showing the aforementioned pattern described in the adult (D–I) Pairs of brightfield and color-coded images of coronal sections from an adult brain The principal landmarks used for their respective identification are 7n for r4 7N for r6 and the rostralmost end of the inferior olive (IO) plus the ambiguous motor nucleus (Amb) for r8 This gene shows also significant expression in VC (D–G) (A–D) Pairs of brightfield and color-coded images of parasagittal sections The rhombomeric expression pattern within the trigeminal column is similar to that described for Mafb in the former figure (E,F) Brightfield and color-coded images of a coronal section from an adult brain at the level of r4 identified by the positions of the fibers of the facial (7n) (encircled by dashed lines) and vestibulocochlear (8n) nerves There is moderate labeling in Sp5 and in the ventral cochlear nuclei (VC) (G,H) Brightfield and color-coded images of a coronal section from an adult brain at the level of r6 as well as the dorsomedial subdivision of the latter (DMSp5) are excluding DMSp5 which lacks expression of this gene (I,J) Brightfield and color-coded images of a coronal section from an adult brain at the level of r8 showing weak expression in the portion of Sp5 in this rhombomere Tac1 (A–C) and Tac2 (D–F) expression In each image Pr5 and/or Sp5 are encircled by dashed lines (A–C) Brightfield image details of a parasagittal (A) and a coronal (B) section from adult brains and of a parasagittal section from a P28 brain (C) processed for detection of Tac1 expression There is expression in the gelatinous layer of the r10 and r11 portions of Sp5 as well as in their caudal continuation in my1 there are sparse positive cells from r7 to r9 There is expression also in the part of the lateral reticular nucleus (LRt) close to Sp5 as well as disperse cells in the solitary nucleus (Sol) and the medullary reticular formation (Rt) (D–F) Counterparts of the former images at similar levels There is a decreasing graded expression in the gelatinous layer of Sp5 from my1 to r10 (C) Brightfield image of a P28 parasagittal section reproducing the aforementioned Calb1 pattern (D–I) Pairs of brightfield and color-coded images of respective coronal sections of an adult brain at the levels of r8 Besides the aforementioned pattern in the trigeminal column in these section planes there is expression in the inferior olive (IO) the trigeminal-solitary transition zone (5Sol) and part of the lateral reticular nucleus (LRt) (J,K) Brightfield and color-coded images of a parasagittal section of an adult brain processed for detection of Calb2 expression There is expression in the r10 and r11 portions of Sp5 showing a laminated pattern with higher intensity in the gelatinous layer together with medium-high intensity in r2 and r3 and scattered positive cells from r4 to r8 (L) Brightfield image of a P28 parasagittal section reproducing the aforementioned Calb2 pattern (M–R) Pairs of brightfield and color-coded images of respective coronal sections of an adult brain at the levels of r8 Note the absence of Calb2 expression in r9 as compared to scattered cells in r8 and a laminar pattern in r10 There is also expression in the inferior olive (IO) the trigeminal-solitary transition zone (5Sol) the lateral reticular nucleus (LRt) and other parts of the medullary reticular formation (Rt) Pde1c (A–D) an Zbtb16 (E–N) expression (A) Brightfield image of a parasagittal section of an adult brain processed for Pde1c detection showing specific expression in the r9 portion of Sp5 (B,C) Magnification detail of the latter image (D) Brightfield image of a parasagittal section from a P28 brain showing the aforementioned expression pattern for Pde1c (E) Brightfield image of a parasagittal section of an adult brain processed for Zbtb16 detection (F,G) Magnification detail of the latter image (H) Brightfield image of a parasagittal section from a P28 brain showing the aforementioned expression pattern for Zbtb16 (I–N) Pairs of brightfield and color-coded images of respective coronal sections of an adult brain at the levels of r8 Note the specific expression of Zbtb16 in the r9 portion of Sp5 as compared to few positive cells in the r8 and r10 portions of this nucleus Other structures close to Sp5 -external cuneatus nucleus (ECu) Amb and LRt- are indicated as reference landmarks (A,D,E,H–N) Scale bars = 500 μm; (B,C,F,G) scale bars = 300 μm The molecular mappings showed that the genes Baiap3 and Camk2a are expressed selectively in KF, while they show no significant expression in Pr5 (Figures 4A,B,D and data not shown). On the other hand, Irx2 is expressed by the Pr5 cells of r2, with no expression in KF (Figure 5A) These patterns thus delimit the r1/r2 boundary as the rostral end of the trigeminal column This region is primarily characterized by the mass of the migrated pontine nuclei aggregated at the ventral surface of r3 and r4; they originate at the rhombic lip from rhombomeres r6-r8 (Di Meglio et al., 2013; Tomás-Roca et al., 2016; Figure 3) As regards our molecular characterization, Irx2 is expressed in the r2 portion of Pr5, with some disperse cells within r3, as can be observed in adult and juvenile sections (Figures 5A–H and Table 1) this gene marks a rhombomeric subdivision of Pr5 in some sections Baiap3 appears expressed in scarce cells in r2 as compared to r3 (data not shown) Similarly, Calb2 is significantly expressed in the r2 and r3 portions of Pr5, together with some positive cells in r4 (Figures 10J–L) Irx2 and Kcng4 display also expression in more caudal hindbrain regions abuts the first myelomere or spinal cord segment (my1) occupied ventromedially by the pyramidal decussation (pyx) The rostrocaudal axis of the medulla oblongata follows more or less a straight horizontal direction Its pronounced curvature (together with possible variations in the angle of the sectioning plane) causes that coronal sections through r11 or even r10 cross obliquely the hindbrain/spinal cord (r11/my1) boundary including thus the pyx in the same sections than the caudalmost rhombomeres the boundary between the interpolar and oral subnuclei of Sp5 is uncertain on a morphological basis so that in the literature it has been located tentatively at different positions (see section “Discussion”) The dorsal horn of the first 2 myelomeres or cervical segments (C1 and C2) is functionally a continuation of the caudal subnucleus of Sp5 as commented above; it receives the terminals of the descending primary trigeminal afferents and has a largely similar cyto- and myeloarchitectural aspect In our molecular mappings we noted a continuous pattern from r10/r11 to my1 concerning the expressed genes and their laminar pattern so that in our figures showing gene expression we have drawn the contour of Sp5 as including the dorsal horn of my1 The eventual continuation of this pattern into my2 could not be ascertained in the studied material since seemingly the analyzed sagittal or coronal brain sections included only down to my1 Tac2 is expressed only in the gelatinous layer of the caudal subnucleus of Sp5 (Figures 9D–F). It displays a regionalized pattern within this subnucleus, with substantially fewer positive cells in r10 than in r11 or my1, as observed across juvenile and adult stages (Figures 9D,F; also compare Figure 9E with Figure 9B from the analyzed genes Tac2 is the only one displaying a rostrocaudal regionalized pattern within the caudal Sp5 as compared with the homogeneous rostrocaudal expression of the aforementioned Baiap3 and Tac1 Considering the set of r7, r8 and r9 Sp5 domains (that is, the medullary part of Sp5, after excluding the caudal subnucleus), it is jointly characterized by Irx2 expression, present as a shared pattern of scattered positive cells, in contrast with the absence of expression in the rest of Sp5 (the retropontine region -r5 and r6- and the caudal subnucleus -r10 and r11) (Figures 5E–H) We have described genoarchitecturally a segmental pattern of the trigeminal column attending to morphologic landmarks of the different rhombomeres and to the regionalized expression of genes that show specificity for one or several rhombomeric domains we comment our results in the context of previous studies and knowledge about trigeminal subdivisions hindbrain segmentation and molecular markers of trigeminal neurons the oral and interpolar Sp5 subnuclei would consist of the r4-r6 and r7-r9 portions of the plurineuromeric trigeminal column Therefore, considering these multiple rostrocaudal subdivisions of Sp5, plus the modern concept of a neuromeric hindbrain (Tomás-Roca et al., 2016; Martínez-de-la-Torre et al., 2018) a rhombomere-based framework needs to be developed that should facilitate the finding and characterization of trigeminal functional units which apparently remain partly hidden if only the tripartite classic schema is assumed An advantage of the rhombomeric model is that it would be underpinned by modern embryological and molecular criteria Such a model would be also a basis for interspecies comparison taking into account the conservation of the rhombomeric (neuromeric) pattern across vertebrates but later the descriptor “cryptorhombomeres” was thought to be more appropriate since these units are not false neuromeres only separated by given molecular properties these data suggest the existence of subtle molecular differences starting with differential Hox gene properties within the medullary and spinal portions of Sp5C which may lead to functional properties that would need to be explored Some of the molecular markers we report (Calb1 Tac2) have been previously described in relation to the regionalization of the trigeminal column A significant novelty of our study is the identification of a segment-like portion of the interpolar subnucleus (r9) that is positive for Calb1 and negative for Calb2 This had not been noticed previously possibly because the scarce use of parasagittal sections in classical neuromorphological analyses Another calcium-related gene, Camk2a (calcium/calmodulin-dependent kinase II α) is functionally involved in nociceptive pathways, as deduced from the study of mutant mice (Zeitz et al., 2004) It was described as expressed in neurons of the marginal and gelatinous layers of the spinal dorsal horn as well as in unmyelinated neurons of the trigeminal ganglion We show now that its expression extends to the superficial layers of the caudal Sp5 subnucleus in r10 and r11 with a pattern similar to that described in the spinal cord in addition to the expression in the non-laminar segment-like portion of the interpolar Sp5 in r9 This latter expression would support the correspondence of the r9 portion with the “caudal/interpolar transition zone,” which is involved in pain processing Tac1 and Tac2 encode, respectively, the neurotransmitters substance P and neurokinin B. Both genes, or their protein products, have been described in the superficial laminae of the dorsal horn and caudal trigeminal subnucleus (Del Fiacco and Cuello, 1980; Ribeiro-da-Silva and Hökfelt, 2000; Mar et al., 2012) We have found that their expression patterns display segment-related differences with Tac1 reaching up to the r10 trigeminal portion in a homogenous pattern and Tac2 showing less positive cells in r10 It would exist also the possibility of a role in the differentiated mature neurons where it is expressed all of which are involved in hindbrain segmentation Our results show that Mafb maintains its expression in the trigeminal derivatives within r5 and r6 in the adult brain suggesting additional roles in these neuronal populations Fn1 encodes the extracellular matrix component fibronectin. Its mRNA expression appears in telencephalic migrating neurons while in the adult brain it is restricted to the subiculum (Sheppard et al., 1995; Kashima et al., 2019) we describe that it is additionally expressed at least within given segmental portions of the trigeminal column Its possible function in these neuronal populations would need to be tested Pde1c encodes the calmodulin-dependent cyclic nucleotide phosphodiesterase and is expressed in olfactory sensory neurons (Yan et al., 1995), in migrating neurons in the cerebellum and cerebral cortex (Gong et al., 2003), and in lamina I neurons of the dorsal horn involved in nociception, principally at lumbar levels (Torsney et al., 2006) our results show a specific expression of this gene in the r9 part of Sp5 delimiting the caudal/interpolar transition zone We have described a novel feature of the expression pattern of this gene namely its specific location in a single rhombomeric domain (r9) We have described a strong and specific expression in the caudal Sp5 trigeminal nucleus so that it might have a role in the nociceptive function of this region we have tentatively traced the interrhombomeric boundaries within the mouse trigeminal sensory column according to current knowledge collected in the prosomeric model based either on descriptive morphologic or molecular gene expression data or on experimental approaches involving fate maps with avian chimeras or transgenic mice Some of the dispersed sparse populations described in some parts of the trigeminal column may correspond to cells migrated tangentially from neighboring rhombomeric modules where a similar labeling is massive in order to ascertain precisely our delimitation of the rhombomeric domains as well as possible cell migration or intermingling across rhombomeric limits it would be necessary to perform future ad hoc experiments These would ideally combine our reported genetic markers of trigeminal subdivisions with experimental fate maps for each rhombomere One difficulty we encountered was the obliquity of some interrhombomeric limits due to the pontine and cervical flexures of the neural tube (Figure 1A). Moreover, it should be remembered that the rhombomeric domains are known to adopt a degree of obliquity with respect to the mediolateral axis (Figure 1B) with their medial parts displaced anteriorly with respect to their lateral portions These circumstances cause that standard coronal sections are necessarily variously oblique in relation to rhombomeric domains and their limits We have tried to solve this handicap in coronal sections by taking into account the well-known rhombomeric landmarks found relatively close to the trigeminal column like the trigeminal and facial motor nuclei or the fibers of the facial and vestibulocochlear nerves our rhombomeric map is consistent with the observations in sagittal sections which display well some of the mentioned deformations An issue that would require further insight is the possible dorsoventral regionalization within each of the trigeminal rhombomeric domains related to mandibular the dorsomedial portion of the Pr5 and Sp5) together with a more exhaustive molecular characterization of the radial layering in the caudal subnucleus of Sp5 It would also deserve further study the transition from the medullary to the cervical portions of the caudal subnucleus considering that it crosses over such a significant landmark as the brain/spinal cord junction we have succeeded in reporting segmental (transverse rostrocaudally ordered) subregions along the trigeminal column based on molecular and fate mapping criteria delimiting rhombomeric domains Our results are reliable since they are reproduced from sagittal to coronal section series of the adult brain We focused this analysis on the adult brain so that the segmental map should not be regarded as a transient developmental state but as an instance of the neuromeric pattern persisting in the mature brain This mapping of the trigeminal system can provide the basis for further functional and/or pharmacological studies considering the molecular and embryological uniqueness of the different rhombomeric subdivisions of this structure The animal study was reviewed and approved by the University of Murcia Committee for Animal Experimental Ethics MM-D-L-T prepared and processed the brain sections from AZIN2-lacZ mice and FM analyzed the data and performed the image analysis and figure preparation All authors contributed to the article and approved the final submitted version This work was funded by a Seneca Foundation grant to LP (Autonomous Community of Murcia reference: 19904/GERM/15; 5672 Fundación Séneca; project name: Genoarchitectonic Brain Development and Applications to Neurodegenerative Diseases and Cancer) IG-G was the recipient of a predoctoral fellowship from the FPU program at the University of Murcia Infrastructure support was provided by the University of Murcia and IMIB-Arrixaca Institute of Murcia The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential 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persistent pain Aroca P and Marín F (2021) Molecular Segmentation of the Spinal Trigeminal Nucleus in the Adult Mouse Brain Copyright © 2021 García-Guillén, Martínez-de-la-Torre, Puelles, Aroca and Marín. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) distribution or reproduction in other forums is permitted provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited in accordance with accepted academic practice distribution or reproduction is permitted which does not comply with these terms *Correspondence: Faustino Marín, bWFyaW5AdW0uZXM= Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher 94% of researchers rate our articles as excellent or goodLearn more about the work of our research integrity team to safeguard the quality of each article we publish almost suburban feel at Mexico City’s Mercado Martínez de la Torre in Colonia Guerrero the market’s surrounding holes-in-the-wall cantinas are populated mostly by men in the laid-back stepdad look of plaid shirts tucked into comfortable jeans with cell phone holsters and tennis shoe/hiking boot hybrids and a few blocks off the market can get a little sketchy but it’s all full bellies and family smiles between Zarco and Calle Soto Martínez de La Torre Market is the cultural and economic center of Colonia Guerrero and one of the best food markets in the city The symphony of scissors snipping chickens into vendible pieces and the smell of piles of ripe guava make their way into the market’s exceptionally wide walkways – a gawking tourist’s dream The quality of meats and sausages is way above par and there are mountains of excellent moles to choose from You can safely get lost wandering the abundance of delicious flavors but we’ve picked out some particular culinary gems at Mercado Martínez de la Torre Named for actress friends of the taquería’s owner “Lola the Truck Driver,” played by Rosa Gloria Chagoyán and “The Bottle Cap,” played by Carmen Salinas La Corcholata is the most publicized eatery in the market and run as such – super attentive staff with pressed uniforms and name tags The buttoned-up look is a little out of place in Guerrero But the local favorites are the beef or chicken tacos with French fries and a perfectly sweet and frothy horchata to balance the salt and heat You don’t have to wait until the weekend for barbacoa at Mercado Martínez de la Torre and their name is accurate – El Mejor is truly the best barbacoa in the market The slow oven-roasted mutton is brought in daily from Mexico City’s agrarian outpost Milpa Alta – the city’s southernmost borough known for its sheep Ignacio Ramírez has manned the butcher block at El Mejor since 2000 and his barbacoa comes off the knife appropriately tender and stringy with the deep buttery barbacoa taquito and a bowl of consommé for only 50 pesos is a wonderful pick-me-up any day of the week and La Güera’s crispy carnitas pork bits are still one of the best reasons to come to the market half-a-pig-sized chicharrón or just sidle up to the counter for a quick taco La Güera won’t let you down on the pork front Just outside the market entrance on Zarco is the tiny fried fish stand The fish – Mexican sierra – are in the mackerel family They’re about the size of a toothpaste tube on the spot with a bit of mayo and hot sauce the ideal last stop for the sweet tooth on the Martínez de la Torre tour Their nostalgic milkshakes and shaved ices will warm your heart and cool your core on a hot day Or go nuts with a fruit frappe rainbow of your design If you’re looking for a drink of the alcoholic variety head across Mosqueta/Eje 1 to La Frida for a pulque – the drinking person’s milkshake fermented agave sap beverage provides a distinctive tingly buzz providing a no-nonsense pulque experience – loud music While the banner promotes over a dozen sweet and savory flavors they’re likely to have only a couple in stock Try celery or oatmeal flavored for something delicious and mellow and ask for some baking soda to shake into it (for the extra bubbles) if you’re really looking to impress Watch traffic whizz by out of the open doors and enjoy your pulque in a cracked glass • Mercado Martínez de la Torre is on Eje 1/Mosqueta This is the 11th in a series on the bazaars ADVERTISE WITH MND COMMUNITY GUIDELINES Subscription FAQ's Privacy Policy Mexico News Daily - Property of Tavana LLC Metrics details Defining the trajectory of cells during differentiation and disease is key for uncovering the mechanisms driving cell fate and identity trajectories of human cells remain largely unexplored due to the challenges of studying them with human samples we investigate the proteome trajectory of iPSCs differentiation to hepatic stellate cells (diHSCs) and identify RORA as a key transcription factor governing the metabolic reprogramming of HSCs necessary for diHSCs’ commitment Using RORA deficient iPSCs and pharmacologic interventions we show that RORA is required for early differentiation and prevents diHSCs activation by reducing the high energetic state of the cells While RORA knockout mice have enhanced fibrosis RORA agonists rescue multi-organ fibrosis in in vivo models RORA expression correlates negatively with liver fibrosis and HSCs activation markers in patients with liver disease This study reveals that RORA regulates cell metabolic plasticity suggesting that understanding the trajectory of HSCs in development and disease may help to identify molecular pathways suitable for preventing HSCs activation or promoting the regression to a quiescent phenotype thus mitigating fibrogenesis in chronic liver diseases we performed a time-resolving proteome characterization of iPSCs to functional HSCs (diHSCs) which mimic phenotypic and functional characteristics of primary human HSCs we identified RORA as a TF regulating both diHSCs differentiation and favouring the maintenance of a quiescent phenotype by modulating the metabolic state of the cells downregulation of RORA mediates a metabolic switch increasing glycolysis and mitochondrial oxidative phosphorylation system (OXPHOS) we confirmed the anti-fibrogenic role of RORA in hepatic and extrahepatic pericytes thus positioning RORA as a potential antifibrogenic multiorgan target this work shows the potential of studying cell trajectories along differentiation and disease progression this broad characterization enabled the construction of a proteomic roadmap detailing the differentiation of diHSCs from iPSCs these data confirm that iPSCs progressively acquire the stellate proteomic phenotype along differentiation thus enabling the use of diHSCs to further investigate their role in cell commitment and disease Principal component analysis (PCA) of the proteome differentiation profile showed a time-dependent distribution of samples along the differentiation process (Fig. 2A). Proteins comprised in PC1 explained stellate cell commitment across time, as cells gained in proteins related to collagen metabolism, ECM organization, or wound healing processes. A Principal component analysis (PCA) of the proteome during differentiation reveals a time-dependent separation of data indicating distinct stages of the differentiation process B Pearson correlation analysis identifies three phases of differentiation towards diHSCs: Phase 1 from day 0 (D0) to day 4 (D4) and the final maturation phase (Phase 3) from day 8 (D8) to day 12 (D12) C Dot plot showing protein enrichment throughout the differentiation process D Volcano plot comparing phases 1 and 2 highlights the enrichment of mesothelial markers (DESP WLS) and transcription factors (TFs) predicted in silico to regulate this transition E Volcano Plot showing the comparison between phases 2 and 3 shows an increase in collagen and extracellular matrix (ECM) proteins (COL5A1 along with predicted TFs modulating this transition F Volcano plot displaying the differentially expressed proteins between phase 3 (diHSCs) and primary HSCs as well as the in silico predicted TFs involved in regulating the adult hepatic stellate cell phenotype G Transcriptomic comparison of primary human activated and quiescent stellate cells with diHSCs focusing on the predicted TFs modulating the adult stellate cell phenotype These results indicate that the diHSCs differentiation takes place in three main phases which mimic key stages of embryonic development During diHSCs differentiation, the gene expression of RORA showed a significant increase at day 4 of differentiation and progressively declined at days 6, 8, 10, lowering the pHSCs expression at day 12 (Fig. 3A). E Principal Component Analysis (PCA) showing transcriptomic differences between cells treated with the RORA agonist SR1078 from day 2 compared to the untreated group F GOs upregulated (red) and downregulated (blue) in cells treated with the RORA agonist G Heatmap of mesoderm markers in treated and untreated cells along differentiation with SR1078 H Heatmap of mesenchymal markers in treated and untreated cells along differentiation with SR1078 I Reactome pathways upregulated (red) and downregulated (blue) in cells treated with the RORA agonist J Representative microscopy images of diHSCs from WT iPSC- RORA+/- and treated iPSC- RORA+/- with the RORA agonist (SR1078) at day 12 K Gene expression of stellate cell markers (LRAT RELN and ACTA2) of WT and treated iPSC- RORA+/- with the RORA agonist (SR1078) at day 12 n = 3 independent differentiations with two replicates L Representative microscopy images of passaged (P1) diHSCs from WT and iPSC- RORA+/- SR1078 treated M Gene expression of activated stellate cell markers (ACTA2 N Immunoflourescence images for COLLAGEN and aSMA in WT passaged cells treated and untreated with RORA agonist SR1078 for 24 h O Gene expression of quiescent (LRAT and LHX2) and activated (ACTA2 and COL1A1) markers in cells treated with the RORA agonist at passage P GOs upregulated (red) and downregulated (blue) in treated cells with the RORA agonist at passage Q Reactome pathways upregulated (red) and downregulated (blue) in cells treated with the RORA agonist at passage R Gene expression of activated (markers in cells treated with the RORA agonist at passage after TGFβ stimulation (10 ng/μL) during 24 h and 7 days n = 3 independent passaged cells with two replicates S Gene expression of activated markers (ACTA2 COL1A1 and LOX) in liver spheroids after SR1078 3 mM treatment during 24 h n = 3; Significant differences are indicated as *p < 0.05 T Gene expression of activated markers (ACTA2 COL1A1 and LOX) in liver spheroids after TGFβ stimuli during 24 h and SR1078 3 mM treatment during 24 h more n = 3 independent spheroids experiments with 10 biological pool replicates each; All data is presented as mean ± SEM **p < 0.01 was determined by One sample t and Wilcoxon test These findings suggest that RORA plays a pivotal role in regulating cellular metabolism during diHSCs differentiation these data indicate that RORA might act as a transcriptional regulator of the mesoderm and mesenchymal specification these results position RORA as a potential transcriptional regulator of mesoderm and mesenchymal differentiation and key for the acquisition of the quiescent phenotype of diHSCs Differentiated cells treated with the RORA agonist SR1078 showed enrichment in the signature of predicted genes these results suggest that RORA plays a role in the deactivation of diHSCs and the maintenance of the quiescent diHSCs phenotype which can be mediated by blocking metabolic adaptations required for HSCs activation A Oxygen consumption rate (OCR) on day 4 WT control iPSC and iPSC RORA+/- treated and untreated from day 2 (B) OCR parameters of day 4 iPSC WT and iPSC RORA+/- treated and untreated from day 2 C Normalized adenosine triphosphate (ATP) production from OXPHOS and glycolysis of day 4 WT iPSCs and iPSC-RORA+/- treated and untreated from day 2 D Glucose consumption at day 4 of WT iPSC and iPSC RORA+/- differentiations treated and untreated from day 2 E Gene expression of key glycolytic and lipid synthesis markers (ALDOB ACACA) and mesoderm and mesenchymal markers (EOMES and VIM) F Immunofluorescence of mesoderm (EOMES) and mesenchymal (VIM) on day 4 of differentiated cells from WT RORA+/- and RORA+/- treated with the RORA agonist from day 2 G OCR passaged diHSCs treated with the RORA agonist for 24 h H OCR parameters of passaged diHSCs treated with the RORA agonist for 24 h I Normalized ATP production from OXPHOS and glycolysis of passaged cells treated and untreated with RORA agonist for 24 h J Glucose consumption at passaged cells treated and untreated with RORA agonist for 24 h mitochondrial respiration and fatty acid beta-oxidation of passaged cells treated and untreated cells with RORA agonist for 24 h L OCR of passaged cells treated with TGFβ 10 ng/mL for 24 h and rescued with RORA agonist treatment for 24 h more M OCR parameters of passaged cells treated with TGFβ 10 ng/mL for 24 h and rescued with RORA agonist treatment for 24 h more N Normalized ATP production from OXPHOS and glycolysis of TGFβ activation model rescued with the RORA agonist for 24 h O Intracellular glucose levels of TGFβ activation model rescued with the RORA agonist for 24 h P Gene expression of key glycolytic and lipid synthesis markers (ALDOB GS6P and ACACA) of the TGFβ activation model rescued with the RORA agonist for 24 h n = 3 independent differentiations; All data is presented as mean ± SEM **p < 0.01 was determined by One sample t and Wilcoxon test in one two one comparisons and ANOVA 1-way test was performed in the comparison of three experimental groups therefore suggesting that RORA’s influence on mesoderm differentiation may be context-dependent these results indicate that RORA acts as a metabolic modulator in diHSCs trajectory which impacts the phenotype of diHSCs at different stages including initial cell commitment and the quiescent cell identity at the end of differentiation These findings suggest that RORA agonist treatment promotes a lipidomic profile consistent with quiescent stellate cells A Schematic overview of a CCl4 fibrotic liver model in staggerer mice (B) Representative images of the livers in 7 staggerer (sg/sg) and 7 WT mice after 4 weeks of CCl4 treatment H&E staining and picrosirius staining for the staggerer and wildtype (WT) counterparts and immunohistochemistry of αSMA C Gene expression of fibrogenic and HSCs activation markers (Acta2 Fn1 and Col1a2) in 7 sg/sg and 7 WT mice after CCl4 treatment Significant differences are indicated as *p < 0.05 D Liver/body ratio of 5 Lrat-Cre-/Rora wt/fl (Cre-) mice vs 5 Lrat-Cre + /Rora wt/fl (Cre + ) after 4 weeks of CCl4 I.P E Hydroxyproline content of the livers of 5 Cre- and 5 Cre+ mice after 4 weeks of CCl4 I.P F Gene expression of Rora and Lhx2 in 5 Cre- and 5 Cre+ after 4 weeks of CCl4 I.P G Representative images of the livers of the five Cre- and five Cre+ mice after 4 weeks of CCl4 I.P these results confirm the role of RORA during fibrosis in vivo therefore supporting our in vitro findings and confirming its specific impact on HSC-driven fibrosis A Illustration of the experimental design of a CCl4 model treated with SR1078 B RORA gene expression of CCl4 model treated with SR1078; n = 14 C Representative images of the livers after 4 weeks of CCl4 I.P H&E staining and Picrosirius staining for the untreated (Vehicle) and treated (SR1078) group and immunohistochemistry of αSMA D Gene expression of fibrogenic and HSCs activation markers in 7 untreated (Vehicle) and 7 treated (SR1078) mice (Acta2 E Illustration of the experimental design of Isoproterenol cardiac injury model performed in two groups of 5 mice each F Representative images of the hearts of vehicle (5 mice) isoproterenol (ISO) (5 mice) and ISO and RORA agonist (5 mice) group of Masson’s trichrome G Rora gene expression of 5 vehicle mice and 5 ISO mice group H Gene expression of Rora and fibrogenic markers (Acta2 and Col3a1) and the hypertrophy marker Acta1 in 5 ISO mice and 5 ISO and RORA agonist mice group suggesting a positive effect of RORA agonism against cardiac fibrosis independently of hypertrophy development These results indicate that pharmacological activation of RORA is a promising strategy to mitigate fibrosis in multiple organs demonstrating the therapeutic potential of targeting RORA to control fibrogenesis Gene expression data is presented as mean ± SEM Correlation analysis data was performed using pearson correlation analysis we aimed to understand the differentiation trajectory of hepatic stellate cells (HSCs) from iPSCs to mature phenotype leading to the identification of key molecular drivers for diHSCs identity and activation Understanding HSCs paths in differentiation and activation is essential to delineate how cells acquire their phenotype and how this is lost during disease thereby providing pathophysiological insights for the development of novel anti-fibrogenic treatments While animal models have advanced our understanding of disease progression translating these findings to humans remains complex and embryonic differentiation trajectories are difficult to fully capture Human iPSC differentiation protocols offer a valuable alternative enabling the study of dynamic cell paths during differentiation we performed a time resolving proteomic characterization of the differentiation of diHSCs leading to the identification of a unique protein signature of this differentiation the comprehensive proteomic data generated in this study will be a useful resource for researchers interested in understanding different aspects of the biology of human HSCs and fibrosis we lacked a detailed understanding of the human development of HSCs and therefore we cannot ensure that diHSCs differentiation is faithfully recapitulating all the features and the natural path of the human embryonic development our results indicate that RORA is not only implicated in diHSCs differentiation and maturation Although their role at the different stages of HSCs maturation is not totally understood these observations are consistent with our findings where we also find RORA upregulated at initial stages of differentiation and downregulated during activation suggesting that they may play a role throughout development and disease This highlights the potential of iPSC-differentiation approaches for investigating cell trajectories and diHSCs are therefore a powerful tool that may be useful for better understanding the molecular drivers of cell differentiation and disease ChIP-Seq analysis of RORA would have been valuable for further insights but despite efforts suboptimal antibody quality for these assays or inefficient capture prevent the analysis A core feature of fibrosis is the activation of fibrogenic cells which undergo significant metabolic changes during activation Our data suggest that diHSCs serve as a model for pericytes in other tissues and that RORA regulates pericyte activation in multiple organs making it a promising therapeutic target for anti-fibrogenic treatments therefore suggesting a downregulation in fatty acid production diHSCs treated with SR1078 shifted their lipidomic profile towards a more quiescent phenotype confirming the role of RORA in regulating lipid metabolism in diHSCs using two in vitro models we found that activated diHSCs showed an increase in OXPHOS and glycolytic flux as compared to the cells treated with RORA agonist thus reducing the metabolic requirements of the diHSCs and preventing their activation our results showed similar results at early stages of differentiation as iPSC-RORA+/- presented a metabolic profile characteristic of undifferentiated cells that was modified when treated with the RORA agonist indicating that RORA regulates the metabolic activity during early stages of differentiation These results indicate that metabolic flexibility is essential throughout the diHSCs differentiation trajectory and impairs the final phenotype of the cell This metabolic regulation is mediated in part by RORA as alterations in RORα transcriptional activation are implicated in modulating the cellular physiological state Our study proposes that changes in RORA expression levels are one of the underlying causes of the observed metabolic state alterations in HSCs and therefore is responsible for the repression of activation being a potential target for anti-fibrogenic therapy our study underlines the importance of examining cell trajectories during both differentiation and disease We show that HSC differentiation and fibrosis mechanisms can be effectively modeled using iPSC-derived cells enabling the identification of key transcription factors like RORA that regulate cell identity and fibrogenesis and can lead to the development of new therapeutic strategies to mitigate fibrosis across multiple tissues iPSCs cell line For the proteome characterization and all the subsequent in vitro assays we have used the human KULi003-A iPSC line (BioSamples Cat#SAMEA110177409); iCas9-FL-BCL were used for the inducible system was obtained from Banco Nacional de Líneas Celulares (BNLC) HepG2 cell line The HepG2 cell line was purchased from Sigma (Cat#8511430) Staggerer mice All animal experiments were approved by the Ethics Committee of Animal Experimentation of the University of Barcelona and were conducted in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals For the fibrogenic experiments in staggerer mice (sg/sg) mice homozygous for the staggerer spontaneous mutation both genders of between 8-10 weeks of age were used Staggerer mice were obtained from the laboratory of Benédicte Antoine The presence of the floxed allele and the Cre transgene in offspring was confirmed by genotyping including Cre- and Cre+ were used to stablish a CCl4 model of 4 weeks with 0.5 mL/kg of CCl4 (diluted in corn oil) twice a week Wild-type (WT) mice All animal experiments were approved by the Ethics Committee of Animal Experimentation of the University of Barcelona and were conducted in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals C57BL/6 J female and male mice of 8-10 weeks of age were used Human samples Liver biopsies from patients diagnosed with liver diseases or controls from liver resections of metastic tumors who were admitted to the Liver Unit at the Hospital Clinic of Barcelona were utilized for this study (HCB/2013/8175) Informed consent was obtained from the patients Cat# 562822) was added to the iPSCs 3 h (h) before nucleofection HiFi Cas9 Nuclease V3 (IDT) was incubated with 120 pmol Alt-R® CRISPR-Cas9 sgRNA (IDT) at 25 °C for 10 min (min) 200,000 cells were dissociated with Accutase (Gibco washed twice with PBS without Ca and Mg and resuspended with 20μl of P3/S1 Buffer RNP complex was added to the cells prior to nucleofection in a 20ul cuvette Cells were nucleofected with the 4-D Nucleofector System (Lonza Nucleofected cells were cultured in a 12-well plate 1000 cells were seeded at a single-cell level in a 100 mm plate to generate single-cell colony Genotyping was performed by PCR and Sanger sequencing from single-cell colonies to analyze the genotype Two rounds of single-cell cloning were performed to ensure a single-cell clone For the experiments concerning the constitutive mutation we used as controls RORA iPSCs wild-type (WT) obtained after the nucleofection process the iCas9-FL-BCL cell line was treated with doxycycline (1 µg/mL) Liver spheroids were generated using diHSCs and HepG2 in a 1:2 ratio 3000 cells were seeded in a non-adherent U-Shape bottom 96-well plate in DMEM Glutamax and experiments started after 5 days of culture liver spheroids were treated with SR1078 at 3 mM for 24 h; 10 ng/mL for TGFβ during 48 h liver spheroids were also treated with SR1078 3 mM Samples were collected for further analysis Rat NCMs were obtained from the ventricles of 1- to 3-day-old Sprague-Dawley rats hearts were digested with a collagenase solution (Collagenase Type I; Life Technologies Cat# 17100017) and differential plating was performed NCMs were plated at a density of 0.5 × 106 cells/well on 6-well culture plates and grown for 24 h NCMs were treated with phenylephrine PE (10 μmol/L; Sigma as a pathological hypertrophic growth factor for 24 h Rat neonatal cardiac fibroblasts (NCFs) were isolated from the ventricles of 1- to 2-day-old Sprague-Dawley rats Passage 1 and 3 cells were plated at a density of 0.3 × 105 cells/cm2 on culture dishes and grown for 24 h day 12 and pHSCs were washed once with DPBS -/- and 500 mL of buffer lysis was added to the culture (7 M urea 2 M thiourea and CHAPS 4%) following the filter aided FASP protocol described elsewhere 22 with minor modifications Trypsin was added at a trypsin:protein ratio of 1:50 and the mixture was incubated overnight at 37oC dried out in a RVC2 25 speedvac concentrator (Christ) Peptides were desalted and resuspended in 0.1% FA using C18 stage tips (Millipore) Samples were analyzed in a hybrid trapped ion mobility spectrometry—quadrupole time of flight mass spectrometer (timsTOF Pro with PASEF Bruker Daltonics) coupled online to a nanoElute liquid chromatograph (Bruker) The sample (200 ng) was directly loaded in a 15 cm Bruker nanoelute FIFTEEN C18 analytical column (Bruker) and resolved at 400 nl/min with a 30 min gradient The column was heated to 50oC using an oven Protein identification and quantification was carried out with MaxQuant software using default settings Searches were performed against a database consisting of human protein entries (Uniprot/Swissprot) with precursor and fragment tolerances of 20ppm and 0.05 Da Only proteins identified with at least two peptides at FDR < 1% were considered for further analysis Data (LFQ intensities) was loaded onto a Perseus platform 25 and further processed (log2 transformation Protein abundances were normalized against the day 0 for each individual The lipidomics assay was conducted in collaboration with Olobion cells were harvested using trypsin and subsequently washed with PBS to eliminate residual media and contaminants We added 275 µL of methanol directly to the cells along with a ball for homogenization 1 mL of MTBE was incorporated into the mixture we collected 100 µL of the upper organic phase and resuspended the residue in 100 µL of methanol containing an internal standard The lipids were separated using an ACQUITY UPLC BEH C18 column maintained at 65  °C and analyzed on a ZenoTOF 7600 system (SCIEX) A sample volume of 4 μL was injected in both ESI positive and negative modes and the lipidomics data were processed using oloMAP 2.0 Human liver sections were included in OCT and maintained at -80 °C They were then washed in PBS for a few seconds and blocked in 100 µL 5% BSA in PBS + 0,3% TritonX100 per tissue section during 1 h Tissue sections were incubated overnight with primary antibodies (RORA 1:50 Cat#ab70061) in 5%BSA/PBS/Triton at 4 °C in a dark moist chamber the sections were incubated 1 h with secondary antibodies in 5%BSA/PBS/Triton at room temperature (RT) in a dark moist chamber Tissue sections were mounted using the Vectashield Mounting medium for fluorescence with DAPI To test the physiological role of RORA upon liver fibrosis in vivo we established a carbon tetrachloride (CCl4) model based on intraperitoneal (I.P.) injection of 0,5 ml/kg of CCl4 diluted in corn oil Mice were injected two days a week during four 4 weeks Blood and liver tissues were collected for further analysis To examine the impact of RORA KO specifically in HSCs in the context of liver fibrosis This was accomplished by crossing RORA floxed (fl/fl) mice with Lrat-Cre mice The presence of the floxed allele and the Cre transgene in the offspring was confirmed through genotyping n = 5 Lrat-Cre+ Rorafl/− and n = 5 Lrat-Cre- Rorafl/− mice were injected i.p with 0.5 mL/kg of CCl4 (0.5 mL/kg of CCl4 diluted in corn oil) twice a week for four weeks Animals were sacrificed 24 h after the last CCl4 injection and blood and liver tissues were collected and analyzed 14 C57BL/6 J mice were injected twice a week with CCl4 0,5 ml/kg (25% corn oil) during 4 weeks mice were divided into two groups: 1) n = 7 mice were injected ip with SR1078 (10 mg/kg) twice a week; 2) n = 7 mice were injected ip with vehicle DMSO at the same concentration SR1078 was diluted in 5% DMSO to a final concentration of 2 mg/ml n = 10 C57BL/6 J mice were administered isoproterenol (Sigma Aldrich Cat#SLC62971) by continuous infusion of 60 mg/kg/day using minipumps (Model 1007D animals were divided into two groups: 1) n = 5 treated with SR1078 (10 mg/kg) every 48 h IP and 2) n = 5 injected IP with the vehicle during 1 n = 19 C57BL/6 J mice underwent ligation of the left ureter and starting 3 days after surgery the mice were divided into two groups: 1) n = 10 treated with SR1078 (10 mg/kg) from day 3 after surgery until day 15.; 2) n = 9 treated with vehicle from day 3 after surgery for 15 Blood and liver heart and kidney tissues were collected for further analysis The amount of collagen in liver tissues was determined by measuring the content of hydroxyproline using a Hydroxyproline assay kit (Abcam Cat#MAK008-1KT) as described by the manufacturer Absorbance was measured at 540 nm using a FLUOstar OPTIMA FL reader (BMG LABTECH) Serum from mice included in the experimental studies was collected and analyzed for alanine transaminase (ALT) phosphatase alkaline (AP) and lactate dehydrogenase (LDH) values GraphPad Prism v8.0 was used for the statistical analyses The D’Agostino-Pearson omnibus normality test Anderson-Darling test and Shapiro-Wilk normality test were performed to assess data distribution For statistical analysis of parametric data the two-tailed unpaired Student’s t test was used for groups of two; one-way ANOVA followed by Sidak multiple comparison posthoc tests were used for comparison of more than two groups the Mann-Whitney U test was used for groups of two while the Kruskal-Wallis test followed by the Dunn multiple comparison posthoc test was used for comparison of more than two groups Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article Building bridges between fields: bringing together development and homeostasis Mesenchymal origin of hepatic stellate cells and perivascular mesenchymal cells during mouse liver development Septum transversum-derived mesothelium gives rise to hepatic stellate cells and perivascular mesenchymal cells in developing mouse liver Targeting metabolic dysregulation for fibrosis therapy Association between fibrosis stage and outcomes of patients with nonalcoholic fatty liver disease: a systematic review and meta-analysis Hepatic stellate cells as key target in liver fibrosis Hedgehog controls hepatic stellate cell fate by regulating metabolism Hedgehog-YAP signaling pathway regulates glutaminolysis to control activation of hepatic stellate cells Acetyl-CoA carboxylase inhibition disrupts metabolic reprogramming during hepatic stellate cell activation A deactivation factor of fibrogenic hepatic stellate cells induces regression of liver fibrosis in mice GATA4 induces liver fibrosis regression by deactivating hepatic stellate cells Age-related phenotypes in the staggerer mouse expand the RORα nuclear receptor’s role beyond the cerebellum The ‘CholesteROR’ protective pathway in the vascular system Melatonin suppresses activation of hepatic stellate cells through RORα-mediated inhibition of 5-lipoxygenase inhibits fibrogenic activation of hepatic stellate cells via suppression of SMAD3 Generation of hepatic stellate cells from human pluripotent stem cells enables in vitro modeling of liver fibrosis Directed differentiation of human induced pluripotent stem cells to hepatic stellate cells Differentiation of hepatic stellate cells from pluripotent stem cells Cell-type-resolved proteomic analysis of the human liver A hepatic stellate cell gene expression signature associated with outcomes in hepatitis C cirrhosis and hepatocellular carcinoma after curative resection Energy metabolism regulates stem cell pluripotency Agonist of RORA attenuates nonalcoholic fatty liver progression in mice via up-regulation of microRNA 122 Lipidomic profiling of rat hepatic stellate cells during activation reveals a two-stage process accompanied by increased levels of lysosomal lipids Liver-specific RORα deletion does not affect the metabolic susceptibility to western-style diet feeding Fate tracing reveals hepatic stellate cells as dominant contributors to liver fibrosis independent of its aetiology Molecular characterization of chronic liver disease dynamics: from liver fibrosis to acute-on-chronic liver failure Single-cell atlas of human liver development reveals pathways directing hepatic cell fates Single-cell transcriptomic analysis reveals a hepatic stellate cell–activation roadmap and myofibroblast origin during liver fibrosis in mice Targeting fibrosis: mechanisms and clinical trials MYC controls human pluripotent stem cell fate decisions through regulation of metabolic flux The power of plasticity—metabolic regulation of hepatic stellate cells Autophagy releases lipid that promotes fibrogenesis by activated hepatic stellate cells in mice and in human tissues Generation of two heterozygous GATA2 CRISPR/Cas9-edited iPSC lines Generation and characterization of a human iPSC cell line expressing inducible Cas9 in the “safe harbor” AAVS1 locus Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide expression profiles Download references is supported by Instituto de Salud Carlos III (ISCIII) “PI20/00765” 23-0356; Dissecting the role of the 14q32 region in hepatoblastoma “Hblast14” Worldwide Cancer Research; PRYCO223102ARME; Scientific Foundation of the Spanish Association Against Cancer (AECC); Q6922 (B-ORG) Plan Complementario de Biotecnología aplicada a la Salud del Plan de Recuperación Ministerio de Ciencia e Innovación and co-founded the European research and innovation program Horizon HORIZON-HLTH−2022-STAYHLTH-02 under agreement no R.A M-G is funded by Instituto de Salud Carlos III “FI18/00215” received a grant from the Ministerio de Educación C.M is funded by Centro de Investigación en Red Enfermedades Hepáticas y Digestivas (CIBERehd) is supported by Competitiveness (MINECO PID2020-15591RB-100) La Marató de TV3 (202001-32) and CERCA Programme/ Generalitat de Catalunya for institutional support is supported by La Marató de TV3 (202001-32) is supported by Deutsche José Carreras Leukämie-Stiftung (DJCLS 13 R/2022) was funded by MCIN/AEI/10.13039/501100011033/FEDER UE through the project grants PID2021-123652OB-I00 and RTI2018-097475-A-100; by MCIN/AEI/10.13039/501100011033 and El FSE invest in your future through the contract RYC 2016-19731; by Pfizer grant #77131383; P.R.B was funded by Ministerio de Universidades fellowships FPU19/05357; M.F.F is supported by Proyecto PID2020-119486RB-100 (funded by MCIN/ AEI/10.13039/501100011033) Proyecto LABAECC2024 (funded by Asociación Española Contra el Cáncer HORIZON-TMA-MSCA-Doctoral Networks 2021 (101073094) and Redes de Investigación 2022 (RED2022-134485-T) This study was funded by Instituto de Salud Carlos III through the Biobanks and Biomodels Platform and co‐funded by the European Union (PTC20/00013 PT23/00009 and PTC23/00002 to N.M.) and by Instituto de Salud Carlos III and European Union—Next Generation EU Plan de Recuperación Transformación y Resiliencia (TERAV/ISCIII RD21/0017/0018) to N.M; M.C was funded by Ramon y Cajal programme from the Ministerio de Ciencia e Innovación RYC2019-026662-I is funded by la Caixa Foundation “100010434” and the European Union’s Horizon 2020 under the Marie Skłodowska-Curie “847648” MCIN/AEI/ 10.13039/501100011033 and FEDER Una manera de hacer Europa The European Union grant agreement 101077312* those of the author(s) only and do not necessarily reflect those of the European Union Neither the European Union nor the granting authority can be held responsible for them”) Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS) Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd) Institute of Biomedical Research of Barcelona Spanish National Research Council (IIBB-CSIC) Maria Fernandez- Fernandez & Anna Moles Departament de Bioquímica i Biomedicina Molecular Institut de Biomedicina de la Universitat de Barcelona CIBER Fisitopatologia de la Obesidad y Nutrición Institute for Research in Biomedicine (IRB Barcelona) The Barcelona Institute of Science and Technology Paula Sànchez-Fernàndez-de-Landa & Antonio Zorzano Institut d’Investigació Biomèdica de Bellvitge (IDIBELL) Damia Romero-Moya & Alessandra Giorgetti Gene Regulatory Control in Disease Laboratory Center for Research in Molecular Medicine and Chronic Diseases (CIMUS) Instituto de Investigación Sanitaria de Santiago de Compostela (IDIS) UCL Institute for Liver & Digestive Health Institute for Bioengineering of Catalonia (IBEC) Barcelona Institute of Science and Technology (BIST) Catalan Institute for Research and Advanced Studies (ICREA) Basque Research and Technology Alliance (BRTA) Center for Cooperative Research in Biosciences (CIC bioGUNE) Department of Pathology and Experimental Therapeutics Alessandra Giorgetti & Marta Varela-Rey Department of Biochemistry and Molecular Biology Oportunius Research Professor at CIMUS/USC Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM) interpreted the results and wrote the manuscript have a patent (EP2016/079464) regarding the hepatic stellate cell differentiation The remaining authors declare no competing interests Nature Communications thanks Scott Friedman reviewer(s) for their contribution to the peer review of this work Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1038/s41467-025-56024-4 Anyone you share the following link with will be able to read this content: a shareable link is not currently available for this article Sign up for the Nature Briefing newsletter — what matters in science Are you ready to join the thousands of companies who rely on Blue Book to drive smarter decisions We’d love to show you what Blue Book can do for you Drop us a line– we’ve been waiting for you Three hundred and fifty banana producers from the Mexican municipalities of San Rafael and Martinez de la Torre are facing the worst marketing crisis in the last 30 years An excess of fruit in Tabasco and a frost that damaged production in this area cause prices to drop so much that farmers have been forced to throw away their crops or feed them to livestock banana market consists of imports from Mexico such as Hurricane Grace which hit Veracruz in August 2021 and resulted in farmers having to spend their savings to rebuild their plantations because there was no federal support; and the increase of 200-300 percent for fertilizer due to global supply chain problems and the conflict between Ukraine and Russia stated that production costs are $2.50-3.00 pesos per kilo while it is sold at the market for 80 cents He said the surplus in Tabasco has flooded the national market and the frosts from December 2021 caused the banana in the region to be affected and unable to compete in quality and that causes it to lose value in the market because it no longer turns yellow Veracruz is the fourth largest producer in the country Get access to all the news and analysis you need to make the right decision --- delivered to your inbox the country’s fastest-growing supplier in Q1 USDA Secretary Rollins says she has been at the negotiating table tirelessly advocating for American agriculture The 2024-25 Chilean season has concluded with shipments of more than 90,000 tons of fresh blueberries Chipotle Mexican Grill announced it has signed a development agreement to open restaurants in Mexico for the first time Peru exported 562,093 tons of table grapes representing a 7.4 percent increase compared to the previous campaign The Department of Commerce’s decision to withdraw from the 2019 U.S.-Mexico Tomato Suspension Agreement is not a victory for anyone This website is using a security service to protect itself from online attacks The action you just performed triggered the security solution There are several actions that could trigger this block including submitting a certain word or phrase You can email the site owner to let them know you were blocked Please include what you were doing when this page came up and the Cloudflare Ray ID found at the bottom of this page South Side Weekly Leer en español This story was published in collaboration with La Voz Chicago the CEO and founder of CashDrop—an app designed to offer businesses a free mobile storefront that allows customers to eat the fees rather than the businesses—outlined a vision for the kind of company he wanted to build The once-undocumented immigrant from Mexico wanted to create an app that would give back to the Latino community “I’m not just interested in the money,” Ruben Flores-Martinez said The CashDrop platform can be adopted by small mom and pop shops and has been marketed to Latino communities in the U.S via Spanish-language ads on social media.  Locally, the app has been used in events like Chi Michelada Fest 2022. Even nonprofits like Healthy Hood Chicago have announced they will exclusively use CashDrop.  Recently, the app hosted a bilingual workshop alongside the Little Village of Commerce at which 22nd Ward Alderperson Michael Rodriguez encouraged street vendors to go cashless with the app as a way to mitigate robberies.  “America has this real obsession with growth,” Flores-Martinez said in the Harvard case study you have to be careful with it.’ Companies can get put into this fasttrack to keep pushing and spending more money until you lose control of where the company is going and just crash CashDrop has been good to Flores-Martinez. It landed him on the Forbes 30 Under 30 2021 Social Impact class list and most recently, in the Crain’s Chicago Business 40 Under 40 class of 2022. By mid-2020, CashDrop had raised around $2.7 million from investors such as Harlem Capital, Long Journey Ventures, and Michelle Phan. According to PitchBook the latest round of investment this past November netted CashDrop more than $11 million.  three former CashDrop employees have collectively taken to social media to denounce workplace bullying and a lack of transparency from Flores-Martinez At least one of these and another former employee said the stress of working at CashDrop led to hair loss South Side Weekly and La Voz Chicago interviewed these and other former employees of CashDrop joined CashDrop as an affiliate in July 2021 and eventually became event manager for the company “Me being Latino and all the employees being Latino was something that I wanted to be a part of,” he said in a testimonial he shared on Instagram.  Trejo was sold on the dream of becoming a millionaire, which was something he said Flores-Martinez would constantly tell his employees that he wanted to see them become. In May, Trejo would bring in one of the company’s biggest promotional events, a car show held in Georgia featuring hundreds of cars and bikes with general admission tickets starting at $250 “That’s where things took a turn,” Trejo stated in the video Trejo said he had made a verbal agreement with Flores-Martinez in which Trejo would keep one hundred percent of commissions from the event But the verbal agreement was apparently not honored—instead Trejo said he received around fifty percent of commissions from the event Trejo said there was never any paper trail for these decisions which according to former CashDrop employees was not out of the norm at the company.  Trejo said he also did not receive reimbursements for some work trips and activation expenses he had paid for out of pocket Multiple employees said company credit cards were often declined forcing them to cover costs on multiple occasions especially during out-of-state trips.  Trejo was laid off and told that the company could not afford to keep him CashDrop’s proposed severance package included only a healthcare stipend for twelve weeks far less than what Trejo felt was owed to him Trejo had to sign the agreement that included a nondisparagement clause that would have prevented him from criticizing CashDrop publicly It’s a really disrespectful offer considering the verbal agreement that Ruben had with me regarding 100% commissions….I won’t be signing anything and I will exercise my right to speak freely about my experiences at CashDrop on social media and to the public Sign up to get the Weekly Digest delivered to your inbox disrespect you and punch down on you daily Trejo wanted to bring attention to his experiences and reached out to other former employees wondering if what he had gone through was shared.  He soon found former colleagues who said that Flores-Martinez had verbally harassed them “They all kept telling me the same thing,” said Trejo “They kind of sparked something inside of me to put a stop to this.” He encouraged them to come forward and build public outcry on social media platforms Ashley De La Torre and Jaqueline Rodriguez who had been at CashDrop under a year and had since left the start-up They came forward recounting instances of verbal abuse and a toxic work environment that they said led them to walk out indefinitely Their videos were liked by hundreds of people and received dozens of supportive comments.  “He would be very stressed out when it was close to paying the bills,” said Rodriguez who remembered Flores-Martinez lashing out as regularly as once a week but especially in the final and first weeks of the month.  Flores-Martinez’s behavior described in the testimonials and interviews took the form of ridiculing interns and in one instance patting an employee on the head.  “It’s unfortunate how much violation became normal—how being violated by a CEO how the emotional abuse just kind of became a part of the day,” said De La Torre in the Instagram video.  In an interview with South Side Weekly and La Voz Chicago Flores-Martinez denied the allegations.At first Flores-Martinez said the allegations were due to “a combination of misunderstanding there’s definitely a lot of emotions involved,” but later called them “completely baseless” and that he was considering legal action.   there’s nothing there,” said Flores-Martinez thirsty for blood to cancel somebody.”“Am I an explosive character It depends on how you slice the apple,” he said where there are disagreements.” But he denied ever threatening employees or making them feel oppressed or controlled Lisa Kowalczyk was part of the company’s customer success team and had joined CashDrop in January 2021 she and other CashDrop employees received a company-wide Slack message alerting them that ex-employees were criticizing the company on social media and demanding that current employees sign an NDA within two hours—or be fired.  Kowalczyk told Flores-Martinez that she had forwarded the document to her lawyer to review.  Before she could get a response from her lawyer Kowalczyk said she was terminated two days later De La Torre and Rodriguez shared their testimonials on Instagram Kowalczyk said she suffered a work-related injury while on a work trip and that she never got the medical insurance to cover it that she was promised She said around $10,000 in reimbursements are still owed to her from instances when company cards didn’t work and that paychecks didn’t come on time on three separate occasions the most recent being just before this past Christmas.  She also recalled not getting support when one of her coworkers was terminated because there was nobody else even qualified that could help me,” Kowalczyk said.  She said the stress from the job eventually led to hair loss and other medical problems.  “There was a lot of blurred lines in the boundaries of work-life balance—where work ends and begins,” said De La Torre adding that it was not out of character for the CEO and employees to drink on the job and constantly be told that they were a family.  stating that she and her colleagues “trauma bonded” and felt as if Flores-Martinez had isolated them from their lives According to a 2021 survey from the Workplace Bullying Institute (WBI) thirty percent of adult Americans are bullied at work a number that at the time of the survey amounted to almost 49 million workers across the country.  The numbers for women working in tech are even more staggering. A 2020 study from Women Who Tech found that close to fifty percent of women working in tech experienced harassment with forty-two percent reporting that harassment was perpetrated by a supervisor.  and therefore we treat it as not worthy of attention and we need to start paying attention because it’s harming millions of workers,” said WBI director and social psychologist Dr WBI is currently lobbying to pass the Healthy Workplace Bill in Massachusetts and New York “to prevent and correct abusive work environments.” The bill was introduced in Illinois in February of 2021 but has not passed the Illinois House of Representatives.  When concerns were brought up to Benjamin Vear the company’s president and the person who handled HR-like responsibilities according to employees employees said Vear reduced Flores-Martinez’s outbursts to the CEO being stressed due to lack of investors.  Vear declined to comment or be quoted on the record Team dinners at the expense of the CEO at notable Chicago restaurants like RPM Steakhouse and Fulton Market Kitchen would later be leveraged in public shamings during company meetings Employees felt stuck because some uprooted their lives and moved to Chicago for the job while others lacked confidence to find a better workplace.  “Ruben was very intentional with who he picked to be around,” said Rodriguez adding that his employees were mainly young Latinos belonging to marginalized communities In response to an email that listed the allegations included in this story Flores-Martinez wrote about the company needing to let go some employees last year for economic reasons “We can’t and will not comment on specific statements made by current or former employees.” In the end he claimed the questions contained “inaccurate” and “verifiable outright falsehoods” but declined to clarify what specifically he objected to “We wish you the best of luck in sorting fact from fiction,” the email ended Flores-Martinez said he wanted to give “opportunities to people that look like [him] in a world where none of them would get opportunities,” adding that “perhaps they think I’m an asshole not everybody’s gonna agree [with] the way that I move Multiple employees said their health declined because of working at CashDrop.  “My mental health deteriorated quickly,” said Rodriguez “I would be extremely anxious because I had not finished a deadline or was nervous about the next day nervous that I was going to get yelled at.” Former employees said deadlines and expectations were often changed last minute and with little notice by Flores-Martinez Kowalczyk recounted a time in which she asked for clarification and Flores-Martinez said,  “that’s not my fucking job Namie said stress and hair loss are real responses to abusive conduct A laundry list of health concerns can stem from verbal abuse including stress-related physical diseases They’re reporting war wounds,” said Namie upon learning of the allegations “Why should you suffer emotional injuries in exchange for a paycheck?”  After going public with their experience at CashDrop, the three employees who shared their stories received words of encouragement, and some vendors like Shop Ankata stopped using CashDrop altogether But they also faced pushback from Flores-Martinez and others at CashDrop in texts and social media posts that attacked the former employees’ characters Flores-Martinez took to his personal Instagram to post a series of stories including one where he wrote “People can say whatever they feel like for their 5 likes and 10 minutes of mini clout.” who worked as a supervisor within the company and had a closer working relationship with Rodriguez left voicemails and text messages invalidating Rodriguez’s testimonial “you did your thing and rode that wave for a free laptop “You’re a slime and don’t do no work,” and “Your child doesn’t deserve his dad to tarnish his image on Ig for likes He doesn’t know he will probably be a slime like you too.” Brewer Flores declined to comment for the story their intent in speaking out is to hold Flores-Martinez accountable.  and realizes that he’s really taking advantage of those around him,” said De La Torre.  Yet Flores-Martinez’s continued denial of any wrongdoing and apparent refusal to engage with the criticisms makes them feel like things won’t change—and if they don’t they believe CashDrop should cease to exist “Abuse is at the fundamental core of it all and Ruben has made it a point to be intertwined with the company so it doesn’t change what I think should happen to CashDrop which hopefully is to be dissolved,” said Rodriguez.  “Even when evidence is tacked against him his ego won’t let him be the better person,” said Trejo CashDrop is used by vendors based in cities around the country Vendors using CashDrop are later reimbursed processing fees.  Kowalczyk said some vendors were still owed those fees when she left CashDrop including a business belonging to a friend she brought on.  She said the apparent financial troubles at the company—delayed paychecks and layoffs—didn’t add up to what she described as Flores-Martinez’s lavish lifestyle.  “There are $15,000 spent on tequila for parties maybe another $20,000 spent at a club in Miami,” said Kowalczyk “There’s no reason for a CEO of a new startup to be staying at the Beverly Hills Hilton.” The former employees have concerns for the vendors still trusting CashDrop with a portion of their business. Customer fees were most recently advertised at five percent with a minimum charge of forty-five cents this is because the fees were changed—a move that has not been reflected in CashDrop’s public-facing platforms.  CashDrop actually raised the rate that they charge the customers their convenience fee,” said Kowalczyk.  Flores-Martinez said the fee is “dynamic” and in some instances “the pricing can be as high at fifteen percent.” He assured the Weekly that CashDrop is completely transparent with its merchants and “if there was cracks in the systems What’s more concerning to the group of former employees who have spoken out is the newly launched Latino Renaissance Fund Flores-Martinez pledged $1 million of his own CashDrop equity to be distributed to 1,000 Latino-owned businesses who use the app.  Something similar was promised to employees at CashDrop who never received paperwork for the equity they were told they would receive in the company “If you’re going to be working under the pretense of being Latino Renaissance you got to treat Latinos right,” said Trejo “We’re not gonna take it especially from somebody that’s not even from Chicago.” Though Flores-Martinez moved to Chicago from Milwaukee he views himself as “a prominent member of the Latino community in Chicago.”  [from] the fucking intern all the way to the mayor,” he said When asked if he was still “not just interested in the money,” he replied it is about creating millionaires and billionaires that look like you and I.” It would be those same words that once drew in employees like De La Torre.  “He might have the gift of gab and say all the right things but he treats the people closest to him awfully,” she said Jocelyn Martinez-Rosales is a Mexican-American from Belmont Cragin As an independent journalist she’s passionate about covering communities of color with a social justice lens and website in this browser for the next time I comment Δdocument.getElementById( "ak_js_1" ).setAttribute( "value" View More View More Volume 11 - 2024 | https://doi.org/10.3389/fvets.2024.1353983 The front-wave velocity of African swine fever (ASF) virus spread is depicted through a retrospective spatial and temporal analyses of wild boar outbreaks from Jan Lithuania and Eastern Poland—regions responsible for more than 50% of all wild boar cases in the EU The study uses empirical semivariograms in a universal kriging model to assess spatial autocorrelation in notification dates and identifies a discernable large-scale spatial trend The critical parameter of ASF front-wave velocity was identified (Mean = 66.33 km/month SD = 163.24) in the whole study area and explored the variations across countries Statistical differences in front-wave velocity values among countries and temporal clusters are explored shedding light on potential factors influencing ASF transmission dynamics The implications of these findings for surveillance and control strategies are discussed ASF is present in wild boar populations in several EU countries (Germany in addition to the Baltic countries and Poland Lithuania and Eastern Poland collectively account for more than 50% of all of wild boar cases in the EU The median annual percentage of notifications in wild boar vs domestic pigs in these four countries has been 99% and has never been below 80% Understanding the dynamics of ASF in wild boar populations is challenging due to limited data on population and movement patterns data as well as logistical and cost constraints in surveillance efforts Spatial modelling becomes crucial for anticipating the spread following confirmed ASF events in wild boar identifying whether the spread is expected to be constant or increasing or if further cases are expected in other areas and at which rate of occurrence we hypothesize that the spatial dynamics of ASF virus in wild boar in the Baltic countries and Eastern Poland where spill-over events at the domestic-wild boar interface have been infrequent along a 7-year period Such rates can be modelled by estimating the velocity of a front epidemic wave we propose a kriging method to interpolate monthly ASF notifications in wild boar This method provides a comprehensive analysis of space and time parameters within the context of ASF spread dynamics Lithuania and Eastern Poland affected by ASF in wild boar from January 2014 until January 2022 We utilized ASF notifications reported to the EU Animal Diseases Information System (ADIS) provide details on the date of event confirmation geographical coordinates where cases are found and any other epidemiologically relevant information as estimated by the reporting country This additional information may include details such as whether the wild boar were found dead or shot or the type of laboratory test employed for confirmation we included information regarding the date and location ASF notifications were mapped using the WGS84 projected coordinate system (ArcGIS Pro 3.0.3.) and depicted through a kernel density map TSA is more often used when there is a known or hypothesized spatial trend that explains the observed variation in the notifications Our approach avoids the need for conditioners or assumptions thereby enabling a holistic investigation of spatial and temporal parameters within the framework of the dynamics of ASF propagation The study area was rasterized into 50km2 cells and the date of the earliest monthly ASF notification in wild boar was extracted for each cell Subsequently, a universal kriging model was applied to the earliest ASF monthly notifications in wild boar per cell throughout the study area. Under the universal kriging model the target variable at a given location si is given by the sum of a trend function, which is a linear combination of p + 1 auxiliary variables fj(si), multiplied by their respective beta coefficient, and a residual which shows spatial autocorrelation (17) (Eq. 1): The kriging weights λi are determined based on the semivariogram model fitted to minimize the prediction error (18) p(Z,s0) is estimated for the pixels covering the study area to yield maps of the predicted date of ASF notifications The predicted kriging time was represented by monthly interval isochrones using ArcGIS Pro 3.0.3 To quantify the ASF front-wave epidemic velocity, the surface of the prediction time resulting from the kriging analysis was included with the 3D Analyst toolbox of ArcGIS Pro as a vector of the map slope (magnitude), following the approach described by Moore et al. (14) The inverse of the slope surface corresponds to the front-wave velocity of ASF at each location within the study area The relationship between velocity (V) and slope (Sp) is given by V = s/t and Sp = rise/s where “s” is space and “t” is time rise was defined as the predicted monthly ASF occurrence time larger values of Sp mean slower velocity (1/Sp) and larger time interval “t” at similar distance result in slower V as well a higher slope value implies slower ASF diffusion while a larger vector of time (lower slope) leads to faster velocity of diffusion Space–time cluster aggregations of velocity of ASF spread were additionally explored using retrospective seasonal and normal models (SaTScan v10.0; 22) These models require the inclusion of case data and temporal information of each case Cluster selection was implemented based with varying temporal window of 3 to 6 months to capture seasonal differences Evaluation of cluster performance was conducted based on ranked relative risk (RR) log-likelihood ratio (LLR) values and p-values obtained from Monte Carlo hypothesis testing (9,999 permutations) The study area contains 9,693 grid cells of which 291 were selected for the universal kriging model, corresponding to 2,305 ASF wild boar notifications (Figure 1) Study area (centroid of each cell grid) depicted in light green overlaid is the kernel density of ASF wild boar notifications from January 2014 to January 2022 based on ADIS data shown in grey-blue shade Red dots are the 291 earliest monthly ASF notifications in wild boar extracted for each cell grid The empirical semivariogram shows strong spatial autocorrelation for notification dates and increasing large-scale semi-variance representing the spatial trend. Figure 2 shows the predicted kriging model fitted (γ) to the residual variogram and the linear combination γ + (β1 ∗ γX)2 (where γX is the variogram of the X coordinate) fitted to the not detrended variogram The one-leave-out cross-validation resulted in a bias of −0.43% and a mean ratio of squared residuals/kriging variance of 1.0031 Predicted kriging time surface of ASF infection in wild boar at 5-month intervals from January 2014 to January 2022 the green color spectrum represents the early phases of ASF prediction events over time while the red color spectrum represents the later months The front-wave velocity of ASF was obtained for 2,277 points with a median of 49.52 km/month (min.: 8.84; max.: 740.39) across the study area (Figure 3) the median velocity was highest for Estonia (75.60 km/month (min.: 21.41; max.: 740.39) n = 397) and lowest for Lithuania [36.52 km/month (min.: 8.84; max.: 344.29) followed by Poland [42.47 km/month (min.: 13.03; max.: 732.96) n = 1,179] and Latvia [53.95 km/month (min.: 11.98; max.: 697.05) Predicted ASF front wave velocity in wild boar (km/month) during the period from January 2014 to January 2022 while the red colors represent higher velocity of the predicted ASF front-wave velocity in wild boar the highest median ASF front-wave velocity in Estonia and Latvia were obtained in autumn [Estonia: 90.28 km/month (min.: 22.9 n = 190; Latvia: 61.11 km/month (min.: 11.98; max.: 329.78) the second highest median in Estonia happened in winter [75.6 km/month (min.: 21.53 while in Latvia it was in summer [59.79 km/month (min.: 16.45; max.: 697.05) the n was much smaller in Estonia in summer (n = 76) and in spring (n = 25) the number of points in Latvia in spring was only 19 the maximum velocities were reached in summer for both countries (640.21 km/month in Estonia and 697.05 km/month in Latvia) the number of predicted points was fewer than for the other countries (n = 258 in total) but the distribution is even among the seasons (although spring has the highest median front-wave velocity was achieved in summer (51.44 km/month) but the highest maximum was in autumn (344.23 km/month) the number of predicted points was much higher in winter (n = 688) than in the other seasons (between 113 and 198) the velocity median was homogeneous across seasons (between 35.45 and 48.34 km/month) although the maximum was reached in winter (732.96 km/month) The results by month shed similar conclusions there is a sufficient number of points to allow comparison for the months between August and January (>10% of the total predicted points per country) but there are fewer points predicted from February to July (<5%) May and June (<2%) to be considered in the comparative descriptive analysis The maximum velocities were reached in October and November in Estonia (706.36 and 740.39 km/month respectively) in Poland in February (732.96 km/month) and in Latvia in August (697.06 km/month) The highest median predicted velocity per year occurred in Estonia in 2015 (89.30 km/month) and in Latvia in 2014 (81.69 km/month) The highest maximum velocity in Estonia was predicted in 2016 (740.39 km/month) in Latvia in 2015 (697.05 km/month) in Poland in 2018 (732.96 km/month) and in 2014 in Lithuania (344.29 km/month) Similar results were obtained with SaTScan analysis showing one higher velocity temporal cluster from August to November (Mean = 93.64 SD = 111.56; p = 0.001) the majority of the predicted values (between 73 and 88%) fell in the maximum quality categories (5 and 6) of available habitat for wild boar making comparisons among the rest of the categories at risk of misinterpretation due to sampling bias The complete output from the descriptive analysis can be found as Supplementary material S1 The identification of a temporal cluster from August to November could indicate a potential period of increased transmission risk but should be interpreted with caution due to potential sampling bias The effect of habitat on wild boar movement was reviewed by Morelle et al. (31) The quality of habitat is influenced by the distribution and abundance of food resources and wild boar movements seem to increase particularly under high population density and low food availability Further investigation into the relationship between habitat characteristics and ASF transmission dynamics may provide valuable insights The velocity of ASF spread identified here allows us to explain the progress of the disease as an event The intensity of this event is not defined here other parameters such as the number of affected animals or serology data should be included For the purposes of policy and prevention practices the economic consequences could be considered similar leads to indirect losses derived from the “news effect” of the disease’s presence (for example in Belgium ASF was only notified in wild boar but domestic pig trade decreased in some sectors) the identification of a velocity front is a sufficient parameter to initiate the evaluation of consequences derived from the arrival of the virus It is important to recognize that our findings may be influenced by bias and uncertainty inherent to the kriging methodology. Kriging, a method that explicitly captures spatial correlation, operates under the assumption that values in close proximity tend to exhibit greater similarity. This allows for the inference of correlation at locations that have not been sampled, based on the points that have been sampled (20) Even though kriging offers certain benefits compared to TSA in terms of capturing spatial dependence and variation it is still susceptible to potential biases Factors such as the quality and quantity of input data which can occur in disease notifications in wild animals whose surveillance is subject to fluctuations can influence the accuracy of kriging predictions due to the possible delay between disease progression and notifications and the interaction between the grid size (which was chosen considering the spatial distribution of notifications) and potential jumps in the advancing front overestimations of the advancement speed may occur it is prudent to interpret our findings with caution recognizing the inherent uncertainties associated with the methodology employed Understanding the spatial and temporal dynamics of ASF front-wave velocity is crucial for designing effective surveillance and control strategies Tailoring interventions based on country-specific patterns and temporal clusters can enhance the efficiency of control measures and resource allocation The original contributions presented in the study are included in the article/Supplementary material further inquiries can be directed to the corresponding author The author(s) declare financial support was received for the research This research was funded by the Horizon 2020 Program of the European Union through the European Project H2020 “VACDIVA-A Safe DIVA vaccine for African Swine Fever control and eradication” project (grant agreement no The authors acknowledge the Ministry of Agriculture in Spain for facilitating ADIS data extraction and sharing The author(s) declared that they were an editorial board member of Frontiers This had no impact on the peer review process and the final decision The Supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fvets.2024.1353983/full#supplementary-material PubMed Abstract | Crossref Full Text | Google Scholar Google Scholar Google Scholar Google Scholar African swine fever in the North Caucasus region and the Russian Federation in years 2007–2012 Google Scholar Assessing the risk of African swine fever introduction into the European Union (EU) by wild boar Reproductive ratio for the local spread of African swine fever in wild boars in the Russian Federation African swine fever: a global view of the current challenge Update on the risk of introduction of African swine fever by wild boar into disease-free European Union countries Google Scholar Estimating front-wave velocity of infectious diseases: a simple PubMed Abstract | Crossref Full Text | Google Scholar Google Scholar Crossref Full Text | Google Scholar Crossref Full Text | Google Scholar Crossref Full Text | Google Scholar Crossref Full Text | Google Scholar Google Scholar A cartographic tool for managing African swine fever in Eurasia: mapping wild boar distribution based on the quality of available habitats Crossref Full Text | Google Scholar African swine fever: lessons to learn from past eradication experiences Eight years of African swine fever in the Baltic States: epidemiological reflections Seasonal occurrence of African swine fever in wild boar and domestic pigs in EU member states Natural inactivation of African swine fever virus in tissues: influence of temperature and environmental conditions on virus survival Behaviour of free ranging wild boar towards their dead fellows: potential implications for the transmission of African swine fever The potential role of scavengers in spreading African swine fever among wild boar Dynamics of the African swine fever spread in Poland Citation: Martínez Avilés M de la Torre A and Iglesias I (2024) Spatial and temporal analysis of African swine fever front-wave velocity in wild boar: implications for surveillance and control strategies Received: 11 December 2023; Accepted: 13 March 2024; Published: 25 March 2024 Copyright © 2024 Martínez Avilés, Montes, Sacristán, de la Torre and Iglesias. 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Volume 15 - 2021 | https://doi.org/10.3389/fnana.2021.787913 The prosomeric model was postulated jointly by L Rubenstein in 1993 and has been developed since by means of minor changes and a major update in 2012 This article explains the progressive academic and scientific antecedents leading LP to this collaboration and its subsequent developments Other antecedents due to earlier neuroembryologists that also proposed neuromeric brain models since the late 19th century as well as those who defended the alternative columnar model The circumstances that apparently caused the differential success of the neuromeric models in the recent neurobiological field are also explored The present special number of BBEV titled ‘Beyond the prosomeric brain model’ offers me the opportunity to present some notes explaining how I came to be involved in the birth of this model I would not have space for reviewing relevant literature in too much detail or even to mention all our publications using this brain model I will rather highlight the major circumstances that Although the account deals initially largely with my own experiences the active participation of various colleagues and collaborators increases in importance afterward strongly influenced by the published work of earlier scientists which also need to be mentioned and placed in context I think that this story began when I first found neuromeric ideas at the school of medicine of Granada (Spain) I was photocopying selections from the Journal of Comparative Neurology During the last year of my medical studies at the University of Granada (term 1970–1971), I started to attend the embryological laboratory of a newly incorporated professor of anatomy, Génis-Gálvez (1924-2003; see biographical notes on Génis-Gálvez in Velasco-Morgado, 2014) He had transferred from Salamanca to Granada in 1968 I had passed my anatomy and embryology subjects with a different professor After 3 years of preclinical subjects and two 2 of clinical studies I had decided to finish my sixth and final year of medicine studies for which I had found I had little vocation I had chosen instead a research career in neurobiology Understanding the mind had been my major interest since adolescence when I first tried to explain to myself volatile adolescent amorous reactions observed within my school class (boys and girls half and half) I was detoured from my consequent strong interest in psychology (then only approached in Spain as a third year subject in medical studies) by the dogmatic verbiage offered by my psychology and psychiatry professors in Granada (third and fourth terms) I had already decided to attempt a formation in neurobiological research After short visits of the physiology and histology/pathology departments (where I found no neurobiological research was done) a professor suggested to try the ‘new’ anatomy professor J The idea was to occupy myself with brain development during the last year of medicine and then apply for entrance at a leading neurophysiology laboratory in Madrid Professor Génis-Gálvez was very receptive He explained to me mainly the possibilities to do experimental embryology in his lab he showed me in the lab how one opens an incubated egg and finds inside the chick embryo this morphological approach with experimental possibilities relegated to the far background my psychological and physiological interests on the mind although they persisted up to the present as a reading hobby that psychology apparently had nothing to offer me at that time point It seemed that there was a lot of preliminary work to be done yet on basic brain structure before higher mental properties could be properly addressed I estimated that the status of neurophysiology probably was not much more advanced in this sense than that of psychology came out of the interview with a handful of reprints on brain development to read and a very vague idea that I wanted to study brain structure hoping to develop more specific plans as I advanced I read immediately the articles Génis-Galvez had given me and entered his office again 20 min later to ask him for more My tutor worked specifically on the developing eye (he was a non-practicing ophthalmologist originally) and thus could not teach me much about the brain proper beyond what one learned then in medical neuroanatomy he was happy to have in the lab somebody who would try to develop a line of work in central aspects of the visual system He helped me by providing guiding literature (he had a good collection of basic books and reprints obtained during his stays in the States) and placed at my disposal a small laboratory I started to work away with maximal dedication and dropped altogether attending my last term classes (I just crammed the subjects 3–4 days before the exams) My intense readings during that first year represented an introduction to modern biology although without systematic botany or zoology complementing partially my standard medical formation in a direction helpful for research I cannot say that I learned much about the brain except what I saw personally at the microscope The theory of evolution apparently was not studied at all in Granada at the time I learned of its major conclusions only years later in books I thus had no idea of comparative neuroanatomy or neuroembryology I simply extrapolated what little I knew from the human brain to the chicken brain I assumed that birds moved their eyes with comparable muscles and nerves as Ramón y Cajal seemed to imply in his treatise He was a pragmatic evolutionist and comparative morphologist accepting a priori that vertebrate brains were comparable but he never discussed theoretical issues such as the concept of homology or Haeckel’s and Baer’s contrasting ideas on developmental recapitulation of evolutive change there was no textbook on comparative neuroanatomy available nor an avian brain atlas at least as regards the visual system and oculomotor mechanisms equivalent structures could be expected in humans (mammals) and birds but my initial rate of advance in learning to recognize chicken brain structures was extremely slow The brain I had in front of me at the microscope was a large complex of structures I could not identify with only a small area illuminated by understanding at the oculomotor nuclei I needed many years of solitary toiling with reprints or photocopies and the microscope before I slowly expanded my neuroanatomic knowledge to neighboring areas helped by avid exploration of literature on the brain of birds compared to those of other vertebrates I expanded to the whole brain thanks to the prosomeric model which allows prediction of structures you are going to see The columnar model defended by Herrick (1910; 1933; 1948) and Kuhlenbeck (1927, 1973) was then absolutely prevalent and was implicitly assumed by all neuroanatomy book authors All of us (experts and beginners alike) used that model often without being aware of its existence or its fundaments (more on this model below) It is like breathing without knowing that air exists Over the summer of 1971, after finishing my medical studies, I returned to my parental home in Tenerife (Canary Islands) and married my girlfriend from preuniversitary studies and subsequent lab collaborator in Granada Margaret Martínez-de-la-Torre. After our honeymoon, I read the well-illustrated textbook of Embryology which had been published by Génis-Gálvez (1970) This stimulating book confirmed my interest in embryology as well as in its experimental possibilities and I started to work on my doctoral thesis project (officially tutored by Génis-Gálvez I also began to teach some anatomy and dissection for medical students as a part-time teaching auxiliary although it was clear to all of us that a research career in the Spain of Franco was highly risky my tutor applied for a transfer closer to his birthplace in Cadix to a position at the anatomy department in the University of Sevilla (Granada is the largest of them; its university also encompassed then the isolated medical school at Cadix) Génis-Gálvez obtained the Sevilla position and in his plans for transferring in 1972 the whole lab (including Margaret and me) he got funding from the Granada faculty dean to photocopy any departmental journal article that was of interest to us The job to decide which articles were to be photocopied in the Journal of Comparative Neurology fell to me (we had an ample collection) I thus spent hours perusing systematically through hundreds of JCN articles since I really knew little of such contents and nothing at all about the possible fame of the authors If I found minimally interesting the title not a result of understanding the need for such a schema but of just noting a new possibility to visualize synthetically brain structure It was the sort of spine-tingling emotion produced otherwise by sublime musical passages which one does not understand technically The neuromeric article thus passed to the collection we brought to Sevilla subsequent calm reading did not show how to relate those interesting theoretical ideas to my daily work in the lab on the oculomotor nuclei and the interstitial nucleus of Cajal (the subject of my thesis) The needed context of being aware of alternative brain models and differential explanatory capabilities was wholly absent without forgetting the impression it had made on me and only returned to it several years later when its implicit content started to become meaningful Figure 1. Neuromeric model of Bergquist and Källén (1954). Note that neuromeres I–VII are superposed by transversal bands 1–7. Also, neuromeres I–II imply an ending of the forebrain axis in the telencephalon (a columnar notion departing from the axial concept of His). In subsequent publications, Bergquist and Källèn corrected this error (see Figure 6) noting that neither the telencephalon nor the eye vesicles represent true neuromeres as well as my first experience with mammalian embryos A major neurohistological figure I met at the INSERM U106 was Constantino Sotelo an expatriated terminal member of Cajal’s school (direct student of Fernando de Castro one of Cajal’s younger collaborators) He was then famed as one of the major experts in brain electron microscopy and also was interested at the time in neuroembryological questions He recommended trying Golgi impregnation methods on the oculomotor migration and particularly the variant of Stensaas (with glutaraldehyde substituting osmium tetroxide in the fixative solution) which he thought worked better in embryos than the standard rapid Golgi procedure (osmium fix) started a collection of Golgi-Stensaas impregnated rat embryos which I brought with me to Sevilla at the end of the stay I also tried out the Stensaas procedure on chick embryos and found it worked much better than in rats (unless the exceptional success was due to the water of Sevilla) I concentrated on the increasing collection of chicken Golgi preparations Apart from my teaching duties (several hours daily) I generally processed one embryo per week (fixation and mounting the thick celloidin sections strictly ordered on slides and finally looking at them at the microscope) Beautifully impregnated developing neurons and many other sorts of cells appeared filled up by the reddish-brown silver chromate precipitate apart from the desired neuronal and glial cells became an autodidact distant pupil of the school of Cajal Figure 2. Selected neuromeric schemata from the work of Tello (1923) showing sagittal sections through chick embryo brains at two different stages wherein diverse reduced-silver-stained tracts clearly relate to neuromeric bulges the growing optic tract is represented as a longitudinal tract relative to the diencephalic neuromeres The school of Cajal had practically disappeared in the post-civil-war period and only the publications remained who was active in his youth in comparative brain studies and Tello (the only pupil of Cajal doing neuroembryology) died in 1958 having been expelled from the direction of the Cajal Institute in 1936 as a collateral result of the Spanish civil war I received some technical advice and psychological support from Constantino Sotelo since he was the first scientist of excellence I met Sotelo also supported my ulterior first publication on the chick neuromeric model (see below) The neuromeric papers of Tello were the second place where I encountered neuromeric concepts although at this point I was concentrated on cellular differentiation details of the tectal neurons and was not attending at all to the issue of neuromeres This gradient allows tentative differentiation stages to be checked rostralwards (where more advanced cells can be seen) and caudalwards (where less advanced cells are found) as long as the preparations are as homogeneously Golgi-impregnated as mine were This project took most of my research time during my last years in Sevilla (up to the end of the 1975/1976 term I left aside momentarily the oculomotor migration problem the tectal work advanced at a slower pace into later stages of development (9–12 days of incubation) with the help of a doctoral student and thereafter also dear friend We jointly discovered an additional more retarded Type III sort of tectal cell prototype She read her thesis on the chick tectum in 1978 when I was already in Cadix after spending a year in Badajoz most of the late tectal data remained unpublished I unexpectedly was offered a contract as full professor and director of the anatomy department at the University of Extremadura (at Badajoz although this implied a transient rupture with Génis-Gálvez my Golgi collection had to remain in Sevilla started a new laboratory and a new Golgi collection in Badajoz where I was wholly on my own as regards research and the sort of neuroanatomy I taught I prepared there under singular conditions (no funding) the manuscript on early differentiation of tectal cell types A comparable result has not been produced yet in anamniotes or mammals although some of the published Golgi data in fish and mammals show neuronal examples comparable to our cell types and the optic tectum is known to be highly conservative in its connections some added elements (perhaps novel cell types) might occur given what we know of divergent superficial tectal stratification and the massive cellular development of the periaqueductal gray compared to that of sauropsids and anamniotes The periaqueductal gray is often figured as if it was independent from the superior and inferior colliculi but its cells underlying the colliculi arise late from the same progenitor domains I think the Puelles and Bendala (1978) tectal report was my first important scientific publication (7 years after I started) it is the most complex histogenetic analysis ever done with the Golgi method I am proud that it was performed without other guidance than the rationale exemplified previously by Ramón y Cajal This study taught me what sorts of differentiative and migratory complexities may be found in the histogenesis of a distinctly delimited progenitor area over time The three distinct postmitotic neuron prototypes diversifying gradually into the various final neuronal forms strongly suggested that the genetic profile present at cell birth was highly important in determining the fundamental behavior of the cell whereas the changing surroundings over time probably exert an epigenetic mechanistic modulatory role on the emerging phenotypes Another paper I submitted jointly to Neuroscience while in Badajoz dealt on the earliest Golgi-impregnated shapes of developing tectal oligodendrocytes (Puelles, 1978a; Figure 3B) It appeared slightly before the tectal one because it was accepted without any changes; this is the only time this happened to me I think that these results still represent the only published visualization so far of young postmitotic oligodendrocytes in the process of enveloping axons with their lamellipodial processes that I obtained part of the multi-volume comparative treatise of Kuhlenbeck (published by Karger between 1967 and 1978; ‘The Central Nervous System of Vertebrates’) which I also studied with enormous interest with whom I have had many mental discussions over the years but he interpreted them as coursing oblique to his axis The hp1 and hp2 domains are the two hypothalamo-telencephalic prosomeres postulated by us in the updated prosomeric model The two cross-sections at left correspond to the section levels marked in the map as 44 and 41 They illustrate the differential histogenetic patterns and the abrupt boundaries corresponding to the interneuromeric borders where Rendahl did not postulate neuromeres He only represented the longitudinal sulcus limitans at p1 and p2 We extend this landmark through p3 and the hypothalamus as well ending under the optic chiasma (blue line) Figure 6. Comparison of the neuromeric models of Bergquist and Källén (1954) (A3) and Rendahl (1924) (C2) with our own chick model, extracted from Puelles et al. (1987a; B4) The precociously populated basal plate neuromeric modules are emphasized The B&K schemata show in (A1) their neuromeres (later corrected) and in (A2) their transversal bands which we take as corresponding most realistically with our neuromeres Our schemata include early stages in the emergence of the neuromeric pattern in the schemata (B1–B3) The Rendahl schemata (C1,C2) reveal that this author postulated the telencephalon as a prosomere I lying rostral to the diencephalon thus showing the influence of the columnar axis it is clearly observed that the three sets of schemata are basically comparable in many aspects I had checked in Badajoz whether the three tectal neuron prototypes were generally present in other parts of the brain as was already suggested by existing Cajal data on the spinal cord substantiating the conclusion that the brain wall was apparently divided into multiple areal progenitor units with differential histogenetic patterns and characteristic cell types (a concept we later used in the prosomeric model Given the success of the Golgi study of the optic tectum I chose to begin in Cadix a Golgi study of neuronal differentiation sequences in the developing chicken diencephalic visual centers The latter were scattered over the hypothalamus and pretectum according to the columnar model I was helped in this project by a new thesis pupil For the diencephalon, we initially applied the widely prevalent columnar model to the analysis of our Golgi data. We followed the columnar embryologic Nissl studies on chick diencephalic development done by Kuhlenbeck (reviewed in Kuhlenbeck, 1973) According to the interpretation of this author were cellularly homogeneous longitudinal masses of neurons displaying similar functional properties along their length (somatic versus visceral; motor versus sensory; somatosensory Since all postulated diencephalic columns clearly received specific retinal input via the optic tract (some of them several distinct inputs) we soon noted that it was absolutely unclear how viscero/somatic or sensitive/motor sorts of signals were distinguished from visual signals in the columnar diencephalon (i.e. apart of entering its visual sensory input through the postulated diencephalic floor and basal plate equivalent region at the hypothalamic chiasma a very odd pattern absent in the hindbrain) the higher resolutive power of the Golgi-Stensaas method (showing differential details of the neuronal axons and dendrites) immediately refuted this assumption Unexpected clear-cut boundaries separated distinct intracolumnar fields having different neuron types crossing obliquely the theoretically homogeneous columns and defining distinct non-columnar blocks of neurons in terms of observable early differentiation sequences Instead of advancing nicely along a gradient the diencephalic Golgi image was a mess of contradictory data we simply could not make meaningful using the Herrick/Kuhlenbeck columnar model started doubting this model and looking around for some alternative model that would explain what we saw Colleagues familiar with my subsequent work think that I moved in the 1990s into neuromeres due to evidence coming from gene expression patterns I first learned about neuromeres some 20 years before but only started to think they might be important for my research due to the problems I had with the interpretation of Golgi impregnations in the chick diencephalon in the late 1970s at which moment I was the expert in neuromeres of the new generation whom I met in his retirement during a private weekend visit in 2002 told me that he had never talked with Holmgren personally and that the members of the ‘school’ hardly interacted or met with each other Even the closer collaborative research relationships of Källén with Bergquist were darkened by the repulse of Källén of the Nazi orientation of Bergquist (they never were friends His former Nazi connection had reduced Bergquist to working after the war as a public school teacher He was a sort of visiting scientist in Tornblad Embryological institute directed by Källén (a casual conversation of Bergquist with Källén’s father He could use there the comparative collection of embryonic preparations of Ivar Broman we hesitated about publishing this conclusion since I feared that an attack on the solid columnar establishment based on ‘mere’ Golgi data in the era of experimental neuroanatomic studies and immunocytochemistry was going to be exceedingly difficult and probably unsuccessful I started preparing instead a photographic documentation of the neuromeric pattern of the developing avian visual centers using Nissl data because I was continually distracted by other preoccupations such as obtaining tenure (in Cadix) and later two academic jobs I accepted in series in Murcia (vice dean of the medical school and later vicerrector of research) an old friend from my German-school days in Tenerife lives in Stockholm and he obtained for me photocopies of all relevant Swedish documents at the Karolinska Institute (all the works by Holmgren (4) Early students of neuromeres did not attempt to investigate their possible modular functional properties in the adult brain These aspects are increasingly being studied today (neuromeric analysis of modular aspects of serotonergic raphe nuclei and still center their attention instead on the connectivity and circuit functions apparently performed by dedicated parts of the columns There is to this day no columnar theory explaining how an embryonic column diversifies into a collection of distinct nuclei the prosomeric model has incorporated dorsoventral and anteroposterior patterning and regionalization effects leading to molecular definition of the component progenitor areas that produce specific cell types or nuclei The columnar model is incompatible with these experimentally demonstrated patterning effects because its arbitrary length axis ending in the telencephalon prohibits it (the implicit meanings of the columnar descriptive terms dorsal and posterior are inconsistent with what we now know of brain patterning) The speculative columnar structure of the forebrain has nevertheless become dogmatically established in the literature after a century as a conventional truth or fact No expert acknowledges that there is an underlying theoretically fallible columnar model dating from 1910 that is responsible of a major scientific impasse Criticisms of the columnar model of Herrick are labeled as unjust attacks on a ‘straw man’ (this is personal experience) since modern neurobiologists no longer are conscious of following the columnar model This factual character of the columnar model in practice was the barrier I had in front once I realized the obsolescence of this model and the need to substitute in its place a neuromeric brain model corrected from earlier errors throughout neuroscience I needed a modern technique which could be used to reverse the status quo what I obviously really needed were gene expression patterns and the experimental analysis of neural patterning These new research instruments could not even be imagined in the late 1970s where no molecular research was performed by anatomists; I was literally thought to be out of my mind when I expressed to colleagues interest in molecular genetics for progress in embryology and anatomy A division of the heads through the midline helped the mapping of the stained neurons through the unstained ventricular zone in the translucent head halves Soon I perfected the method by dissecting after the reaction the skin and meninges away under an operating microscope using sharp tungsten needles Frozen or cryostat sections habitually used for histochemistry did not allow an overview unless graphic reconstruction was attempted a method hampered by the usually incomplete cryostat section series Our study of AChE wholemounts accordingly increasingly concentrated most of my efforts during the last period in Cadix (1978 in which endeavor I was helped considerably by a new collaborator and thesis pupil The same study still continued for several years more in Murcia before we first attempted publication in 1986 We proposed the introduction in the neuroanatomic terminology of the term pre-isthmus alluding to this novel midbrain anatomic domain just in front of the isthmus proper and caudal to the inferior colliculus At the beginning of the 1980–1981 term (September) in whose medical school and anatomy department Margaret and I worked thereafter for 40 years The laboratory had to be organized from zero No silver or Golgi method worked because double distilled water contained too much organic material so that silver and chromium salts systematically precipitated This was the reason why I ceased to use those techniques and concentrated on the AChE material We had to install a system for de-ionized water which took some time and the arrival of national funding We first obtained in 1981 a grant from the ministerial central funding agency in Madrid (I had not applied before I do not know exactly why: perhaps no public grants were available during my previous formative years or at least they were not known to me; Génis-Gálvez had been funded habitually by a private foundation) we continued having sufficient national grant support on a 3-year renovation basis up to my retirement in 2018 (the last 5–6 years we had an extremely hard to get Excellency grant—only two were given per year in Spain) We also had extra help in parallel from several European projects we also obtained some significant funding from the local regional government in Murcia (Séneca Foundation) All this funding helped us to expand and modernize the lab to international standards and we established infrastructure and know-how for molecular biology procedures as well as automatized scanning of our slides we studied neurogenetic patterns in the chick brain using thymidine autoradiography This accompanied our whole-mount AChE visualization of differentiating young neurons Parts of the autoradiographic data on the isthmus and diencephalon (thalamus) were published where an incipient but remarkably incomplete brain Bauplan was found it had to be capable of application in all vertebrates and its early forms might be already detectable in cephalochordates (this was indeed what we found in the cited Amphioxus paper with unsuspected novel collateral aspects) we worked much (demonstrating the wide usefulness of the prosomeric model in such studies as noted also by other laboratories that incorporated to the quest) and obtained large amounts of histological material of various types It was all very useful for advancing my evolutionary ideas although we did not publish large parts of it since we first had to introduce the neuromeric model into the field (we only achieved this by publications in 1987 and 1993) and then we had to lift our lab to the capacity to perform molecular mapping studies The first published gene pattern I noticed that showed a neuromeric pattern appeared in the report of Gaunt et al. (1986) on Hox1.5 expression in mouse embryonic hindbrain The authors showed that Hox1.5 expression had a distinct rostral limit at a constriction of the hindbrain neural wall They apparently did not realize (as I immediately did) that this constriction was an interrhombomeric boundary I noted the enormous relevance of that sort of data for our project since all interneuromeric and intraneuromeric boundaries now probably could be explained and visualized as patterned gene expressions I visited the laboratories of friends in Paris (Marion Wassef at L’ École Normale) and Madrid (Angela Nieto at the Cajal Institute) in order to learn personally in situ hybridization procedures and associated molecular concepts and techniques we looked preferentially at intrinsic patterns of neurons inside the brain wall it represented altogether a stronger basis for our attempt to attack the columnar establishment with our revamped neuromeric model for the forthcoming morphologic interpretation of relevant gene expression data in the early 1990s The Puelles et al. (1987a) manuscript on AChE data in a neuromeric pattern was first submitted in 1986 after first showing it to Sotelo in Paris who passed it on with a favorable comment to S the editor-in-chief of the Journal of Comparative Neurology The paper evaded criticizing the columnar model; it just showed abundant photographs of what we had found and stated that the patterns were clearly consistent with the neuromeric models of the past Palay sent back reviews suggesting that perhaps because we worked in a Spanish provincial city we were not aware that this subject of neuromeres had been discarded many years before and that we should examine modern literature in university libraries in Madrid or Barcelona The rather supercilious reviewers apparently thought that we somehow were using ideas and literature citations left behind by our grandfathers Palay nevertheless asked us to send back a shortened and appropriately modified version of the text I highlighted the point that none of the reviewers had found anything to criticize or reinterpret about our photographic data so that we did not see in which sense we should change our interpretation Our results seemed indeed to revitalize old forgotten neuromeric notions but the age of such notions and their conventionally disregarded status were not our responsibility We just stood on a novel sort of evidence—unobjectionable wholemount AChE reactions—that strongly agreed with these old ideas obviously disagreeing with the contrary columnar ideas a la mode I made known to Palay the vast preliminary analysis of relevant literature I had performed over the previous 10 years (including not only everything in English but also in other important scientific languages) I included a synopsis of the journal and book material obtained from the libraries of the Karolinska Institute in Stockholm the Max Planck Institute in Göttingen and the second version was accepted without any further discussion and he probably acted against the opinion of the reviewers in agreement with the important supporting position of Sotelo in our first decade working in Murcia (1981 to 1991) three of them dealing with neuromeric subjects Margaret and I traveled to La Jolla (California) to work 1 month on fish and amphibian brains with the comparative neuroanatomist Glenn R Northcutt at the Scripps Institute of Oceanography using his own material (previously unknown to us) evidence in favor of the neuromeric brain model We first tried some Ambystoma tigrinum (salamander) brain specimens cut sagittally, which we immunoreacted with calretinin (note that Ambystoma was the amphibian species most studied by Herrick in his columnar studies and also the object of his recapitulative book “The Brain of the Tiger Salamander,” Herrick, 1948) I thought that the calretinin—CR—marker (used before by us in chick and lizard embryos) might be favorable for identifying a positive dorsal thalamus against the CR-negative ventral thalamus and pretectum domains (these represent the three diencephalic neuromeres in the alar plate) Such differential staining should also show that the corresponding limits are transversal (i.e. contrary to columnar expectations postulating at least one of them—the interthalamic zona limitans limit—to be longitudinal) the yearly meeting of the American Society for Neuroscience was held nearby in San Diego and I attended it as an observer (this was my first time at the SfN) I heard an oral intervention by a young researcher from San Francisco—John R Rubenstein—who presented the diencephalic and telencephalic expression pattern of a newly discovered gene then called Tess1 (for his daughter Tess; this was his first output in gene mapping) This gene subsequently was reclassified as Dlx2 (a member of the important Dlx family functionally involved in the generation of forebrain GABAergic neurons among other properties; distalless refers to lack of terminal leg parts in Dlx-mutant Drosophila) Rubenstein presented at the SfN a columnar interpretation of the diencephalic expression pattern of Dlx2 which seemed to me problematic in several aspects I thought that a more significant interpretation was possible using the neuromeric model Somebody he had recently consulted had given it to him as a possibility to explore alternative interpretations he needed its application to mouse embryos I assured him that this posed no difficulties Figure 7. First version of the prosomeric model, from Bulfone et al. (1993). The genes mapped are color-coded. Note that, at variance with the bipartite chick hypothalamus of Puelles et al. (1987a; see Figure 6B4) we postulated three hypothalamic prosomeres p4–p6 for the mouse This was an error that we corrected subsequently Figure 10. Comparison of the prosomeric models conceived by Puelles and Rubenstein (2003) (A) and Puelles et al. (2012a) (B) to visualize the significant changes introduced in the updated version, which were explained in detail in Puelles and Rubenstein (2015) Equally important for the progress of the prosomeric model were the experimental embryologic contributions of my pupil Salvador Martínez on the analysis of the isthmic and mid-diencephalic secondary organizers as well as on clone-isolating properties of interrhombomeric and forebrain limits (B) Schema representing the whole set of prosomeric units in the adult brain (the cerebellum belongs to r0—the vermis—and r1—the hemispheres) Note the large evaginated telencephalic development corresponding to hp1 (red) while the rostral end of the brain corresponds to the acroterminal domain within hp2 (orange) The bipartite hypothalamus is divided into peduncular hypothalamus (PHy) within hp1 and terminal hypothalamus (THy) within hp2 The axis of the brain clearly bends ventrally at the cephalic flexure where a number of interneuromeric boundaries converge pialwards and also shows a less marked dorsal bending at pontine levels causing there also some convergence of neuromeric boundaries at the ventricular surface apart what was already mentioned in the text but I believe there are many possibilities implicit in the modular serial arrangement of neuromeric components as well as in the dorsoventral microzonal structural aspects I expect a revolution in neurophysiology and a step forward in cognitive studies the model has been already extraordinarily successful in the fields of developmental neurobiology (including particularly fate-mapping and patterning studies) and comparative evo-devo studies It is difficult at the present stage to envisage what sort of neural model could be still better in order to improve studies on the nervous system although time and the nature of things will probably modify sooner or later this impression An eye needs to stay open in this direction to ensure that the prosomeric model does not become a centenary dogma The validity of a model is limited by its utility and the credibility of its assumptions The support of the Spanish Ministry of Economy and Competitiveness grant BFU2014-57516P (with European Community FEDER support) and the Seneca Foundation (Autonomous Community of Murcia) Excellency Research contract reference: 19904/GERM/15; project name: Genoarchitectonic Brain Development and Applications to Neurodegenerative Diseases and Cancer (to LP) by Seneca Foundation (5672 Fundación Séneca) are acknowledged The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest The handling editor declared a past co-authorship with the author LP Molecular regionalization of the developing amphioxus neural tube challenges major partitions of the vertebrate brain Compartimentación y dinámica neurogenética del tubo neural del embrión de pollo Manifestación heterocrónica del plan 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This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) *Correspondence: Luis Puelles, cHVlbGxlc0B1bS5lcw==; orcid.org/0000-0002-9541-7073 Asia Eng and Antonia Aguilar Cole [courtesy photo]With grit and dedication the Villa Parke boxing program’s Golden Girls have punched and jabbed their way into the Golden Gloves State Championship this weekend Asia Eng and Antonia Aguilar Cole will be fighting on April 1 and 2 in Concord If they win the upcoming state championship they will advance to the Golden Gloves nationals tournament to be held in Pennsylvania from May 8-13 and could potentially earn a spot at the Olympic qualifiers The proud boxing instructor of the quartet head boxing instructor at the Villa Parke Boxing Gym said it is their dedication that sets these women apart from others “They have put so much work and dedication into their workouts and into the sport,” he said who will be fighting at the 178-pound division has been with the Villa Parke boxing program since she was 11 “She lives and breathes boxing for all her life That’s her,” De La Torre said of Martinez having been listed as a finalist for the Olympic trials in the past Martinez said she picked boxing as her sport because she experienced bullying when she was young She said the sport strengthened her not only mentally but as well as physically head boxing instructor at the Villa Parke Boxing Gym [Eddie Rivera/Pasadena Now]“What I love about this sport is that you’re able to express yourself through the sport You’re able to leave it all in the ring but it’s about being a smarter fighter being able to show and display your hard work “This sport has taught me so much discipline It has taught me that I have to also prepare for life I have to also make sure that I was able to get an education through the sport of boxing It just helps you become a better person.” Martinez’s goal is to make it to the Olympic trials again and get a gold medal “I wasn’t able to get a gold medal a few years back but now I want to try it again and test myself again “She tries really hard to keep up with the team,” he said “She doesn’t have a lot of experience Machabie will be fighting in the Golden Gloves State finals at 146 pounds She started boxing in 2018 but it was only over summer last year that she got into competitive boxing and truly dedicated herself to the sport There’s so many layers to the sport that it really challenges you as an athlete “And just like the intricacies of the art to really exercise discipline see how far you can progress in something and learn the footwork like the different striking techniques and build your endurance.” Machabie admitted being in male-dominated sports is hard at the beginning because as a female no one will take you seriously until you have proven yourself you need to be a competitor or no one’s going to pick you up just because you’re a woman Then everybody will skip on you to play a five-on-five game but then when you prove yourself that you’re a competitor and you can score And I feel like it’s the same in boxing.” “You prove that you love the craft and you can compete and then more men are likely to spar with you It’s kind of a ceiling because you are a woman but we’re trying to break through that.” The most important thing she learned from the sport is the importance of consistency and teachability Machabie hopes to win on Sunday and make it to the Olympic team “I think that’s the first goal — to see if I can try to make the Olympic team It’s going to take a lot of training.” Eng has been in the sport for about 10 years now but she has been with Villa Parke boxing program only for four years Described by De La Torre as a “very strong” and “seasoned” fighter will be fighting the State finals at 139 pounds boxing for Eng is a “violent dance.” so it’s really important for my mental [health] “I feel like I give a little piece of myself in the ring and that person also gives a little piece of themselves in the ring and we just share like this very unique experience There’s a lot of reasons why I like boxing and those are just a handful.” Eng finds it satisfying to constantly grow in the sport so I know I’m a [stronger] person this year than I was last year.” She has also learned that while boxing is a one man sport when inside the ring winning is impossible without the community “I couldn’t be there without my coach or my stablemates I also can’t do it without the people who helped me in the gym.” “I’d be really nice if I could learn how to give back to teach it at the gym I would like to become a referee or judge or just see where the sport can carry me.” Described by De La Torre as a committed and hardworking boxer Cole will be fighting in the 125-pound flyweight division “I really love the sport,” said Cole who started boxing when she was in college People say boxing is the loneliest sport because it’s you in that ring But what I found is that I have my coach in the corner and it’s really been a journey of finding yourself and finding your capabilities.” Cole has also tried other sports including basketball and weightlifting “For me [boxing] has become a way of life So it has really become the foundation for how I live my life and the way I train and the things I interact with.” just fight the good fight and have fun,” Cole said when asked about her goal De La Torre said the four are now undergoing rigorous training in preparation for their upcoming fight The four go to the gym two to three times a day for workouts and training “Three times out of the week we’re out sparring So we go to different gyms and we spar with other girls or other males,” De La Torre added “They can either be male or female just to keep them prepared Golden Gloves is one of the most prestigious amateur boxing in the sport of amateur boxing which features both male and female amateur boxers ages 18 and up from across Southern California will serve as a qualifying tournament for the 2024 Olympics in France For more information about the Parks, Recreation and Community Services Department and its programs, visit www.cityofpasadena.net/parks-and-rec or call (626) 744-6530 More » The ‘Humming-Byrd’ House: A Mid-Century Storybook Ranch Surrounded by Nature Pasadena Now has been published daily since April 2004 and is among the very oldest continuously operated community news websites in the U.S Pasadena Now strives to publish a full spectrum of news and information articles in service to the entire community The publication will remain free to readers and will not erect paywalls Copyright © 2025. Pasadena Now | Terms of - Service | Privacy Policy | CA Notice of Collection RSS (Really Simple Syndication) feeds offer another way to get Pasadena Now content. Subscribe to our feeds to get the latest headlines, summaries and links back to full articles – formatted for your favorite feed reader and updated throughout the day. Hypophosphatasia (HPP) is an inborn metabolic error caused by mutations in the ALPL gene encoding tissue non-specific alkaline phosphatase (TNSALP) and leading to decreased alkaline phosphatase (ALP) activity. Although the main characteristic of this disease is bone involvement, it presents a great genetic and clinical variability, which makes it a systemic disease. The two new mutations have been classified as pathogenic. At the clinical level, this study suggests that both mutations not only lead to pathognomonic symptoms of the disease, but may also play a role at the systemic level. Volume 14 - 2023 | https://doi.org/10.3389/fendo.2023.1320516 Introduction: Hypophosphatasia (HPP) is an inborn metabolic error caused by mutations in the ALPL gene encoding tissue non-specific alkaline phosphatase (TNSALP) and leading to decreased alkaline phosphatase (ALP) activity Although the main characteristic of this disease is bone involvement it presents a great genetic and clinical variability Methods: Patients were recruited based on biochemical assessments Diagnosis was made by measuring serum ALP and pyridoxal 5-phosphate levels and finally by Sanger sequencing of the ALPL gene from peripheral blood mononuclear cells Characterization of the new variants was performed by transfection of the variants into HEK293T cells where ALP activity and cellular localization were measured by flow cytometry The dominant negative effect was analyzed by co-transfection of each variant with the wild-type gene measuring ALP activity and analyzing cellular localization by flow cytometry Results: Two previously undescribed variants were found in the ALPL gene: leucine 6 to serine missense mutation (c.17T>C L6S) affecting the signal peptide and threonine 167 deletion (c.498_500delCAC T167del) affecting the vicinity of the active site These mutations lead mainly to non-pathognomonic symptoms of HPP Structural prediction and modeling tools indicated the affected residues as critical residues with important roles in protein structure and function In vitro results demonstrated low TNSALP activity and a dominant negative effect in both mutations The results of the characterization of these variants suggest that the pleiotropic role of TNSALP could be involved in the systemic effects observed in these patients highlighting digestive and autoimmune disorders associated with TNSALP dysfunction Conclusions: The two new mutations have been classified as pathogenic this study suggests that both mutations not only lead to pathognomonic symptoms of the disease but may also play a role at the systemic level Figure 1 Main functions of TNSALP in the body (A) shows the correct function of TNASLP: PPi is hydrolyzed to Pi for subsequent conversion to hidroxyhapatite crystals (HAP) in microvesicles via the action of TNSALP PLP is hydrolyzed to PL by TNSALP to cross the blood-brain barrier where it is subsequently reconstituted into PLP and leads to the formation of neurotransmitters (B) shows the molecular effects related to defective TNSALP: Loss of function in TNSALP produces an accumulation of its substrates PPi inhibits bone mineralization while PLP cannot cross the blood-brain barrier decreasing the production of neurotransmitters (C) shows clinical manifestations related to defective TNSALP: The accumulation of PPi alters calcium/phosphate homeostasis while increased levels of PLP lead to a decrease in B6 supply at the neurological level the autoimmune and digestive implications have not been explored to date although there appears to be a relationship between low in vitro TNSALP levels and disease severity there are other variables that influence the development of clinical manifestations in affected patients two previously undescribed mutations are presented in two patients recently diagnosed with childhood-onset HPP with no familial relationship and with completely different clinical features The aim is to characterize each of the new mutations at the genetic structural and functional levels to establish a relationship with the clinical features Clinical characteristics and biochemical parameters have been useful in establishing a correct diagnosis of the rest of the relatives it is worth emphasizing the importance of establishing a geno-phenotypic relationship for each newly identified mutation to provide more information and better patient management Two fifteen-year-old male patients were evaluated in the Endocrinology Unit of the University Hospital Clínico San Cecilio of Granada following the algorithm developed by García-Fontana et al. (4). Neither of the two patients took vitamin B6 supplements. Patients with secondary causes of hypophosphatasemia such as malnutrition, magnesium and zinc deficiencies, haemochromatosis or certain therapies, were excluded (2, 27) Two venous blood samples were taken from each patient at the Clinical Analysis Unit of the University Hospital Clínico San Cecilio; one was for ALP and PLP determinations and the other one was used for ALPL gene sequencing Written informed consent was obtained from their legal guardians and an individualized and personal interview was conducted on potentially related HPP symptoms This study was approved by the ethics committee of Granada following the principles of the World Medical Association Declaration of Helsinki (Project ID: 0777-M1-20 Research Ethics Committee of Granada Center [(CEI-Granada) on 8 May 2019] Plasma PLP levels were measured by high-pressure liquid chromatography (HPLC) at the Clinical Unit of the University Hospital Niño Jesús (Madrid) Chromatographic determination was determined using an isocratic HPLC system an emission laser at 320 nm and a fluorescence detector was used Reference values (3.6-18 ng/mL) were established by University Hospital Niño Jesús and for fifteen-year-old males a copy variant number study was performed by multiple ligation probe amplification (MLPA) (MRCHolland) and the results were analyzed using the SeqPilot program (JSI Medical System) The Biomedical Diagnostic Center of the Clinic Hospital of Barcelona provided the sequencing results After confirmation of the presence of mutations in the ALPL gene in the two patients available relatives were recruited to perform ALP blood measurements the reference values for adult males were 43-115 IU/L while for females were 33-98 IU/L MutPred, PROVEAN and Mutation Taster algorithms were used to predict the consequences of protein mutations. Combined annotation-dependent depletion (CADD) was used to rank mutations according to impact and was compared to the mutation significance cut-off (MSC) obtained for CADD scores (30) Both mutations have already been introduced into the VarSome database with references NM_000478:c.17T>C for the L6S variant and NM_001369805.2:c.498_500del for the T167del variant For 3D modelling, the complete atomic model was predicted using AlphaFold2_advanced (32). The models with the highest scores in the local distance difference test (pLDDT) were chosen. Finally, the visualization and preparation of the figures were performed using Chimera X software (33) Human embryonic kidney cells 293T (HEK293T) were used cells were cultured at 37°C and 5% CO2 with Dulbecco’s Modified Eagle Medium (DMEM) High Glucose (pH 7.2) (Biowest) supplemented with 10% fetal bovine serum (Capricorn scientific) 5% Ham’s F12 Nutrient Medium (Biowest) and 1% of 100X Antibiotic-Antimycotic (Biowest) The vectors used were constructed by modifying the pcDNA3.1 plasmid The ALPL gene with the study variants L6S (pcDNA3.1:ALPL c.17T>C) and T167del (pcDNA3.1:ALPL c.498_500delCAC) was inserted in this plasmid The pcDNA3.1 plasmid with the WT ALPL gene insertion (pcDNA3.1: ALPL) was used as a positive control to functionally characterize the identified variants The empty vector (EV) without insertion (pcDNA3.1) was used as a negative control to monitor the basal expression of the ALPL gene at the cellular level The different variants of the ALPL gene were inserted between the HindIII and BamHI restriction sequences belonging to the multi-cloning site 150000 cells were grown per well in 24-well plates transient transfection was performed by adding to each well 50 µL of serum-free DMEM containing 1.5 µL of LipoD293 DNA in vitro transfection reagent (SignaGen Laboratories) and 500 ng of the corresponding plasmid Co-transfections were carried out by mixing 250 ng of each variant with 250 ng of WT obtaining 500 ng of total reaction The cells were incubated for 18 hours with the mixture and after that time 2 mL of DMEM supplemented with serum was added the cells were incubated for 24 hours and harvested for the different assays The term homozygous will refer to cells transfected with a single plasmid while the term heterozygous will refer to cells co-transfected unless otherwise indicated the results were analyzed by using the ΔΔCt method Cell viability was determined by using FITC Annexin V Apoptosis Detection Kit I (BD Biosciences) following the manufacturer’s protocol Cells that were negative for both propidium iodide and Annexin V expression were considered viable cells while cells that were positive for Annexin V individually or together with propidium iodide Antigenic density was performed following the protocol developed by Lopez-Perez et al. (34) the cells were washed with PBS and incubated with 2 µL of BV421 Mouse Anti-Human Alkaline Phosphatase (BD Biosciences) for 20 minutes the cells were fixed with 4% formaldehyde for 20 minutes the cells were washed twice with PBS and resuspended in 100 µL To standardize the expression of TNSALP variants on cell membranes 5 µL of CountBright™ Absolute Count Beads (Invitrogen) were resuspended in 100 µL of PBS All results were obtained with the BD FACSAria III Cell Sorter flow cytometer (BD Biosciences) on a logarithmic scale Antigenic density was calculated as the ratio of the median intensities of the TNSALP-positive cells versus the median intensity obtained by the CountBrigh Absolute Count Beads TNSALP activity was measured at a wavelength of 450 nm by spectrophotometry (Dynex Technologies) using the Alkaline Phosphatase Detection Kit (Abnova) from cell extracts according to the manufacturer’s recommended protocol Each experiment was performed in triplicate Saphiro-Wilk test was used to test the normal distribution of data To evaluate the differences between groups the one-way ANOVA test was used followed by Tukey HSD P-values below 0.05 were considered significant All tests were conducted with GraphPad Prism 9.5.1 Table 1 shows the results of the biochemical analyses of each patient The blood ALP activity in Patient 1 (P1) and 2 (P2) had persistently low ALP activity (73 IU/L and 45 IU/L respectively) while the PLP concentration were 6.5 and 2.5 times higher (118 µg/L and 45.5 µg/L respectively) than the normal values gastroesophageal reflux disease (GERD) and dyspepsia vitamin D deficiency and high blood pressure (HBP) while P2 presented clinical manifestations related to autoimmune diseases such as Crohn’s disease and inverse psoriasis in addition to vitamin D deficiency P2 was treated with calciferol and immunosuppressants none of the patients presented pathognomonic symptoms of HPP both patients maintained persistently low levels of ALP activity (P1: 42 IU/L; P2 38 IU/L) Table 1 Anthropometric and clinical parameters of HPP patients P1 presented a missense mutation in heterozygosis in the second exon of the ALPL gene (c.17T>C) changing a leucine for serine in position 6 of the protein sequence (L6S). P2 was found to be heterozygous for a three-nucleotide in-frame deletion (c.498_500del) that resulted in the loss of the threonine residue at position 167 (T167del) in the TNSALP sequence. Figure 2A shows a schematic context of the newly identified variants at genomic Both mutations have not been previously described in the scientific literature as they have not been found in any database such as genomeAD LOVD or Clinical relevant Variation (ClinVar) (Web References) The results of the pathogenicity predictions are shown in Table 2 P1 showed a disparity of results regarding the prognosis of the severity of the disease caused by the mutation the P2 mutation was classified as pathogenic in all the programs used Table 2 Features of new mutations found in patients 1 and 2 Table 3 Clinical results of relatives affected with HPP To know the degree of conservation of the mutated amino acids, MSA was performed using TNSALP sequences. Twenty animal TNSALP sequences were chosen for MSA (Supplementary Figure S1) The results obtained demonstrated that the beginning of the sequence is quite poorly conserved except for three amino acids, M1, L6 and L8, which match all the sequences analyzed. Regarding the mutation of P2, the T167 matches throughout all aligned sequences (Figure 2C) After simulation in AlphaFold2_advanced, the highest-ranked predictions based on the pLDDT score were chosen for WT (Figure 3A) and both new variants Figure 3 TNSALP 3D modelling based on the algorithm developed in AlphaFold2_advanced and visualized in Chimera X (Web References) (A) shows the visualization of TNSALP WT top and bottom structure (B) shows the representation of the WT TNSALP and variant L6S signal peptide (C) shows the representation of the structural characteristics of the T167del mutation The shortening of the loop due to the deletion of T167 causes the loss of most of the hydrogen bonds including those that affect the catalytic site Results of ALPL gene expression by RT-qPCR showed that cells transfected with the mutated vectors and with the WT vector had a 45-55 fold change compared to EV (p<0.001). There was no significant difference between the mutations and WT (Figure 4A) Figure 4 In vitro characterization of TNSALP activity of studied variants Results are expressed as mean with standard deviation ANOVA was used for comparisons between groups (***p<0.001) (A) shows relative mRNA expression after overexpression of new ALPL gene variants in HEK293T cells The results were normalized using the housekeeping RPL13 (B) shows cell viability results obtained by flow cytometry The results of survival and apoptosis were represented as a percentage concerning the total count of the cells in each culture (C) shows TNSALP activity determination of the new ALPL variants The quantitative results of the ALP assay were expressed in International Units per milligram of protein (IU/mg) (D) shows determination of the dominant negative effect of the study variants Determinations were made by TNSALP activity and normalized per mg of protein Cell survival was not compromised in any of the cases. Figure 4B shows that all populations had high survival rates (92-89%) and low apoptosis rates (5-7%) no statistically significant differences were observed between any of the study groups TNSALP activity in transfected cells showed a decrease in two variants compared to the WT protein. As shown in Figure 4C with no significant differences with respect to EV whereas the L6S variant had a statistically significant higher activity compared to EV and the T167del mutation Regarding the measurements of the TNSALP activity in the co-transfections, lower activity is observed in those in which the mutations were used compared to those that used EV (Figure 4D) EV: WT presents a percentage of activity of 53.06% which represents half of the total activity obtained in cells transfected in homozygosis with the WT plasmid while L6S: WT and T167del: WT in heterozygosis presented an average activity of 36.33 These results confirm that although both variants generate a DNE the T167del variant exerts a greater effect than the L6S variant on the WT monomer The expression of TNSALP in the cell membrane was analyzed by flow cytometry in homozygosity and heterozygosity of each variant The WT variant expressed a significantly higher amount of surface protein (0.749) than the other variants There were no differences between the populations that expressed the T167del (0.27) and L6S (0.24) variants; however the cells that expressed T167del showed a greater amount of TNSALP on the surface than those cells that expressed EV (0.17) Figure 5 Determination of TNSALP localization on the cell surface of HEK293T cells (A) shows an example of flow cytometry gating and histogram in cells homozygous and heterozygous for TNSALP variants The internal control is represented by the histogram in pink C) show the percentage of TNSALP positive cells (B) and TNSALP relative expression on cell surface (C) in homozygosis ANOVA was used for comparisons between groups (***p<0.001 E) show the percentage of TNSALP positive cells (D) and TNSALP relative expression on cell surface (E) in heterozygosis previously undescribed variants in the ALPL gene have been identified in two 15-year-old male patients leading to the genetic screening of the patients’ relatives for better understanding and personalized management for affected families For T167del variant T167 could play an important role as a stabilizer of the active site since we found that it has a structural function providing stability to E332 a direct Mg2+ ligand that is part of the catalytic center Based on our results and previous scientific literature T167del variant has been classified as pathogenic This agrees with the results obtained for T167del (located close to the active center of the protein) which showed more severe DNE We have observed a decrease not only in the percentage of cells that express TNSALP but also in the amount of protein in the membrane the amino acid L6 could play an important role in the process of exporting TNSALP to the cell membrane which could explain the low antigenic density of TNSALP although the percentage of cells expressing this protein is higher the underlying mechanisms in the alteration of the cellular localization of the protein are unknown to date we suggest that the mutated protein could be taken to the proteasome for its complete degradation which would explain the low activity and antigenic density of both homozygous and heterozygous The computational 3D modeling and alignment linked to clinical results and functional analyses suggest that L6S variant could be classified as likely pathogenic associated with a mild HPP phenotype all the data collected suggest the classification of this variant as likely pathogenic with a moderate phenotype offering valuable insights for forthcoming research endeavors although it is difficult to establish a gene-phenotypic relationship of each variant described in HPP due to the participation of several external factors that enhance phenotypic variability it is important to identify and characterize new variants that serve as a starting point for future research and patient management we have identified two new previously undescribed variants that produce clinical manifestations of HPP more related to systemic diseases than to bone disorders Considering the results shown in this study linked to previous scientific evidence we suggest low ALP activity could be related to worsening of inflammatory/autoimmune disorders in HPP patients In this study we show that both new identified mutations could be classified as likely pathogenic and have a DNE that affects both enzymatic activity and cell location through overexpression in HEK293T cells The original contributions presented in the study are included in the article/Supplementary Material Further inquiries can be directed to the corresponding authors The studies involving humans were approved by Research Ethics Committee of Granada Center (Project ID: 0777-M1-20 approved on 8 May 2019) The studies were conducted in accordance with the local legislation and institutional requirements Written informed consent for participation in this study was provided by the participants’ legal guardians/next of kin This research was funded by the Instituto de 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This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) *Correspondence: Manuel Muñoz-Torres, bW10QG1hbXV0by5lcw==; Cristina García-Fontana, Y2dmb250YW5hQHVnci5lcw== Volume 15 - 2021 | https://doi.org/10.3389/fnana.2021.806815 The vestibular column is located in the hindbrain between the sensory auditory (dorsal) and trigeminal (ventral) columns It contains the vestibular nuclear complex that receives sensory innervation from the labyrinthine end organs in the inner ear Gene expression studies and experimental manipulations of developmental genes particularly Hox genes and other developmental patterning genes are providing insight into the morphological and functional organization of the vestibular nuclear complex we will review studies of the classical vestibular nuclei and of vestibular projection neurons that innervate distinct targets in relation to individual rhombomeres and the expression of specific genes Studies in different species have demonstrated that the vestibular complex is organized into a hodological mosaic that relates axon trajectory and target to specific hindbrain rhombomeres and intrarhombomeric domains with a molecular underpinning in the form of transcription factor signatures which has been highly conserved during the evolution of the vertebrate lineage descending (spinal) vestibular nucleus; IC descending (inferior) vestibular nucleus; Is Their results suggest a plurisegmental subdivision of the vestibular column from r7 to r11 Expression of selected molecular markers in the cytoarchitectonic vestibular nuclei of the mouse according to existing literature in mammals (see main text) Sagittal sections were obtained from ISH data of the Allen Brain platform Anatomical landmarks with known rhombomeric locations are indicated and rhombomeres are delimited based on Reference Atlas (Allen Developing Mouse Brain Atlas) (A) Phox2b expression (experiment 100085299; image 7 of 18); (B,C) Slc6a5 expression (experiment 100057240; images 10 and 11 of 19 lateral vestibular nucleus (dorsal Deiterśnucleus); LVe (v) descending (inferior) vestibular nucleus; LVe/VeL Other types of vestibular projection neurons (vestibulo-vestibular vestibulo-thalamic) remain to be characterized according to rhombomeric organization Most of the analyzed vestibular projection neuron groups are plurisegmental in origin with some exceptions such as the lateral vestibulospinal group (LVST group) and the ipsilateral medial vestibulospinal group (iMVST group) which derive from r4 and r6 in the mouse and chicken (see below) The vestibular “hodological mosaic” has been corroborated both in amniotes and anamniotes [see below; reviewed in Glover (2000; 2003, 2020a,b); Díaz and Glover (2002); Di Bonito et al. (2013); Straka and Baker (2013); Díaz and Puelles (2019)] This implies that the patterning of the hodological mosaic reflects an evolutionarily conserved mechanism that links position gene expression differences between rhombomeric domains contribute to creating functional diversity among vestibular projection neuron subpopulations has not yet been characterized developmentally Table 1. Transcription factor signatures of the vestibulospinal neuron groups relative to the rhombomeres [based on Lunde et al. (2019)] Vestibulo-ocular projections to the abducens nucleus have not yet been defined in the same hodological context With respect to the relationship between the hodologically defined vestibulo-ocular groups and rhombomeric domains, the cR-VO group relates to a single rhombomere (r1), whereas the iR-VO and cC-VO groups derive from multiple rhombomeres (r2-r3 and r5-r7, respectively; Pasqualetti et al., 2007) so the cC-VO group should contain predominantly excitatory neurons The cR-VO group is partially located in the ventral part of the caudal cerebellar peduncle in r1 (Figure 5E). This hodological cR-VO subpopulation was correlated with the infracerebellar nucleus by Pasqualetti et al. (2007) based on a comparison with mammalian literature Perhaps this r1-related cR-VO subpopulation whose axons project in the brachium conjuctivum is actually patterned as part of the cerebellar anlage by the isthmic organizer rather than as part of the vestibular column per se The relationship between hodologically defined vestibular groups with classically defined nuclei is summarized in Figure 6 contralateral vestibulo-ocular group; cMVST contralateral medial vestibulospinal group; DVe (SPVe) descending (spinal or inferior) vestibular nucleus; iMVST ipsilateral medial vestibulospinal group; iR-VO The vestibulo-thalamic and vestibulo-reticular projections have yet to be explored in any species in a rhombomeric context contralateral caudal vestibulo-cerebellar group; cC-VO contralateral caudal vestibulo-ocular group; cMVST contralateral medial vestibulo-spinal group; cR-VC contralateral rostral vestibulo-cerebellar group; cR-VO contralateral rostral vestibulo-ocular group; iC-VC ipsilateral caudal vestibulocerebellar group; iC-VO ipsilateral caudal vestibulo-ocular group; iMVST ipsilateral rostral vestibulocerebellar group; iR-VO ipsilateral rostral vestibulo-ocular group; LVST other hodologically defined cell groups are plurisegmental (for example Interspecies variations have also been described in the mediolateral axis (Figure 7); indicating that comparative studies of the dorsoventral sources of these neuron groups are needed a segmental interpretation of morphological and developmental data has contributed to clarifying the anatomical and functional organization of the vestibular column wherein each rhombomere has a unique gene expression signature correlates with hodologically defined vestibular neuron groups with specific functional identities related to axon trajectory and synaptic target The hodologically defined vestibular neuron groups are unisegmental or plurisegmental in origin; in contrast the classical cytoarchitectonic nuclei are mainly 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This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) *Correspondence: Carmen Diaz, Y2FybWVuLmRpYXpAdWNsbS5lcw==; Joel C. Glover, am9lbC5nbG92ZXJAbWVkaXNpbi51aW8ubm8= Post hoc analysis of the ISAACC study, including 723 patients admitted for a first ACS (NCT01335087) in which RP was performed. To identify specific RP patterns, a principal component analysis (PCA) was performed using six RP parameters: AHI, oxygen desaturation index, mean and minimum oxygen saturation (SaO2), average duration of events and percentage of time with SaO2 < 90%. An independent HypnoLaus population-based cohort was used to validate the RP components. A RP component, mainly characterized by intermittent hypoxemia, is associated with a high risk of recurrent cardiovascular events in patients without previous CVD who have suffered a first ACS. Volume 9 - 2022 | https://doi.org/10.3389/fmed.2022.870906 Introduction: Obstructive sleep apnea (OSA) severity is based on the apnea-hypopnea index (AHI) The AHI is a simplistic measure that is inadequate for capturing disease severity and its consequences in cardiovascular diseases (CVDs) Deleterious effects of OSA have been suggested to influence the prognosis of specific endotypes of patients with acute coronary syndrome (ACS) We aim to identify respiratory polygraphy (RP) patterns that contribute to identifying the risk of recurrent cardiovascular events in patients with ACS Methods: Post hoc analysis of the ISAACC study including 723 patients admitted for a first ACS (NCT01335087) in which RP was performed a principal component analysis (PCA) was performed using six RP parameters: AHI average duration of events and percentage of time with SaO2 < 90% An independent HypnoLaus population-based cohort was used to validate the RP components PCA showed that two RP components accounted for 70% of the variance in the RP data These components were validated in the HypnoLaus cohort with two similar RP components that explained 71.3% of the variance in the RP data The first component (component 1) was mainly characterized by low mean SaO2 and obstructive respiratory events with severe desaturation and the second component (component 2) was characterized by high mean SaO2 and long-duration obstructive respiratory events without severe desaturation component 2 was associated with an increased risk of recurrent cardiovascular events in the third tertile with an adjusted hazard ratio (95% CI) of 2.44 (1.07 to 5.56; p-value = 0.03) compared to first tertile no significant association was found for the risk of recurrent cardiovascular events mainly characterized by intermittent hypoxemia is associated with a high risk of recurrent cardiovascular events in patients without previous CVD who have suffered a first ACS Cardiovascular diseases (CVDs) are one of the leading causes of death worldwide, and more than half are consequences of coronary heart disease, which is often manifested for the first time as acute coronary syndrome (ACS). Ranging from unstable angina to myocardial infarction, ACS is the main cause of morbidity and mortality worldwide (1, 2) The AHI does not provide information about apnea depth and length and is a simplistic measure that has many downsides and could result in inadequate capture of the severity of the disease Respiratory polygraphy (RP) allows the evaluation of a large number of physiological signals associated with respiratory disorders during sleep Polygraphic recordings include at least oronasal flow Dimensionality reduction technique allows the transformation of data from a high-dimensional space into a low-dimensional space so that the low-dimensional representation retains some meaningful properties of the original data The dimensionality reduction analysis of the signals of RP could contribute to the construction of models that would relate OSA to cardiovascular risk Available evidence supports that the indicators of cardiovascular risk in patients with OSA we aimed to evaluate the RP patterns that could contribute to identifying the risk of recurrent cardiovascular events in patients with a first ACS and without previous CVD Acute coronary syndrome was defined as the acute presentation of coronary disease with or without ST elevation infarction The ethics committee of each participating center approved the study (approval number in the coordinating center: 2010-852) and patients provided written informed consent The RP components in the ISAACC cohort was externally validated in the HypnoLaus population-based cohort (3), a nested-study of CoLaus/PsyCoLaus (15, 16) This study was designed to assess the prevalence of sleep-disordered breathing in a general population aged 35–75 years who were selected at random and were enrolled in the study Questionnaires to record demographic and anthropometric characteristics medical history and usual pharmacological treatment were administered the day before the sleep study along with questionnaires associated with quality of life (EuroQol-5D questionnaire) and the degree of daytime sleepiness (Epworth Sleepiness Scale test) All patients were evaluated at baseline and 1 month All patients were monitored and followed up for a minimum of 1 year sociodemographic and anthropometric variables previously related to increased cardiovascular risk were recorded Each follow-up visit included assessments of the rate of a composite of cardiovascular events [cardiovascular death or non-fatal events (acute myocardial infarction hospital admission for heart failure and new hospitalizations for unstable angina or transient ischemic attack)] we explored RP parameters to identify specific components We explored the contribution of RP components to the risk of recurrent cardiovascular events The cardiovascular risk variables explored were age, sex, current cigarette smoking, current alcohol consumption, obesity, hypertension, previous cerebrovascular disease, diabetes mellitus, and dyslipidemia (low-density lipoprotein cholesterol ≥100 mg/dL or the use of lipid-lowering drugs). Moreover, on the basis of cardiovascular risk prediction models (17, 18) we explored the following other cardiovascular variables: antihypertensive drugs no in-hospital percutaneous coronary intervention troponin and mean SaO2 Participants underwent full polysomnography at home. Details of the sampling and procedure methodologies have been described elsewhere (3, 15) we analyzed the data from 1941 HypnoLaus patients The ethics committee of the University of Lausanne approved the CoLaus/PsyCoLaus cohort study and the HypnoLaus Sleep Cohort study We obtained written informed consent from all participants The main characteristics were described using medians (25th percentile and 75th percentile) or percentages. The P value for trend was evaluated using Spearman’s rank correlation coefficient when data were continuous and non-normally distributed and the chi-square test for trend when they were categorical (19) Statistical analyses were conducted using R, version 3.6 (22) and statistical significance was set at 0.05 Baseline characteristics of patients from the ISAACC cohort The external validation cohort was 2,168 individuals from the HypnoLaus population-based cohort. Finally, 1941 individuals were selected without previous CVD and available data. The median age was 56.9 years, 47% were males, and the median AHI was 9.8 events per hour (Supplementary Table 3) The mean time of follow-up (or CV event) was 4.25 (standard deviation: 1.77) years Correlations between respiratory polygraphy parameters and components derived from the PCA accounted for 43 and 26% of the total variance accounted for 49.2 and 22.1% of the total variance Dashed lines represent –0.5 and 0.5 correlations Description of the respiratory polygraphy parameters by tertiles of the components derived from the PCA in the ISAACC cohort ISAACC patients were characterized using sociodemographic and clinical characteristics by tertiles of the two RP components (Supplementary Table 7) Patients with high adherence in component 1 had more comorbidities as the third tertile showed a greater proportion of patients with obesity Similar characteristics were shown by tertiles of component 2 In the same way, HypnoLaus subjects were described using sociodemographic and clinical characteristics by tertiles of the two RP components (Supplementary Table 8) Subjects with high adherence in component 1 had more comorbidities as third tertile showed a greater proportion of subjects with hypertension dyslipidemia and a higher body mass index (BMI) the third tertile showed a greater proportion of subjects with hypertension a lower proportion of subjects with dyslipidemia and no differences regarding BMI and diabetes In the present study, from the ISAACC study, we explored specific RP parameters and components (Supplementary Table 9) in the subgroup of patients admitted by a first ACS and without previous CVD (n = 723) In the subgroup of patients without previous ACS, OSA severity (measured by AHI) showed a trend toward statistical association [HR (95% CI) of 1.62 (0.98–2.66)] with an increased risk of recurrent cardiovascular events (Supplementary Table 10) In the analysis of RP components and the risk of recurrent cardiovascular events, it was observed that component 2 showed a significant independent association with the risk of recurrent cardiovascular events in patients with the highest adherence (3rd tertile) of component 2 with an adjusted HR (95% CI) of 2.44 (1.07 to 5.56). For component 1, no significant contribution was found to the risk of recurrent cardiovascular events (Table 3 and Figure 3) The discriminatory power of the models showed a C-statistic for the risk of recurrent cardiovascular events of 72.3% for patients without previous CVD Similar results were found exploring components from PCA as continuous [HR (95%CI): for Component 2: 1.38 (0.99–1.94) and for Component 1: 0.82 (0.6–1.13)] Cox proportional hazard model for the primary composite endpoint in the ISAACC cohort Hazard ratio for recurrent cardiovascular events by tertiles of respiratory polygraphy components in the ISAACC cohort and lines represent 95% confidence intervals hazard ratio (95% confidence interval); ACS *Significant p values (p < 0.05) In the HypnoLaus cohort the risk of recurrent cardiovascular event in patients with first ACS could not be assessed due to its population-based design the RP components identified in the ISAACC cohort were also identified in the HypnoLaus population-based cohort which captures only one aspect of this heterogeneous disease The ability to dissect OSA heterogeneity using additional features is necessary for incorporating personalized approaches in disease management we observed that the effect of OSA severity showed a trend toward a statistical association (with an HR of 1.62) with the risk of recurrent cardiovascular events in the group of patients without previous CVD indicating an increase in the risk of recurrent cardiovascular events we suggest that the analysis of other parameters from RP could contribute to the determination of RP components and their relationship with the development of recurrent cardiovascular events in patients who have suffered an ACS an estimation of the depth and duration of respiratory-related desaturation is an alternative metric associated with an increased risk of CVD mortality the authors of that study admit that this measure does not fully characterize the components of hypoxic stress because it does not distinguish short and deep desaturations from long and shallow desaturations we found that the component associated with recurrent cardiovascular events was component 2 The highest adherence to this component (3rd tertile) showing the highest risk of recurrent cardiovascular events higher mean SaO2 and longer event duration This tertile would suggest an intermittent hypoxemia component the lowest adherence to this component was characterized by lower AHI which would suggest a sustained hypoxemia component The results of the present study suggest that from available variables from RP it is possible to improve the evaluation of the risk of recurrent cardiovascular events with an HR of 1.61 exclusively with AHI to 2.44 considering additional variables The patients with the highest adherence to component 2 (3rd tertile) which represents an intermittent hypoxemia component strengthen the hypothesis that OSA exerts a risk in specific subgroups of patients with ACS the patients with the lowest adherence to this component would have a lower risk of recurrence of a cardiovascular event the same RP components were found in the HypnoLaus cohort suggesting that these components are not specific to the ISAACC population it was not possible to estimate the predictive nature of the identified RP components for recurrent cardiovascular events in this cohort the low number of available cardiovascular events due to its population-based design The existence of specific RP components that could be associated with detrimental effects in specific phenotypes of patients with ACS invites us to reconsider the design strategies of new interventional studies to demonstrate the possible beneficial effect of CPAP treatment on secondary CVD prevention. These preliminary results raise the possibility that, in studies such as the ISAACC study (8) no beneficial effect was seen because of the inclusion of patients with different phenotypes of ACS where OSA could have different effects The contribution of information about sleep phases should be explored in further studies the results from the present study may not be extrapolated to a population different from that of patients admitted to the hospital for ACS This fact makes it necessary to specifically identify phenotypes in patients with OSA who are attended in different clinical settings in patients who have suffered a first ACS and without previous CVD that is associated with a greater risk of recurrent cardiovascular events These first exploratory results must be confirmed in future studies which will evaluate the effect of RP components of specific ACS populations and the possible beneficial effect of CPAP treatment for patients with a first ACS and without previous CVD in whom a deleterious effect of this RP component has been found The original contributions presented in the study are included in the article/Supplementary Material further inquiries can be directed to the corresponding authors The patients/participants provided their written informed consent to participate in this study Concepcioìn Rodriìguez-Garciìa Francisco Javier Goìmez de Terreros Caro Abel Jesuìs Martiìnez-Garciìa and MS-d-l-T contributed to the study concept and design and RH contributed to the data acquisition and MS-d-l-T contributed to the data analysis and interpretation All authors contributed to the drafting of the manuscript critically revised the manuscript for important intellectual content and approved the final version MS-d-l-T is the guarantor of the manuscript Instituto de Salud Carlos III (ISCIII; PI10/02763 “Una manera de hacer europa,” IRBLleida – Fundació Pifarré Associació Lleidatana de Respiratori (ALLER) and Sociedad Española de Sueño (SES) AZ is the recipient of a predoctoral fellowship “Ajuts 2021 de Promoció de la Recerca en Salut-9a edició” from IRBLleida/Diputació de Lleida JD acknowledges receiving financial support from ISCIII (Miguel Servet 2019: CP19/00108) co-funded by the European Social Fund (ESF) “Investing in your future.” MS-d-l-T has received financial support from a “Ramón y Cajal” grant (RYC2019-027831-I) from the “Ministerio de Ciencia e Innovación – Agencia Estatal de Investigación” co-funded by the European Social Fund (ESF)/“Investing in your future.” FB received funding from from ResMed (an Australian company that develops products related to sleep apnea) The funders were not involved in the study design or the decision to submit it for publication We thank the individuals who participated in this trial and their families the clinical and research teams of the various sleep and cardiology departments and the Spanish Sleep Network for their work on the ISAACC study The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmed.2022.870906/full#supplementary-material Acute coronary syndromes: diagnosis and management CrossRef Full Text | Google Scholar Central sleep apnoea is related to the severity and short-term prognosis of acute coronary syndrome Prevalence of sleep-disordered breathing in the general population: the HypnoLaus study Effects of moderate-to-severe obstructive sleep apnea on the clinical manifestations of plaque vulnerability and the progression of coronary atherosclerosis in patients with acute coronary syndrome Upcoming scenarios for the comprehensive management of obstructive sleep apnea: an overview of the spanish sleep network Obstructive sleep apnoea and cardiovascular disease CrossRef Full Text | Google Scholar Long-term cardiovascular outcomes in men with obstructive sleep apnoea-hypopnoea with or without treatment with continuous positive airway pressure: an observational study Effect of obstructive sleep apnoea and its treatment with continuous positive airway pressure on the prevalence of cardiovascular events in patients with acute coronary syndrome (ISAACC study): a randomised controlled trial The effect of sleep apnea on cardiovascular events in different acute coronary syndrome phenotypes Effect of positive airway pressure on cardiovascular outcomes in coronary artery disease patients with nonsleepy obstructive sleep apnea: the RICCADSA randomized controlled trial CPAP for prevention of cardiovascular events in obstructive sleep Apnea The hypoxic burden of sleep apnoea predicts cardiovascular disease-related mortality: the osteoporotic fractures in men study and the sleep heart health study On the rise and fall of the apnea-hypopnea index: a historical review and critical appraisal rationale and methodology of the impact of continuous positive airway pressure on patients with ACS and nonsleepy OSA: the ISAACC trial The CoLaus study: a population-based study to investigate the epidemiology and genetic determinants of cardiovascular risk factors and metabolic syndrome The PsyCoLaus study: methodology and characteristics of the sample of a population-based survey on psychiatric disorders and their association with genetic and cardiovascular risk factors An international model to predict recurrent cardiovascular disease A validated prediction model for all forms of acute coronary syndrome estimating the risk of 6-month postdischarge death in an international registry Building bivariate tables: the comparegroups package for R CrossRef Full Text | Google Scholar The application of electronic computers to factor analysis Google Scholar Multivariable prognostic models: issues in developing models doi: 10.1002/(SICI)1097-0258(19960229)15:43.0.CO;2-4 R Core Team.A Language and Environment for Statistical Computing Vienna: R Foundation for Statistical Computing (2019) Google Scholar Predictive models for short- and long-term adverse outcomes following discharge in a contemporary population with acute coronary syndromes Obstructive sleep apnea during REM sleep and cardiovascular disease Heinzer R and Sánchez-de-la-Torre M (2022) Respiratory Polygraphy Patterns and Risk of Recurrent Cardiovascular Events in Patients With Acute Coronary Syndrome *Correspondence: Manuel Sánchez-de-la-Torre, c2FuY2hlemRlbGF0b3JyZUBnbWFpbC5jb20=; Geoffroy Solelhac, Z2VvZmZyb3kuc29sZWxoYWNAY2h1di5jaA== †These authors share first authorship ‡These authors share senior authorship Volume 7 - 2020 | https://doi.org/10.3389/fvets.2020.00155 This article is part of the Research TopicAfrican Swine FeverView all 11 articles A correction has been applied to this article in: Corrigendum: Evolution of the ASF Infection Stage in Wild Boar Within the EU (2014–2018) African swine fever (ASF) is one of the most important emerging transboundary diseases of pigs Nine countries in the continental European Union (Estonia and Hungary) have been affected by ASF from 2014 to 2018 and it keeps spreading despite the efforts to control it we have witnessed high case-fatality rates in wild boar found dead particularly in new infected areas which is typical of the peracute and acute forms of the infection at the beginning of an ASF epidemic Experimental evidence with currently circulating strains indicates that some infected animals can remain asymptomatic and might even survive the infection An increased presence of virus of moderate virulence can complicate ASF diagnosis as well as the mitigation and control of the disease We analyze the ASF surveillance data in wild boar in the four EU countries where ASF has been present for longer comparing the spatial density of antibody positive notifications with the time ASF has been present per region Results indicate an increasing annual distribution of notifications based on antibodies over nucleic acid detection in hunted wild boar in Estonia and Poland seem to have experienced more acute forms in 2017 and 2018 than Latvia and Estonia Overall there was a positive statistical correlation between time with infection (TWI) and antibody positive density The increasing trend in potential survivors (hunted wild boar with confirmed PCR negative and antibody positive results) enhances the importance of surveillance design to sample and test shot wild boar surveillance data based on ASFV detection by PCR and serology can be used to assess the status of the epidemic in wild boar Despite the surveillance and control actions taken in the EU ASF has since 2018 also quickly expanded in up to 10 countries in Asia including China causing severe consequences within the pig industry Of the 24 known genotypes of ASF virus (ASFV) only two have caused epidemics outside Africa: genotype 1 (1960–1990's affecting mainly Spain and Portugal in Europe and reaching some countries in Central and South America) and genotype 2 (current epidemic in Europe and Asia) It has been hypothesized that under stressing conditions, like hunting, drought, lack of food or concomitant infections, survivors that have apparently cleared the infection (negative to virus detection but antibody positive) can become infectious again (11) A prolonged shedding together with a higher percentage of survivors may therefore constitute a prolonged source of infection for other susceptible animals possibly indicating the late phase of the epidemic they recognize that ASF could become endemic instead of fading out it is now clear that comprehensive surveillance and laboratory results based on ASFV detection by PCR and serology can be used to assess the status of the epidemic in wild boar The aim of this study is to analyze the ASF surveillance data notified through the EU Animal Disease Notification System (ADNS) with the objective of characterizing the infection in wild boar in those areas in which ASF has been present for longer (Estonia one would expect to find a higher density of seropositive wild boar in those areas in which the infection has been present for longer Each notification (confirmed ASF) in the ADNS database contains at least information on the host (wild boar/domestic pig) There is space to add free text and countries generally include here other useful information in a non-systematic way: test results We restricted the study to wild boar notifications we were able to assign the category of dead/hunted for each wild boar notification we searched for key terms like “hunted,” “shot,” “hunting,” “executed,” “killed,” “shoot,” to assign the “hunted” category and “dead” or “found” for dead wild boar some notifications included both the words hunted and dead in the text and it was necessary to classify these on a one-by-one basis When in the same notification there was information about both dead and hunted wild boar (n = 62) we favored the category “hunted” since our interest is primarily to analyze the evolution of infection when the disease might be unnoticed if there was no information on whether the wild boar were either hunted or dead we favored the category “dead” (n = 1,213) Notifications were also classified according to whether the confirmation of infection had been performed by PCR which we assumed represented the initial stages of infection (Stage 1); by PCR and an antibody test (ELISA and/or IPT) which we assumed would represent animals which had the infection for some time longer (Stage 2); or which were positive to the antibody test and the nucleic acid detection test was either not specified or negative which we assume would represent the latest stage of infection when ASFV detection decreases but immunity mounts leading to an increased percentage of survivors (Stage 3) For 1,160 notifications (<10% of the total 12,661) with no information on whether the wild boar was hunted or found dead or on the test used we assumed they were dead wild boar tested with PCR Wild boar notifications estimated to be in Stage 3 of infection comprise those with a positive antibody result together with either those that specifically state that a negative PCR has been obtained or those in which we assume the PCR has been negative because this diagnostic test result is not specified The estimated TWI was explored spatially by representing the distribution of natural breaks (Jenks) classification in a choropleth map in each administrative unit We explored whether there could be a correlation between the number of notifications in which antibodies were detected and the estimated TWI per administrative unit by computing Spearman's correlation coefficient Rho in R Core Team (24) where a p <0.05 was considered statistically significant The same analysis was also carried out with the proportion of notifications in which antibodies were detected and TWI The ASF wild boar notifications with positive serology were fitted a kernel density function in a map using geodesic distances between points and an output cell size of 0.034 sq km Both maps were developed in ArcGIS 10.2 (ESRI) and were compared qualitatively Lithuania and Poland mainly notified ASF in wild boar from dead animals Lithuania and Poland have increased the number of notifications in wild boar each year while Estonia's notifications in wild boar peaked in 2016 and Latvia's in 2017 Annual number of ASF notifications in wild boar The annual distribution of notifications by diagnostic test used and estimated stage of infection is shown in Table 1 9,882) were based on PCR results (Stage 1) The remaining 22% comprise 393 notifications that include both PCR and antibody positivity results (Stage 2) and 2,386 notifications based on antibody results only (Stage 3) Annual distribution of ASF notifications in wild boar by diagnostic test/s used and estimated stage of infection The apparent increase in notifications in Stage 3 can be better observed in Figure 2 where the proportion of notifications in each stage over the total ASF notifications in wild boar per year has been stratified by dead/hunted and by country the predominant diagnostic result is obtained by PCR In fact there are very few notifications (n = 15) in Stage 3 in dead wild boar: 2 from Latvia (one in 2015 the other in 2016) and the rest from Bialski there are some differences by country but not statistically significant according to a factorial ANOVA test the % of notifications based only on PCR results (Stage 1) has decreased since 2014 to give rise to the notifications based on antibody detection (Stage 3) A factorial ANOVA test for hunted wild boar in Stage 3 showed statistically significant differences by year particularly from 2016 onwards (Tukey's honest significant test Only Lithuania has not increased the % of antibody notifications by year 1,218 Stage 3 notifications are truly PCR negative antibody positive and in 1117 PCR is not mentioned 1,106 are from Latvia and exhibit an annual increasing trend (2015 = 158; 2016 = 282; 2017 = 297; 2018 = 369) and the remaining 108 are from Poland (2016 =1; 2017 =19; 2018 = 88) A Poisson regression model on the apparent annual increase of notifications in Stage 3 in Latvia indicates that it is statistically significant (p <0.01) The correlation test indicated an overall strong positive statistical association between ASF serology notifications and TWI by administrative unit (rho = 0.77 the correlation was weaker when considering the proportion of notifications based on antibody detection (rho = 0.35 Proportion of ASF wild boar notifications by estimated stage of infection over total ASF annual wild boar notifications Both Estonian virus were isolated from 2015 outbreaks In both regions ASFV has continued to circulate since 2015 since Valga is classified in the longest TWI category (3.5–5 years) and Tartu in the second longest (2.5–3.5 years) Both fall in an area corresponding to the second highest seroprevalence density category the virus recovered from a 2017 outbreak in Engures was non-hemadsorbing (non-HAD) This area has a TWI of only 1.5–2.5 years however it also falls into an area with the second highest seroprevalence density category Figure 4. Kernel density map of antibody-based ASF notifications in wild boar classified by natural breaks (darker color indicates higher density). Points indicate the location of ASF virus of attenuated virulence characterized at the EURL, from top to bottom: circle: ES15/WB/Tartu14 (5); square: ES15/WB/Valga6 (5); diamond: LV17/WB/Rie1 (6) The analyses of the evolution of wild boar ASF notifications to the European Union (EU) surveillance database (ADNS) in Estonia the four countries which have had ASF since its introduction in the EU in 2014 reveal a progressive and statistically significant increase in the percentage of notifications based on antibody positive results with either negative or assumed negative PCR result in the period 2014–2018 in hunted wild boar (Stage 3) even if the number of notifications in hunted wild boar has remained relatively stable and much lower than notifications of wild boar found dead across the whole period The annual increase in “truly” Stage 3 (PCR negative antibody positive) notifications was tested only for Latvia since it was the only country with consistent data across the study period that in pigs surviving acute or subacute infections viral DNA has been detected in blood for up to 78 days since there has been up to now an active component of surveillance for hunted boars in infected areas this offered an opportunity to evaluate the likelihood of ASFV spread by “healthy” animals more antibody positive and PCR negative field samples are to be expected if the surveillance design still contemplates hunting to test wild boar for control and eradication purposes at least This is because antibodies for ASF are assumed to remain for life it is with intermittent peaks and therefore easier to miss under surveillance conditions the probability of finding more animals surviving the infection should not be considered a rare event in the current epidemiological situation particularly if there are only a few hours of light like is the case in the Baltic countries in winter it can also be difficult to find dead animals under harsh weather conditions Wild boar surveillance data is imperfect by nature and its epidemiological interpretation is of utmost importance to understand the extent of the infection in the field Both aspects could explain the increase in ASF wild boar found dead there has been an increase in the proportion of antibody positive notifications concentrates a high number of notifications with serology there is a strong statistical correlation between the number of notifications with an antibody positive result and TWI per administrative unit which is what was expected given the common regulatory framework that harmonizes surveillance efforts among countries The continuous presence of ASF in certain areas together with the never-ending threat of reintroduction from endemic areas or with a tendency to become endemic should be considered to update the surveillance and control plans the TWI provides a relatively fast and easy tool to assess the evolution of ASFV infection by geographical area even with limited population data Surveillance based on ASFV detection by PCR and serology is a powerful source of data to assess the status of the epidemic in wild boar despite its imperfect nature and allows to follow up the evolution of further potential survivors Data were from the EU Animal Disease Notification System database. The authors don't have permission to share the datasets. Requests to access these datasets should be directed to Marta Martínez-Avilés, bWFydGEuc2FuaWRhZGFuaW1hbC5pbmZvQGdtYWlsLmNvbQ== This study was funded by the INIA project AT2015_002 CrossRef Full Text | Google Scholar African Swine Fever in Europe (Eastern Europe & Belgium) Google Scholar Self-Declaration of the Recovery of Freedom From African Swine Fever in All suids by the Czech Republic Self-declaration submitted to the OIE on 01 April 2019 by Dr Zbynek Semerád Director General State Veterinary Administration Google Scholar Gaps in African swine fever: Analysis and priorities Evolution in Europe of African swine fever genotype II viruses from highly to moderately virulent Attenuated and non-haemadsorbing (non-HAD) genotype II African swine fever virus (ASFV) isolated in Europe Third wave of African swine fever infection in Armenia: virus demonstrates the reduction of pathogenicity Deletion at the 5'-end of Estonian ASFV strains associated with an attenuated phenotype Biological characterization of African swine fever virus genotype II strains from north-eastern Estonia in European wild boar Analysis of Estonian surveillance in wild boar suggests a decline in the incidence of African swine fever an essential tool in the epidemiological investigation PubMed Abstract | Google Scholar PubMed Abstract | CrossRef Full Text | Google Scholar Isolation and identification in metropolitan France African Swine Fever Eradication and Development of the Swine Industry in Haití [ES] Report of the Inter-American Institute for Cooperation on Agriculture Google Scholar Epidemiological situation in France and in Europe of the most important viral contagious diseases of swine [FR] Google Scholar CrossRef Full Text | Google Scholar CrossRef Full Text | Google Scholar African swine fever eradication: the Spanish model Trends in Emerging Viral Infection of Swine Google Scholar Monitoring of African swine fever in the wild boar population of the most recent endemic area of Spain No evidence for long-term carrier status of pigs after African swine fever virus infection Lack of evidence for long term carriers of African swine fever virus- a systematic review 24. 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African Swine Fever (Infection With African swine fever). Chapter 3.8.1 of the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. (2019). p. 18. Available online at: https://www.oie.int/en/standard-setting/terrestrial-manual/access-online/ (accessed October 18 Google Scholar Google Scholar Citation: Martínez-Avilés M Iglesias I and De La Torre A (2020) Evolution of the ASF Infection Stage in Wild Boar Within the EU (2014–2018) Received: 30 October 2019; Accepted: 03 March 2020; Published: 01 April 2020 Copyright © 2020 Martínez-Avilés, Iglesias and De La Torre. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) *Correspondence: Marta Martínez-Avilés, bWFydGEuc2FuaWRhZGFuaW1hbC5pbmZvQGdtYWlsLmNvbQ== NOTE: When a new stake is created or a stake presidency is reorganized in an existing stake leaders are given time to report the changes and provide brief biographical information for each member of the new stake presidency A list of stakes reporting changes is compiled and published at the end of each month The actual date of the stake’s creation or reorganization is in parentheses following the name of the stake DIBINDI DEMOCRATIC REPUBLIC OF THE CONGO STAKE: (Dec A new stake has been created from the St George Utah Boulder Ridge Stake The St George Utah Boulder Ridge East Stake ST GEORGE UTAH BOULDER RIDGE EAST STAKE: (Nov sales director and business development employee; wife A new stake has been created from the Toamasina Madagascar District Counselors — Jean Marius Andriatsitoherina A new stake has been created from the Orem Utah Geneva Heights Stake Church physical facilities manager; succeeding Jose L end user computing manager; succeeding Dennis O KINSHASA DEMOCRATIC REPUBLIC OF THE CONGO MOKALI STAKE: (Dec 2022) President — Iyelenge Jean Paul Balakompandje general director; succeeding Vally Tshimanga Kapepula; wife 2022) President — Danny Joel Vasquez Aguirre 2022) President — Osvaldo Elias Martinez Carreño Seminaries and Institutes of Religion director; succeeding Juan C Counselors — Flavio Cristian Manfred Henriquez Sanchez warehouse manager; succeeding Modesto Mendez; wife Counselors — Luis Alberto Hernandez Moreno MBUJI-MAYI DEMOCRATIC REPUBLIC OF THE CONGO STAKE: (Dec aviation administration virtualization administrator; wife 2022) President — Iván Israel Rodríguez Figueroa health ministry archives manager; succeeding Gonzalo A Counselors — Rodrigo Antonio Huaiquimilla Arcos lawyer and Municipality of Sete Lagoas coordinator; succeeding Márcio Batista Souza; wife You are about to access Constant Contacts (http://visitor.constantcontact.com) You are now leaving a website maintained by The Church of Jesus Christ of Latter-day Saints We provide the link to this third party's website solely as a convenience to you and security practices that differ from those on our website By referring or linking you to this website we do not endorse or guarantee this content If you would like to stay on the page you are viewing please click Cancel To download media files, please first review and agree to the Terms of Use Download a photo or video by clicking or tapping on it To download all photos or videos related to this article select the links at the bottom of each section IDIBAPS researchers have published in Nature Protocols the process for the directed differentiation of human induced pluripotent stem cells (iPSCs) into hepatic stellate cells (iPSC-HSCs) Raquel Martínez García de la Torre and Julia Vallverdú Receive the latest news from our institution directly to your email in a few moments you will receive a confirmation email INDIANAPOLIS &#151; Sometimes obsession pays off.For the last 3 1/2 months has been obsessed with catching Kerry Silvers.On Wednesday was caught in Mexico as the result of a multi-agency effort Wojdylo coordinated Police say Silvers may have critical information about the disappearance and death four years ago of Jill Behrman a 19-year-old Indiana University student.Silvers was spotted in Mexico less than a week ago and Mexican authorities closed a net around him and snatched him up Silvers was working as a teacher and computer expert in his new home town little Martinez de la Torre on the Gulf of Mexico."The last 109 days this case has been my life," Wojdylo said of the search for Silvers whom he wanted to catch because Silvers might be able to provide key information in the Behrman case although he is not a suspect himself.Jill Berhman\'s parents attended the news conference.Wojdylo had good reason to seemed pleased with himself and his law enforcement colleagues Friday at a news conference in Indianapolis They\'d apprehended perhaps Indiana\'s most-sought-after felon-at-large.Silvers was wanted for escape from the Lawrence County Jail in May 2000 and from the Orange County Jail in April 2002 even if meant a life on the lam: The escape forestalled Silvers serving a 63-year prison sentence in Lawrence County on his conviction there for multiple felonies And it delayed his prosecution in Orange County on more than a dozen felony charges &#151; including attempted murder &#151; for the 2002 jailbreak in Paoli.Orange County Prosecutor Kelly Minton said at the news conference that any idea of cutting Silvers a deal to help with the Behrman case was it was a huge frustration to Indiana State Police Detective Rick Lang.Lang heads the Behrman investigation and previously had interviewed Silvers about a conversation Silvers had with Uriah Clouse the Ellettsville man who police have named as a suspect in the Behrman case.Two women told authorities that Clouse had hit Jill Behrman\'s bike with his truck then had taken her to Salt Creek North Fork stabbed her to death and dumped her in the creek But the story crumbled when Behrman\'s bones were found in a forest in Morgan County in March 2003.Clouse and Silvers had been in jail together at one point and investigators hoped Clouse may have told Silvers something about the case Lang said he\'d taken "great interest" in things Silvers had told him before his escape and flight to Mexico but Lang said he was not free to say what more he wants to learn from Silvers.Lang said since Silvers arrived in Indianapolis "I can say at this point that he is cooperative It remains to be seen what he will say to us Whether or not he will continue to speak with me is entirely up to him."As for Silvers\' capture Wojdylo outlined the process that led to his apprehension in Mexico &#151; as well as Silvers\' path there.Silvers and two other men had escaped from the Orange County Jail in Paoli on April 18 allegedly attempting to kill a dispatcher on the way out but Silvers evaded capture in the forests of Orange County for two days.He then made his way to Louisville just across the Rio Grande from Brownsville then moved to the state of Veracruz east of Mexico City eventually settling down in Martinez de la Torre.Wojdylo said every criminal makes "small but critical mistakes," and Silvers\' was that he was getting money from his mother in Lawrence County Wojdylo said Vickie Silvers sent her son $15,000 over 15 months &#151; something she may come to regret.Aiding a criminal in flight is a federal offense and Vickie Silvers will be the subject of discussion for possible prosecution by U.S and she knows I know it," Wojdylo said.He added that Silvers\' family had doubted marshals could catch him So Wojdylo said he took great pleasure in calling Vickie Silvers late Thursday to inform her that he had her son in custody.Before his capture Silvers had become a big man around town in Martinez de la Torre &#151; and hardly for bad things He was teaching English to children in the local school and his computer expertise was a major asset driving a new Volkswagen Jetta."He was very prominent in the community and was doing well," Wojdylo said.Until a week ago marshals quickly put together a two-country They then drove three hours to Martinez de la Torre where they began to stake out places Silvers routinely visited.The fugitive was spotted tooling around in his Jetta When Silvers stopped at a video store late in the day Wednesday followed by the marshals.Silvers was taken "after a brief struggle," Barfield said at the news conference decades of prison face him.Wojdylo said marshals flew back to the United States with Silvers early Thursday after a night of doing the paperwork Mexico required for his extradition They arrived in Indianapolis Thursday afternoon.Silvers initially was held in a cell at the Federal Building he was transferred to the Indiana Department of Correction.Wojdylo praised all the officials and agencies He and others also lauded the "America\'s Most Wanted" show for being instrumental in making the public aware of Silvers and the Behrman case.Another U.S former Bloomington Police Chief Jim Kennedy Just results," he said.Wojdylo said he was happy for Lawrence and Orange counties because Silvers "owes 61 years (of unserved time) to the county of Lawrence and I\'m sure Orange County would like to add to that."And Wojdylo whose obsession with catching Silvers began when he saw the "America\'s Most Wanted" account of the Behrman case said he hopes the capture leads to its resolution."It was \'America\'s Most Wanted\' that brought the case into my living room," he said adding he hopes it will lead to "closure" for Eric and Marilyn Behrman whom he said "deserve peace."He said it now falls to Lang who heads the ongoing Behrman investigation to wrap that up."My job is done," Wojdylo said.<i>Reporter Kurt Van der Dussen can be reached at 331-4372 or by e-mail </i>at kvd@heraldt.com IL — A new restaurant that serves up authentic Mexican food has opened in Lake in the Hills Rinoconcito de Vercruz is located in the former Cachas Taco Bar at 2122 W. Algonquin Road. Restaurant owner Luis Landa has been thinking about opening a restaurant for a decade and decided on Lake in the Hills after realizing the village did not have an authentic Mexican restaurant Business has been picking up at the new restaurant which has an extensive menu and serves up seafood dishes that are popular in Veracruz and the business is also working on acquiring a liquor license from the village of Lake in the Hills Once that is approved, cocktails such as margaritas, pina coladas and Veracruz-inspired drinks, including a cantarito — which is served in a clay jar and includes orange juice, grape juice, lime and spices with tequila —will be added to the menu. More via Shaw Local News Network Get more local news delivered straight to your inbox. Sign up for free Patch newsletters and alerts. MEXICO CITY — A weathered pastel image of the Virgin of Guadalupe hangs from German Herrera Hernandez's dashboard, watching over his passengers from her perch next to his cigarettes, gum and the handful of coins he uses to make change. "We believe in her," said Herrera, a 55-year-old who has been driving a cab in Mexico City for about a decade. "She protects us, wherever we go." When Pope Francis visits this week he'll make an emotional stop at the Basilica of the Virgin of Guadalupe — patron saint of Mexico and "empress of the Americas" — where millions of pilgrims flock each year to pray before the shroud that bears her image. But she's also an ever-present part of life for millions of people like Herrera, not just at the basilica. Across the country, in private homes and public marketplaces, she gazes down beneficently from the walls of taco stands and police stations, from hair salon mirrors and even outside no-tell motels. In poor barrios and posh shopping districts, perhaps nothing unites Mexicans more than their reverence for the Virgin. Grieving families light candles beneath her likeness in shrines to dead relatives, while young hipsters shell out big bucks for shirtsleeve tattoos of the Virgin. "There's the old refrain in Mexico that Mexicans are 90 percent Catholic and 100 percent Guadalupan," said Andrew Chesnut, chair in Catholic studies at Virginia Commonwealth University. "If there's one main constituent element of Mexican-ness, it's Guadalupe, because she obviously transcends the religious realm. ... So she sells products, she's tattoos, (even with) people who aren't necessarily her devotees." According to tradition, the dark-skinned virgin appeared before the Indian peasant Juan Diego in 1531 at Tepeyac, a hillside near Mexico City where Aztecs worshipped a mother-goddess, and her image was miraculously imprinted on his cloak. The image helped priests inculcate Catholicism among indigenous Mexicans during Spanish colonial rule, and the church later made her patron of all the Americas. Juan Diego was canonized as the hemisphere's first Indian saint in 2002 during the papacy of John Paul II. Juan Diego's cloak is on display today behind glass at the basilica, the most visited Marian shrine on the planet, where signs admonish against flash photography and long lines of worshippers are carried past along a self-moving walkway. Chesnut called Francis "a very strongly Marian pontiff" and said his visit to the basilica will be one of the defining moments of his trip to Mexico, which has more Catholics than any other Spanish-speaking country. Each year on Dec. 12, millions of people visit the basilica in northern Mexico City, many of them crawling or bearing statues of Guadalupe on their backs, to beseech all manner of favors: to be healed of an illness, freed from an addiction, for the welfare of loved ones. The rest of the year, many Mexicans make the sign of the cross when they pass Guadalupe shrines found in just about every nook and cranny of the country. Custodians make sure the shrines are always well-kept with offerings of fresh flowers or candles. At the Martinez de la Torre market in central Mexico City, a portrait of Guadalupe framed by neon tubes in the red, white and green of the Mexican flag graces a wall overlooking a meat counter. "Why have a Virgin? Because our faith in her is very great, very important," said butcher Erlinda Olivares Zuniga, who choked up when asked about Guadalupe ahead of Francis' visit. "We are God's chosen, because the Virgin appeared here." Volume 5 - 2018 | https://doi.org/10.3389/fvets.2018.00077 African swine fever (ASF) has spread from the Caucasus region to eastern European Union countries affecting domestic pig and wild boar populations mitigation measures targeting both populations have been established ASF has been reported in thirteen different countries (Georgia In the absence of an effective vaccine or treatment to ASF introduction and spread of ASF onto domestic pig farms can only be prevented by strict compliance to control measures This study systematically reviewed available measures to prevent the spread of ASF in the EU domestic pig sector distinguishing between commercial The search was performed in PubMed and using a common browser A total of 52 documents were selected for the final review process 37 measures were identified as preventive measures for the introduction and spread of ASF these measures were assessed by ASF experts for their relevance in the mitigation of ASF spread on the three mentioned types of farms All experts agreed that some of the important preventive measures for all three types of farms were: the identification of animals and farm records; strict enforcement of the ban on swill feeding; and containment of pigs so as to not allow direct or indirect pig–pig and/or pig–wild boar contacts Other important preventive measures for all farms were education of farmers and operators; no contact between farmers and farm staff and external pigs; appropriate removal of carcasses and food waste; proper disposal of manure and dead animals and abstaining from hunting activities during the previous 48 h (allowing a 48 h interval between hunting and being in contact with domestic pigs) all experts identified that the important preventive measures for non-commercial and outdoor farms is to improve access of those farms to veterinarians and health services The Directive lays down the measures to be taken in the infected area and the provisions to apply on the holdings of that area All control and eradication measures applicable are based on classical disease control methods These measures are applied in combination with strict quarantine and biosecurity measures on domestic pig holdings and animal movement control The Directive also requires that Member States develop and implement plans for the eradication of the disease the aim of this study is to review described measures to prevent the introduction and further spread of ASF in the domestic pig sector focused on the EU scenario An additional aim of this review was to assess the importance of these identified measures depending on the different pig farming systems (see materials and methods section) Adequate identification of relevant measures will allow for the creation of guidelines for pig producers to prevent the spread of ASF which is one of the identified goals of the COST Action 15116 Understanding and combating African swine fever in Europe (ASF-STOP) supported by COST (European Cooperation in Science and Technology) Scientific papers written in English (for reviewing convenience) between the last 39 years (1978 and 2017) were reviewed A list of key words was combined into a Boolean query to identify titles and/or abstracts of documents of interest The key words used (and any word containing the stem presented) were “African swine fever,” “Preventive measure/s,” “Biosecurity,” “Risk,” and “Pig farm.” The search terms applied were “African swine fever” AND [Preventive measure* OR Biosecurity OR Risk OR Pig farm] To make sure other relevant documents such as technical guidelines the literature search was performed following the same query on the internet using a common browser A two step-process was followed to select the literature relevant for the aim of this review A primary exclusion criteria was applied when reading title and abstract of found literature (abstract when available): (i) published before 1978; (ii) not related to the theme of this review; (iii) not related to the European scenario; and (iv) repeated document (already selected among retrieved results) the piece of literature would be kept for the next stage the full text of each selected piece of literature was screened documents (v) which full text was not available; (vi) no preventive measures were described; (vii) described preventive measures were not focused on ASF; or (viii) information on the theme was insufficient The explained process was individually performed by three reviewers following the mentioned exclusion criteria in order to cross-check selected literature and resolve any disagreement A group of experts was invited to participate in an expert opinion session to assess the preventive measures identified in this review Participants belonging to the COST (European Cooperation in Science and Technology) action: “Understanding and combating African swine fever in Europe” (ASF-STOP) supported by COST (COST Action 15116)2 were encouraged to suggest experts with relevant expertise in ASF prevention ASF epidemiology and the EU domestic pig sector the list of measures were reviewed by authors to ensure measures were accurate and clear 20 experts were invited to participate and contacted by email Experts were asked to assess the relevance of each described preventive measure by answering yes or no to the closed question: “Is this measure important for commercial and outdoor-farms?” “Importance” was defined as the perceived need for each measure Experts were asked to perform this assessment within the EU context experts were encouraged to suggest additional measures if they thought they were missing Results were recorded in an Excel datasheet (Microsoft Corp. Figure 1 shows the literature selection process and Table 1 compiles the selected literature The search made on PubMed database returned 168 scientific papers After applying the primary exclusion criteria 69 were selected for the second step of the review the full text was not available for 10 of them 59 scientific articles were selected for the second screening round The same search on a common browser returned 5,100 results of potential interest By applying the primary exclusion criteria 58 results were selected for the second round Flowchart summarizing the literature selection process (A) on PubMed database and (B) on a common browser Pieces of literature included in the review process After applying the second exclusion criteria and completion of the screening rounds 34 articles (25 retrieved from PubMed and 9 retrieved from the browser search) 4 official recommendations (meaning information coming from governmental authorities) and 5 technical guidelines were included in the review The rest of the documents including reports and guidelines were retrieved from the browser search Preventive measures described hereinafter were obtained from the 52 pieces of literature selected during the systematic review These measures were classified in four different groups: general prevented measures suggested for all types of farms (as some of them were common for commercial and three groups of suggested measures for each of the identified types of farms Specific preventive measures based on biosecurity have been proposed depending on the type of farm: commercial, outdoor, or non-commercial (15) Commercial farms are significantly larger in size and number of animals (18) and so, the economic and animal health impact of ASF is greater than on outdoor and non-commercial farms (24, 29) Finally, proper disposal of manure as well as dead animals and other removable material should be ensured (5, 58, 65). Containers and storage basins should accomplish with the minimum requirements for storage capacities recommended by the Best Available Techniques (71) Table 2 compiles the general preventive measures and specific preventive measures for commercial and outdoor farms described in this review General measures to prevent African swine fever spread on domestic pig farms plus specific measures focused on commercial (CM) A total of 12 experts participated in the assessment of the importance of identified preventive measures All of them completed the questionnaire and therefore their responses were included in the analysis Around 3% of assessed measures (2.85%) were categorized as “not applicable” preventive measure There was 100% agreement among experts (12 experts out of 12) that the identification of animals and farm records including animal movements; enforcement of the ban on swill feeding; and containment of pigs to not allow contact with pigs from other farms were important preventive measures for the three types of farms (commercial Other important preventive measures identified for all farms were education of farmers slaughter residues and food waste; proper disposal of manure and dead animals; and a 48 h (minimum) interval between hunting and being in contact with domestic pigs for all farm staff particularly those who work in an infected wild boar area all experts identified as important preventive measures for non-commercial and outdoor farms to improve access of those farms to veterinarians and health services Between eight and nine of experts considered that logistical arrangement for the entry and exit of animals including protocols regarding entrance of vehicles loading areas and role of pig transporters; quarantine period for purchased animals and quarantine rooms; and internal audits and evaluations to enforce biosecurity measures were not important preventive measures for non-commercial farms 10 experts concluded that control measures against flies were not an important preventive measure on outdoor farms Additional preventive measures were suggested by some experts such as the use of nets on animal facilities; establishment of pest control programs on farms; use of carbonic dioxide traps to check the presence of Ornithodoros ticks and change of boots before entering the farm and units several respondents wanted to emphasize the importance of measures already included in the questionnaire establishment of double fencing perimeter on outdoor farms; education of swine veterinarians and farmers paying especial attention to clinical signs and transmission routes; and discouragement of using the same injection syringes and instruments on different farms unless thoroughly disinfected sterilized Figure 2 and Table 2 summarize the results obtained for preventive measures on commercial Figure 2. Results of the assessment of identified preventive measures represented as percentage of yes (blue bars), no (red bars), and not applicable (gray bars) to (A) commercial farms, (B) non-commercial farms, and (C) outdoor farms. Listed preventive measures are described in Table 2 prevention is the main tool to avoid further spread of ASF or an endemic situation Both the systematic literature review as well as the expert opinion elicitation highlighted three main areas where preventive measures would be very relevant to halt ASF spread in the domestic pig population: (1) control of entries into the farm and (3) improvement of health services and education which contains the majority of measures reviewed in the systematic literature review aim at the improvement of biosecurity measures dealing with the replacement of animals in particular in relation with cleaning and disinfection facilities Very few outbreaks have led to secondary spread in the EU and there has been a significant progress in EU advice to improve preventive measures against ASF in non-commercial farms Farm location far from suitable wild boar areas and close to geographical barriers was classified as important by more than half of experts such measures were relevant to more experts on outdoor farms (9 experts) This slight difference might be explained because the likelihood of wild boar being in contact with pigs would be higher on outdoor farms (where biosecurity is intrinsically lower) than on commercial or non-commercial farms Experts who declined to consider it important refereed that this measure is almost unfeasible considering the ecological characteristics of the European continent some of the experts who considered it important wanted to highlight that such a measure would only be applicable to new holdings wild boar cases have continued being notified in the area suggesting that there is still room for improving the strategy or insufficient enforcement are the most common reasons to fail to comply with these measures and are directly related to the third main area of ASF prevention: improvement of health services and education to be able to effectively influence farm workers it is essential to analyze the “at-risk” practices that depended on human behavior which can perpetuate ASF spread and find out measures tailored to each specific situation prevention of ASF becomes very challenging for the European pig sector a safe and effective vaccine is still lacking control and eradication of this disease still relies on rapid detection in field followed by the application of strict sanitary measures biosecurity is the only tool farms have to prevent the introduction of ASF joined efforts focusing on the domestic pig sector and wild boar need to be applied in parallel we will move forward to the final goal of eradicating ASF from the second largest world’s pork producer African swine fever is currently one of the major threats to the pig production in the EU biosecurity is key to prevent its spread between and within domestic pig farms This study identified thirty-seven preventive measures aimed at reducing the spread of ASF among domestic pigs These measures were also assessed by ASF experts within the framework of the EU scenario the most important preventive measures for commercial and outdoor farms were the identification of animals and farm records; enforcement of the ban on swill feeding; and containment of pigs to not allow contact with pigs from other farms other measures were considered relevant in preventing ASF introduction farm staff and external pigs; appropriate removal of carcasses slaughter residues and food waste; proper disposal of manure and dead animals and abstention from hunting activities for a period of 48 h prior to any contact with domestic pigs all experts considered important to facilitate and promote the access of veterinarians and health services to non-commercial and outdoor farms Adequate implementation of these measures can lead to significant advances in ASF prevention and control and possibility contributing to the eradication of ASF from the EU pig sector All authors contributed to the literature review performed to build this review CJ compiled the whole information and wrote the manuscript and SB designed the questionnaire for the assessment of preventive measures CJ analyzed results from the expert opinion and AT participated in the creation of the argument line of this the text All authors contributed to the critical review of the manuscript and approved the final version HF was employed by company GD Animal Health Research Institute All other authors declare no competing interests The reviewer AB and handling Editor declared their shared affiliation This article is based upon work from COST Action 15116 Understanding and combating African swine fever in Europe (ASF-STOP) supported by COST (European Cooperation in Science and Technology) CJ is the recipient of a Spanish Government-funded PhD fellowship for the Training of Future Scholars (FPU) given by the Spanish Ministry of Education MM-A has been financed by the INIA funded project AT-2015-002 Carla Rowe for her help during the review of the manuscript as well as the three referees for their valuable comments and great ideas suggested to get to the final version of this article Transmission routes of African swine fever virus to domestic pigs: current knowledge and future research directions Google Scholar African swine fever: how can global spread be prevented Philos Trans R Soc Lond B Biol Sci (2009) 364(1530):2683–96 Preventive measures aimed at minimizing the risk of African swine fever virus spread in pig farming systems Thirty-five-year presence of African swine fever in Sardinia: history evolution and risk factors for disease maintenance Transbound Emerg Dis (2016) 63(2):e165–77 7. 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General Veterinary Inspectorate Poland. African Swine Fever in Poland. Update on Epidemiological Situation (2017). 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This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited *Correspondence: Cristina Jurado, Y2pkaWF6QHVjbS5lcw== Already a member? Log in Not a member? Sign up Explore our Food Tours → Mexico City is blessed with great weather all year round Summer is not very hot because it’s the rainy season things cool down very fast as soon as the rain starts falling and even early afternoons in summertime can make us break out in a sweat Two of the many delicious ways we have to cool down are paletas (popsicles) and raspados (shaved ice) Paletas and raspados are popular treats all over Mexico but they’re especially popular among schoolkids It’s common to see paleta and raspado vendors around schools waiting for eager little customers at the end of any school day Paletas in Mexico are divided into two main categories: de agua (generally made with fruit juice) and de leche (made with milk) and the flavors are usually those that complement milk arroz con leche (rice pudding) and chongos zamoranos (a typical dessert from the state of Michoacán that is made by boiling milk and adding sugar until only the curds are left) Paletas de agua flavors can be anything from the classic lime and tamarind flavors to “exotic” ones such as kiwi One of our favorite places to get a taste of these kinds of flavors not only in paletas but also in ice cream and sorbets a small shop in Colonia San Rafael that imports its products from Pachuca It’s a great place to sit down for a while and enjoy a good exotic paleta Ice cream parlors are abundant in the neighborhood of Coyoacán One of our favorite parlors is Helados Siberia which has been in business for almost a century and sells a delicious yogurt paleta When you walk the streets of Mexico City, or any other Mexican city for that matter, the brand you see popping up everywhere is La Michoacana This ice cream maker is one of the most recognized in Mexico when a little ice cream parlor started selling popsicles and ice creams in the town of Tocumbo and by the 1990s there was at least one La Michoacana in every plaza of Mexico’s most important cities There are more than 30,000 stores nationwide sometimes several in the same neighborhood But is it worth stopping at one for a paleta or ice cream La Michoacana products are made with natural ingredients and the paletas de agua and aguas frescas have a high fruit content Raspados are another much-loved after-school treat for kids you’ll see vendors pushing carts with big blocks of ice all over the city They usually shave the ice by hand and place the shavings in a plastic bag or cup One of our favorite places to get raspados is at Raspados Sonia in the Mercado Martinez de la Torre in the Guerrero neighborhood Editor’s note: It’s Beat the Heat Week at Culinary Backstreets we’re sharing some of our favorite spots to visit when the summer temperatures soar loading map - please wait...Map could not be loaded - please enable Javascript!→ more information volume 5 - 2011 | https://doi.org/10.3389/fnana.2011.00011 This article is part of the Research TopicAdaptive Function and Brain EvolutionView all 20 articles The expression of the Nkx2.2 gene is involved in the organization of the alar-basal boundary in the forebrain of vertebrates Its expression in different diencephalic and telencephalic regions helped to define distinct progenitor domains in mouse and chick Here we investigated the pattern of Nkx2.2 protein distribution throughout the development of the forebrain of the anuran amphibian We used immunohistochemical and in situ hybridization techniques for its detection in combination with other essential territorial markers in the forebrain No expression was observed in the telencephalon Nkx2.2 positive cells were scattered in the suprachiasmatic territory but also in the supraopto-paraventricular area as defined by the expression of the transcription factor Orthopedia (Otp) and the lack of xDll4 In the basal hypothalamus Nkx2.2 expressing cells were localized in the tuberal region In the diencephalon it was expressed in all three prosomeres (P1–P3) and not in the zona limitans intrathalamica The presence of Nkx2.2 expressing cells in P3 was restricted to the alar portion whereas in P1 the Nkx2.2 expressing cells were located in the basal plate and identified the alar/basal boundary These results showed that Nkx2.2 and Sonic hedgehog are expressed in parallel adjacent stripes along the anterior–posterior axis The results of this study showed a conserved distribution pattern of Nkx2.2 among vertebrates crucial to recognize subdivisions that are otherwise indistinct and supported the relevance of this transcription factor in the organization of the forebrain particularly in the delineation of the alar/basal boundary of the forebrain herein we have analyzed the pattern of distribution of Nkx2.2 a functionally and anatomically related transcription factor in vertebrates We have characterized phenotypically the developing Nkx2.2 expressing forebrain subdivisions and neurons by means of the combination of Nkx2.2 expression with forebrain essential regulators and markers The results of this study showed an extremely conserved distribution pattern of Nkx2.2 among vertebrates crucial to delineate subdivisions that were otherwise indistinct and supported the relevance of this transcription factor in the establishment and organization of the forebrain For the present study, adults, juveniles, and tadpoles of X. laevis were used. Embryos and larvae were classified according to Nieuwkoop and Faber (1967) and metamorphic (59–65) stages were used minimizing as much as possible the number of animals used All animals were treated according to the regulations and laws of the European Union (86/609/EEC) and Spain (Royal Decrees 1201/2005) for care and handling of animals in research after approval from the University to conduct the experiments described Adult Xenopus were purchased from commercial suppliers (Xenopus Express; France) and the different developing specimens were obtained by in vitro fertilization and maintained in tap water at 20°C throughout their development embryos and larvae were deeply anesthetized in a 0.4-mg/ml solution of tricaine methanesulfonate (MS222 and late larvae were perfused transcardially with 0.9% sodium chloride followed by cold 4% paraformaldehyde in a 0.1-M phosphate buffer (PB The brains were removed and kept in the same fixative for 2–3 h they were immersed in a solution of 30% sucrose in PB for 4–6 h at 4°C until they sank embedded in a solution of 20% gelatin with 30% sucrose in PB and stored for 6 h in a 3.7% formaldehyde solution at 4°C The brains were cut on a freezing microtome at 40 μm (adults) or 20–30 μm (juveniles and late larvae) in the transverse or sagittal plane and sections were collected and rinsed in cold PB The embryos and premetamorphic larvae were fixed by immersion overnight at 4°C in MEMFA [0.1 M MOPS (4-morpholinopropanesulfonic acid) 2 mM ethyleneglycoltetraacetic acid then they were processed in toto and finally sectioned at 14–16 μm thickness in the transverse A immunohistofluorescence procedure was conducted with the primary antibody on the free-floating sections that was diluted in 5–10% normal serum of the species in which the secondary antibody was raised in PB with 0.1% Triton X-100 (Sigma) and 2% bovine serum albumin (BSA as follows: (1) Incubation for 72 h at 4°C in the dilution of the primary antibody mouse anti-Nkx2.2 (1:500; Developmental Studies Hybridoma Bank Clone: 74.5A5) and (2) the second incubation was conducted for 90 min at room temperature with the labeled secondary antibody Alexa 488- conjugated goat anti-mouse (1:500; Molecular Probes; catalog reference: A21042) the sections were mounted on glass slides and coverslipped with Vectashield mounting medium (Vector Laboratories The cocktails of primary antibodies were diluted in PB with 0.1% Triton X-100 and used for 60 h at 4°C. They always included mouse anti-Nkx2.2 (1:500; DSHB) in combination with: rabbit anti-Otp (1:1000; produced by “PickCell laboratories” Amsterdam, The Netherlands; according to the protocol described in Lin et al., 1999) rabbit anti-MST (diluted 1:2000; donated by Dr Guerné Université de Strasbourg rabbit anti-Nkx2.1 (1:500; Biopat Immunotechnologies rabbit anti-Tbr1 (1:250; Santa Cruz Biotechnology rabbit anti-TH (diluted 1:1000; Chemicon International and rabbit anti-GABA (1:3000; Sigma-Aldrich The secondary antibodies were used in appropriated combinations and were: Alexa 488-conjugated goat anti-mouse (1:500 Molecular Probes) and Alexa 594-conjugated goat anti-rabbit (1:500 secondary antibodies were diluted in PB with 0.1% Triton X-100 for 90 min at room temperature the sections were mounted on glass slides and coverslipped with Vectashield For double histofluorescence labeling experiments, we combined the immunohistochemistry for Nkx2.2 with in situ hybridization for the following markers: xShh (provided by Dr. Randal Moon. University of Washington; Ekker et al., 1995) and xDll4 (provided by Dr. Nancy Papalopulu. University of Manchester; Papalopulu and Kintner, 1993) Hybridization step was done with 3 μl/ml of a DIG-labeled RNA probe in a 50% formamide containing medium overnight at 55°C The solution used for prehybridization (at 60°C for 1 h) and hybridization contained 50% deionized formamide (Fluka 5× standard saline citrate (Sigma-Aldrich 0.5% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS; Sigma-Aldrich) 5 mM of ethylenediaminetetraacetic acid (Sigma-Aldrich) and 50 g/ml of heparin (Sigma-Aldrich) in water Hybridization was detected using an alkaline phosphatase coupled anti-DIG antibody (Roche Diagnostics Alkaline phosphatase staining was developed with Fast red tablets (Roche Diagnostics) The in situ hybridization was followed by the immunohistochemistry for mouse anti-Nkx2.2 (1:500; DSHB) revealed with Alexa 488-conjugated goat anti-mouse (diluted 1:500 embryos and early larvae were immersed in a solution of 30% sucrose in PB until they sank embedded in a solution of 20% gelatin and 30% sucrose in PB and stored overnight at 4°C in a solution of 4% formaldehyde and 30% sucrose in PB Sections were cut at 14–25 μm thickness in the frontal and horizontal plane on a freezing microtome The sections were analyzed with an Olympus BX51 microscope that was equipped for fluorescence with appropriate filter combinations Selected sections were photographed by using a digital camera (Olympus DP72) Contrast and brightness of the photomicrographs were adjusted in Adobe PhotoShop CS3 (Adobe Systems CA) and figures were mounted in Canvas 11 (ACD Systems Photomicrographs of sagittal (A–D) and transverse (E–P) sections through the Xenopus forebrain subdivisions along the different representative developmental stages The sagittal sections show the almost continuous Nkx2.2 expression from anterior hypothalamic areas to the most caudal regions of the forebrain (A–D) Nkx2.2 is not expressed in telencephalic areas Nkx2.2 expression starts in the SPV territory of the alar hypothalamus (E–H) In the basal hypothalamus Nkx2.2 expression is restricted to the tuberal hypothalamus (I–L) Nkx2.2 is observed in the three prosomeres (P1–P3) and the Zli lacks Nkx2.2 expressing cells (I–P) preoptic commissural area/commissural septo-preoptic area; PO 100 μm (D,H) and 50 μm (A,B,C,F′,G′,I) In Xenopus, Nkx2.2 immunoreactive (Nkx2.2-ir) cells were not observed in telencephalic areas, neither evaginated nor non-evaginated territories, from early developmental stages through the juvenile, when the brain morphology is close to that observed in adults (Figure 1) In order to further characterize the localization of Nkx2.2-ir cells within the forebrain, we carried out double labeling experiments throughout development, using different prosencephalic markers (Figures 2–5) Figure 5. Photomicrographs of transverse (A,B) and horizontal (C) sections through the forebrain illustrating Nkx2. 2 expression in combination with xShh. The expressions of Nkx2.2 and xShh extend along the anterior–posterior axis in parallel adjacent stripes, forming longitudinal columns along the forebrain. Abbreviations as in Figure 1 All the results obtained from the double labeling analysis confirmed the localization of the Nkx2.2-ir cells in the regions described above and summarized in Figure 6 Figure 6. Schematic drawings of sagittal and coronal sections through a premetamorphic brain of Xenopus laevis illustrating the distribution of Nkx2.2 expressing zones (green regions in the sagittal view) and cells (green dots in the coronal view) along the forebrain. The appropriated levels of the coronal sections are indicated in the sagittal view. Abbreviations as in Figure 1 To fully understand the precise topological distribution of Nkx2.2 expression its combination with the respective expression of different forebrain markers has been shown to be extremely useful we have analyzed the distribution of Nkx2.2 in combination with the localization of Nkx2.1 and in situ hybridization for the detection of xShh and xDll4 Shh and Nkx2.2 are not always expressed in the same regions Nkx2.2 expression is not detected in a comparable region given that the most anterior expression found coincides with the Otp SPV expressing zone within the hypothalamus (see below) On the basis of combined expression analysis, at least two different longitudinal alar domains have been proposed in the chick hypothalamus: the Dlx- and Shh-negative SPV area, which lies under the border of the FoxG1-positive telencephalic field, and the subparaventricular area, which lies under it and is adjacent to the Shh-positive basal plate, and expresses Dlx5 and Nkx2.2 (Bardet et al., 2010) the most anterior Nkx2.2 positive cells were localized in a Shh−/Nkx2.1−/xDll4−/Otp+ territory forming a thin strip of cells that delimit the region just anterior to the SC (present results) and their localization within this distinct region is highlighted because some of these cells contain both Otp and Nkx2.2 given the close spatial relationship between Shh and TH expressing neurons in the SC of Xenopus a possible implication of Shh/Nkx2.2 may exist for the acquisition of the dopaminergic phenotype in this region suggesting that Nkx2.2 could be implicated in the acquisition of the GABAergic phenotype Interestingly, also in Xenopus, Nkx2.2 expression is observed in the prethalamus, and its precise localization is corroborated by the colocalization of the Nkx2.2 expressing cells in the territory of P3 that is xDll4+/Nkx2.1+/Pax6+/TH+/xShh+ (present results; Bachy et al., 2002; Brox et al., 2003; Moreno et al., 2008a,b; Domínguez et al., 2010) and that xShh-expression in the Zli could lead to establish the Nkx2.2 expression pattern in the P2/P3 territory of amphibians This work supported by grants from Spanish MICINN and the UCM (Grant numbers: BFU2009-12315 and BSCH-UCM GR58/08) Ruth Morona for the fruitful discussions about the diencephalic regionalization and to Dr López for the critical reading of the manuscript Pubmed Abstract | Pubmed Full Text Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Pubmed Abstract | Pubmed Full Text Pubmed Abstract | Pubmed Full Text Pubmed Abstract | Pubmed Full Text Pubmed Abstract | Pubmed Full Text Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Pubmed Abstract | Pubmed Full Text Pubmed Abstract | Pubmed Full Text CrossRef Full Text Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Pubmed Abstract | Pubmed Full Text Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Pubmed Abstract | Pubmed Full Text Pubmed Abstract | Pubmed Full Text Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Pubmed Abstract | Pubmed Full Text | CrossRef Full Text CrossRef Full Text Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Pubmed Abstract | Pubmed Full Text Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Pubmed Abstract | Pubmed Full Text Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Pubmed Abstract | Pubmed Full Text CrossRef Full Text CrossRef Full Text CrossRef Full Text CrossRef Full Text Pubmed Abstract | Pubmed Full Text Pubmed Abstract | Pubmed Full Text Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Pubmed Abstract | Pubmed Full Text Pubmed Abstract | Pubmed Full Text Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Pubmed Abstract | Pubmed Full Text Pubmed Abstract | Pubmed Full Text Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Pubmed Abstract | Pubmed Full Text Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Pubmed Abstract | Pubmed Full Text Pubmed Abstract | Pubmed Full Text CrossRef Full Text CrossRef Full Text Pubmed Abstract | Pubmed Full Text Pubmed Abstract | Pubmed Full Text Pubmed Abstract | Pubmed Full Text Pubmed Abstract | Pubmed Full Text Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Pubmed Abstract | Pubmed Full Text Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Pubmed Abstract | Pubmed Full Text Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Pubmed Abstract | Pubmed Full Text Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Pubmed Abstract | Pubmed Full Text Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Pubmed Abstract | Pubmed Full Text Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Pubmed Abstract | Pubmed Full Text Pubmed Abstract | Pubmed Full Text Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Pubmed Abstract | Pubmed Full Text Pubmed Abstract | Pubmed Full Text Pubmed Abstract | Pubmed Full Text Pubmed Abstract | Pubmed Full Text Pubmed Abstract | Pubmed Full Text Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Pubmed Abstract | Pubmed Full Text Pubmed Abstract | Pubmed Full Text Pubmed Abstract | Pubmed Full Text Pubmed Abstract | Pubmed Full Text González A and Moreno N (2011) Ontogenetic distribution of the transcription factor Nkx2.2 in the developing forebrain of Xenopus laevis Copyright: © 2011 Domínguez This is an open-access article subject to an exclusive license agreement between the authors and Frontiers Media SA provided the original authors and source are credited *Correspondence: Nerea Moreno, Faculty of Biology, Department of Cell Biology, University Complutense of Madrid, C/José Antonio Novais 2, Madrid E-28040, Spain. e-mail:bmVyZWFAYmlvLnVjbS5lcw== COVID-19 Resources. Read more! DOWNEY – Sarah Sarofeem was crowned Miss Downey 2023 at the Downey Rose Float Association’s 66th annual Miss Downey Pageant on Saturday Jessica Frometa was crowned Miss Teen Downey The title of Junior Miss Downey went to Alexis Givens Clover Curtice was crowned Little Miss Downey Little princesses include Mavis Cortez and Jacqueline Ruelas “We are extremely proud to have been hosting the Miss Downey Pageant for 66 years,” the Downey Rose Float Association said in a statement “Downey produces amazing youth who are smart They serve as ambassadors for the Downey Rose Float and the city in many local events throughout their reign “We would like to thank all of the volunteers parents and contestants for another successful year.” Below is a complete list of competition winners: Academic Little Miss Downey Contestant Clover Curtice Junior Miss Downey Contestant Sophia Anaya Athletic Little Miss Downey Contestant Jacqueline Ruelas Miss Teen Downey Contestant Denise Cantero Volunteer Little Miss Downey Contestant Mavis Cortez Junior Miss Downey Contestant Leilah Simonian Miss Teen Downey Contestant Jessica Frometa Miss Downey Court (l-r) Princess Ruth Palmquist Little Miss Downey Court (l-r) Princess Jacqueline Ruelas Queen Clover Curtice and Princess Mavis Cortez Junior Miss Downey Court (l-r) Princess Sophia Anaya Queen Alexis Givens and Princess Emmarie Perez Miss Teen Downey Court (l-r) Princess Miah Mount Queen Jessica Frometa and Princess Aleezah Mackewicz Copyright © 2023 The Downey Patriot Newspaper Please enable JS and disable any ad blocker INDIANAPOLIS A fugitive who escaped from Lawrence and Orange County Indiana jails - and who may have information on the murder of IU student Jill Behrman - has been captured in Mexico and brought home Behrman went missing while on a bike ride in Monroe County in May 2000 Her remains were found in a secluded area of Morgan County north of Paragon in March 2003 but investigators have said they believe that the escapee has had conversations with a primary suspect in the case 1,900 miles away from the Orange County Jail where police had last seen him Silvers\' was profiled twice on the FOX TV program "America\'s Most Wanted" in segments on Jill Behrman\'s death Police have said that Silvers is not suspected of being involved in Behrman\'s disappearance and death but police said he was being sought for questioning.While in jail Silvers reportedly had conversations with Uriah Clouse an Ellettsville man who had been named as a suspect in the Behrman case.Remains belonging to Behrman were found by a father and son out tracking game in early March 2003 in a wooded area near Duckworth and Warthen roads.Clouse has never been charged in her disappearance or death Silvers had eluded authorities for nearly 1,000 days and Orange County Sheriff Doyle Cornwell is happy to see him behind bars once more."That was a good Christmas present for me," Cornwell said "He was a very dangerous type individual and I was scared to death that someone else would get hurt before we got him off the streets he\'ll have 150 years to decide if it was worth it."Silvers escaped from the Orange County Jail on April 18 Two years earlier he had disappeared from Lawrence County Jail.Authorities finally caught up with Silvers late Wednesday in the small village of Martinez de La Torre Marshals returned him to Indianapolis Thursday.Police officials held a press conference in Indianapolis this morning announcing Silvers\' capture That press conference did not happen before press deadline this morning.Earlier Cornwell said there is a chance that Silvers will return to his jail in the future."Extra extra precautions will be taken if and when that person is incarcerated at my facility," he said "During visitation is when it (the escape) happened He will not have those privileges and will be in solitary confinement and will have no access to other inmates He is an extremely intelligent con-artist."Cornwell said his department had learned from the experience and changes have been implemented CAMAGÜEY.-The Ballet of Camagüey (BC) premiered this Friday a suite of the ballet The Nutcracker allows us to follow the thread of the Christmas story around a broken gift and Clara's dream that motivated the work premiered in 1892 with choreography by Lev Ivanov book by Marius Petipa and music by Piotr I a former BC dancer and current artistic director of the Kentucky Ballet Theater but the COVID-19 pandemic slowed down the process I feel satisfied because it is a dream come true they cannot be trusted because there is no perfection in ballet,” he declared to Adelante He was a professional dancer at BC from 1988 to 1993 then he went to Mexico to work as a principal dancer and since 2017 he has been collaborating as a guest choreographer for the company founded by Vicentina de la Torre and which counted Fernando Alonso “Artistic maturity occurs with the same experience that dancers can use on stage Shirley has to keep trying harder because her role is technically demanding and difficult to achieve,” he added children from the Fernando Alonso Dance and Ballet Promotion Center appear in the first act and in the party and snow scenes which promotes the active role of the family and their union Due to the injury of the prima ballerina Rosa María Rodríguez Armengol assumed the leading role of Clara and she has been with the company for three years “It is a great honor and a joy to have the opportunity that I have had dealing with adverse situations but as long as there is passion and dedication also with the aspiration of other roles in the repertoire She looked elegant together with the couple Harold Báez Corona and claims to appreciate the choreography from the first moment: “It is a very beautiful version but enjoyment must take precedence over everything and that was the case” as well as a bit of nerves knowing that an audience eager to see good results awaited us Former dancer and rehearsal teacher Liuba Corzo prefers to relate Shirley's first time as a leading lady with how new The Nutcracker is for the company: “We are very happy because we have been trying to do it for years therefore we cannot compare ourselves with ourselves” the prevalence in the current BC of members at the beginning of their career is evident: “There is a missing link between when you leave school and enter the company because they come with the technique but details need to be polished” the maître Hilda María "Lila" Martínez de la Torre and Regina Balaguer received recognition from cultural institutions higher education and the Union of Cultural Workers in the territory president of the Provincial Council for the Performing Arts described the 55th anniversary as an "act of sincere reverence for such commendable dedication," and highlighted the collective's understanding of art "as an event of vital relevance in the ethical Regina Balaguer praised the dedication of BC workers technicians and administrators to overcome obstacles in the country's circumstances exemplified in sessions without electricity: "If six rehearsals have been done with music That reason for discomfort in daily life made her doubt about the attendance of the public due to the blackouts however she received the satisfaction of a full Principal Theater it is not the complete choreography because the conditions did not allow it We will continue perfecting our work because our public from Camagüey and invited to the next performance of The Nutcracker suite this Sunday Reproduction is allowed when mentioning the source The chief of the Police Regional Office-Davao (PRO-11) has been relieved from his post effective Friday PRO-11 said the relief of Brigadier General Aligre Martinez may have something to do with the serving of arrest warrants against Kingdom of Jesus Christ (KOJC) leader Pastor Apollo Quiboloy and five others on June 10 “It is one of the considerations,” PRO-11 Spokesperson KOJC and former President Rodrigo Duterte have condemned and called the service of the warrants ‘excessive and overkill.’ However police maintained that it was a lawful operation Police Brigadier General Nicolas Torre III has replaced Martinez “Confirmed na na-relieve po ang current Regional Director ng Police Regional Office 11 Police Brigadier General Aligre Martinez effective June 14 At siya po ay papalitan ni Police Brigadier General Nicolas Torre III,” Dela Rey said Martinez was installed as PRO-11 chief on April 25 replacing Brigadier General Alden Delvo who retired from the police service Martinez will be reassigned to the Police Holding and Accounting Unit in Camp Crame was the former chief of the Quezon City Police District He resigned from his post amid the investigation into the road rage incident involving a former policeman and a cyclist He will leave his current post as director of the PNP Communications and Electronics Service to serve in PRO-11 The formal turnover of command is yet to be scheduled gmaregionaltv.com is home to the latest stories produced by news teams of GMA Regional TV from stations in key areas across the Philippines gmaregionaltv@gmanetwork.com About us Advertise with us OrganisationAlhaurín de la Torre, first ‘Malaguistas to the Core’ of 2023 The nearby Málaga municipality was the star of the show during #MálagaTenerife last weekend at La Rosaleda, with representation in the stands and in the box. Alhaurín de la Torre kicks off the New Year for Malaguistas to the Core, repeating last season’s Copy linkLast Saturday’s #MálagaTenerife fixture corresponding to LaLiga SmartBank matchday 22 was dedicated to the ‘Malaguistas to the Core’ from Alhaurín de la Torre.  La Rosaleda once again hosted a large number of residents from the nearby Costa del Sol municipality who travelled the 19 kilometres to experience the unique atmosphere of a stadium that enjoyed the second best attendance of the season The 50 ‘Malaguistas to the Core’ from Alhaurín de la Torre a national and international benchmark location in terms of quality of life arrived at La Rosaleda by bus to enjoy the warm-up and receive the team's home jersey as soon as they entered the ground One featured the 11 boys and girls who accompanied the players on the pitch within the Escort Kids initiative The other group was located in the stands at the corner of Tribuna with Fondo Norte Alto the Councillor for Sport in Alhaurín de la Torre María del Mar Martínez and others enjoyed the warm-up together with Sebastián Fernández 'Basti' from the MCF Foundation’s Social Department Alhaurín de la Torre’s presence was highlighted throughout Saturday afternoon A promotional spot was featured in the run-up to play and at half-time the town’s residents received a warm welcome via the public address system as well as the ongoing appearance on the ‘U’ television during the match The municipality was also represented in the VIP box by Councillor for Sport together with Málaga CF’s board of directors and the political institutions from the city and the province The municipality also installed a stand on the first floor of the VIP Area of Tribuna featuring typical regional festive goods The support of the 'Malaguistas to the Core’ was crucial during their second visit to La Rosaleda for the team to enjoy the necessary energy to go level Exactly the same result as their previous invasion on 16th October 2021