Assemblyman Matt Slater and Somers Town Supervisor Robert Scorrano announced today that a $281,500 state grant will fund important restoration and weather proofing improvements designed to preserve the town’s landmark Elephant Hotel which is celebrating its 200th anniversary.  “The Elephant Hotel holds a significant place not only in the history of Somers and New York but also in American history,” Harckham said “This important investment in the preservation and restoration of the cherished national landmark is vital so it can continue to serve the community as a town hall home of the Somers Historical Society and as an archive for vital historical records The improvements will help to preserve this structure for another 200 years as a testament to the rich history and heritage it represents.” “The Elephant Hotel is an iconic piece of Somers history and I am proud to work alongside our partners in government to support critical funding to preserve it,” Slater said “The Hudson Valley is full of historic landmarks like the Elephant Hotel and it is our responsibility to ensure future generations can see Congratulations to Supervisor Scorrano and the great town of Somers!” “This year marks the 200th anniversary of the historic Elephant Hotel a treasured landmark listed on both the New York State and National Register of Historic Places,” Scorrano said “This  grant will support critical repairs to preserve not only the building itself but also the invaluable historic artifacts and essential town documents housed within I am proud to support a state capital plan that makes key investments like this This funding ensures that the Elephant Hotel remains a cornerstone of our community for generations to come.” The funding will enable the town to repair rehabilitate and weatherproof the building so that it will remain a National Historic Landmark and continue to provide a space for town offices The goal is to prevent further damage to the site.  the Elephant Hotel was named in honor of a particular pachyderm named “Old Bet,” which was owned by circus entrepreneur Hachaliah Bailey The structure is noted by architectural historians as being representative of a rare distinctive example of Federal period domestic architecture and a  rural turnpike hotel from its era.  The work is part of an ongoing restoration project of the structure which was designated a National Historic Landmark in 2005 was replaced by a new bronze sculpture created by local artist Luigi Badia and funded by the Wittmann family The grant was funded through the state Regional Economic Development Councils (REDC) and sourced from the state’s from the Environmental Protection Fund A handful of people in Pompeii that were killed by the devastating eruption of Mount Vesuvius in 79 are not who experts thought they were according to a team of researchers that recently collected DNA from the individuals’ remains The team’s findings—published today in Current Biology—spotlight previous incorrect conclusions about relationships between the residents of Pompeii and reveals new insights about the demographics of the Ancient Roman port city “We show that the large genetic diversity with significant influences from the Eastern Mediterranean was not only a phenomenon in the metropolis of Rome during Imperial times but extends to the much smaller city of Pompeii which underscores the cosmopolitan and multi-ethnic nature of Roman society,” said Alissa Mittnik an archaeogeneticist at the Max Planck Institute for Evolutionary Anthropology and Harvard University The people weren’t so lucky. They died while being bombarded by pyroclastic flows—fast-moving clouds of superheated gas, ash, and dust—though some of them may have lived for hours before final succumbing to the extreme conditions But they left behind human-shaped voids in the hardened ash which early investigators of Pompeii learned to fill with plaster giving them an eerie cast of the person who died there The researchers behind the new study extracted DNA from 14 of the 86 plaster casts currently undergoing restoration Despite the volcanic conditions that killed off the Pompeians traces of their genetics remain in the bones they left behind The team found that some residents were different sexes than previously thought and had different genetic relationships with one another One particularly famous set of remains revisited by the team is that of an adult with a golden bracelet and a child—the child being on the adult’s lap the remains actually belong to an unrelated male and a child Another duo—long thought to be sisters who died together—included at least one male but they weren’t two closely related females “This study illustrates how unreliable narratives based on limited evidence can be, often reflecting the worldview of the researchers at the time,” said co-author David Caramelli, a researcher at the Universita di Firenze, in a Cell release “Most narratives spun around the victims take into account that they were likely attempting to flee the city but these stories often link them to their discovery place,” Mittnik said the man found at the Villa of the Mysteries was portrayed as the custodian of the villa who dutifully remained at his post.” “Our research demonstrates that such interpretations are often unreliable and instead we should consider a wide range of scenarios that could explain the evidence we find,” she added Previous genetic studies of the ancient city’s residents revealed how people moved to Pompeii from other parts of the Mediterranean. One 2022 paper found evidence that at least one man who died there had Sardinian ancestry in addition to bacteria associated with spinal tuberculosis the team found that five individuals in Pompeii weren’t so genetically associated with modern-day Italians and Imperial-period Etruscans as they were to groups from the eastern Mediterranean and North Africa—specifically North African Jewish populations Pompeii was an important port in first-century Rome so it’s not a huge surprise that it had representation from across the Mediterranean—but the genetic stories of the studied individuals verifies it these findings highlight the potential of ancient DNA analysis When integrated with bioarchaeological records it can offer a more nuanced understanding of Pompeii’s victims,” said Gabriele Scorrano a geneticist at the University of Rome Tor Vergata and a researcher involved in the 2022 paper “Regarding the genetic makeup of the Pompeian population the new data aligns with previous genomic study suggesting an ancestry strongly influenced by recent migration from the eastern Mediterranean.” “Despite the challenges of DNA preservation in Pompeian remains the authors did an impressive job of retrieving genetic information providing insights into specific aspects of Pompeian life The study also shows that genetic research of the people in Pompeii is an opportunity to right the wrongs of the past The team wrote that “it is possible that the exploitation of the casts as vehicles for storytelling led to the manipulation of their poses and relative positioning by restorers in the past.” previous research and restoration work in Pompeii may have distorted the ground truth at the site—where individuals were relative to one another when they died so they give modern experts an opportunity to correct narratives that may be borne out of previous attempts to dramatize the final moments of Pompeii residents in specific ways Pompeii is one of the most horrifying—but amazing—examples of how a disaster can provide a portal into the past New research methods are making it possible to see more through that portal than before As genetic testing of the Pompeii remains continues—and indeed excavation of the many still-buried parts of the city—we’ll get a more complete portrait of the city swallowed by a volcano ' + scriptOptions._localizedStrings.webview_notification_text + ' " + scriptOptions._localizedStrings.redirect_overlay_title + " " + scriptOptions._localizedStrings.redirect_overlay_text + " Metrics details The archaeological site of Pompeii is one of the 54 UNESCO World Heritage sites in Italy thanks to its uniqueness: the town was completely destroyed and buried by a Vesuvius’ eruption in 79 AD we present a multidisciplinary approach with bioarchaeological and palaeogenomic analyses of two Pompeian human remains from the Casa del Fabbro We have been able to characterize the genetic profile of the first Pompeian’ genome which has strong affinities with the surrounding central Italian population from the Roman Imperial Age despite the extensive connection between Rome and other Mediterranean populations a noticeable degree of genetic homogeneity exists in the Italian peninsula at that time palaeopathological analyses identified the presence of spinal tuberculosis and we further investigated the presence of ancient DNA from Mycobacterium tuberculosis our study demonstrates the power of a combined approach to investigate ancient humans and confirms the possibility to retrieve ancient DNA from Pompeii human remains Our initial findings provide a foundation to promote an intensive and extensive paleogenetic analysis in order to reconstruct the genetic history of population from Pompeii Geographic location of the Pompeii site, Campania (Italy). Map source: SINAnet ISPRA – Dem75 (QGIS 3.22 ‘Biatowieza’) https://www.qgis.org/it/site/ have dramatically increased the amount of data that can be obtained from previously unsuitable samples for genetic research and may open new avenues to substantially increase the knowledge of the genetic diversity in the ancient Pompeian population In this work, we present a multidisciplinary approach with bioarchaeological and palaeogenomic analyses of two human remains from the Casa del Fabbro (House of the Craftsman: Supplementary Fig. S1) from Pompeii The successful recovery of aDNA from one individual enabled us to reconstruct its genetic history and to investigate the presence of blood-borne pathogens this data can also give us an overview of the genetic diversity outside of Rome during the Roman Empire Both skeletons have been discovered in anatomical position They were both leaning on a low relief in a corner of what probably was the dining room a sort of couch or chaise longue used in Roman buildings during meals Individual A was in left lateral recumbent position with flexed limbs with the left arm and leg on the ground and right limbs on the triclinium The individual B had the arms gathered in front of the skull and legs on the ground flexed on the right side with the back leaning against the triclinium The low coverage obtained for individual B prevented us from reaching any further assessments of quality parameters we report the details of the analyses only for individual A Point estimates and ± 3 standard errors for the top twenty populations with significantly (Z < 3) more allele sharing with the ancient Pompeian in comparison to 24 ky old Mal’ta based on the statistic D(Mbuti, Test; Pompeian, Russia_MA1_HG). All results can be found in Supplementary Table S5 indicating that in individual A of Pompeii no further contribution by Iranian-related ancestry occurred after the Iron Age We set a minimum threshold of 100,000 SNPs and only considered results when p > 0.05 Photography and digital radiograph of the fourth lumbar vertebra (L4) affected by tuberculous spondylodiscitis of the individual A Extrapulmonary tuberculosis can cause characteristic skeletal changes such as collapse of the vertebrae (Pott's disease) the two individuals had never been in contact with the soil during the diagenesis process because they were entirely covered by volcanic material This makes the finding of the Mycobacterium tuberculosis DNA more likely to be endogenous our results represent the first successfully sequenced Pompeian human genome This makes it likely that this male lineage arrived in the Italian peninsula through an Anatolian source during the Neolithic but very likely he is not part of the large external migrations related to the practice of enslavement our study—albeit limited to one individual—confirms and demonstrates the possibility of applying palaeogenomic methods to study human remains from this unique site Our initial findings provide a foundation to promote an intensive analysis of well-preserved Pompeian individuals Supported by the enormous amount of archaeological information that has been collected in the past century for the city of Pompeii their paleogenetic analyses will help us to reconstruct the lifestyle of this fascinating population of the Imperial Roman period Periapical digital radiographs were taken using a NOMAD hand-held dental X-ray device (Aribex All radiographs were taken with a Rinn-type digital sensor holder with 0.05 s exposure time and 60 kV a form of osteo-articular tuberculosis (TB) was examined by morphological approach and digital radiograph of the fourth lumbar vertebra (L4) a DR Fujifilm machine was used with an exposure (100 ms) at 55 kV to 100 mA The otic capsule was targeted and around 100–200 mg bone powder was used for DNA extraction In order to remove the surface contaminants the samples were pre-digested using a digestion buffer (0.46 M of EDTA pH = 8; 10 nm of TE buffer 100×; 0.14–0.22 mg/ml of Proteinase K; 0.5% of N-laurylsarcosine; 1/1000 vol of Phenol red) for 45 min at 37 °C the samples were centrifuged at 2000g for 2 min and the supernatant was discarded new digestion buffer was added for a 24-h digestion at 37 °C The samples were then centrifuged at 2000g for 5 min and the pellet was stored for later re-extraction The aDNA extraction was performed on the digested solution using Silica powder-based DNA extraction protocol 100 μl silica suspension and 10 × volume of binding buffer (4.88 M GuHCl and 29.3% 2-propanol; 1/1000 vol of phenol red; 24.88 mM of NaCl; 87.6 mM of Na Acetate; final pH = 4) was added and adjusted to pH 4 with 37% HCl The solution was incubated for 1 h at room temperature after which the samples were centrifuged for 2 min at 2000g and the supernatant was discarded The silica was re-suspended in 1 ml of binding buffer transferred on a new 2 ml tube and the aDNA was washed using ice-cold ethanol the DNA was eluted in 80 μl Quiagen EB buffer For ligation NEB Quick Ligation module (E6056L) was followed and the mix was incubated at 20 °C for 15 min the mixture was purified using Qiagen MinElute spin columns and DNA was eluted in 20 μl EB buffer adapter fill-in reaction was performed incubating at 65 °C for 20 min and 80 °C for 20 min 30 μl of reaction mix The quantification of the library was conducted using SYBER green mix according to manufacturer’s instructions and using IS8 and IS7 primers The amount of DNA library was used to assess the optimal number of PCR cycles required for DNA library indexing The indexing was performed on 20 μl DNA library using 2X Kapa U (following the manufacture’s temperature instruction) and 1 μl of each primer (10 mM inPE forward primer and indexed reverse primer) The indexed amplified DNA libraries were then purified using Qiagen MinElute Kit and eluted in 50 μl EB buffer To quantify the DNA libraries an Agilent Bioanalyzer 2100 was used and the libraries were sequenced on Illumina HiSeq 2500 using v3 chemistry and paired end (PE) 100 cycles The mitochondrial genome mapping was performed against the revised Cambridge Reference Sequence (rCRS) using base and reads mapping quality > 30 The RY parameter represents the fraction of the total number of reads aligned with the Y chromosome (nγ) divided the total number of reads mapped with both sex chromosome (nγ and nγ): RY = ny/(nX + nY) A RY parameter value above 0.077 is consistent with male individuals while a value lower than 0.016 with female ones projecting the ancient individuals onto the components calculated for modern Western Eurasian populations using “lsqproject” and “shrinkmode” options of smartpca While these reads could stem from actual circulating Mycobacterium tuberculosis found in the individual the low amount of data does not provide enough resolution to authenticate or to perform downstream analysis on it The allignment bam file generated in this study has been deposited in the Zenodo database under the permanent DOI: https://doi.org/10.5281/zenodo.6468368 Impact of the AD 79 explosive eruption on Pompeii Causes of death of the inhabitants inferred by stratigraphic analysis and areal distribution of the human casualties A re-evaluation of manner of death at Roman Herculaneum following the AD 79 eruption of Vesuvius Methodological strategies to assess the degree of bone preservation for ancient DNA studies Degradation of DNA in dried tissues by atmospheric oxygen Ancient DNA in human bone remains from Pompeii archaeological site 2000 Year-old ancient equids: An ancient-DNA lesson from pompeii remains Genetic characterization of Pompeii and Herculaneum Equidae buried by Vesuvius in 79 AD Recovery and amplification of ancient DNA from Herculaneum victims killed by the 79 AD Vesuvius hot surges Ancient DNA and family relationships in a Pompeian house Histological analysis and ancient DNA amplification of human bone remains found in Caius iulius polybius house in pompeii Enzymatic repair of selected cross-linked homoduplex molecules enhances nuclear gene rescue from Pompeii and Herculaneum remains The genomic history of southeastern Europe The genomic history of the Iberian Peninsula over the past 8000 years Genome flux and stasis in a five millennium transect of European prehistory Comparing ancient DNA preservation in petrous bone and tooth cementum The impact of pyroclastic density currents duration on humans: The case of the AD 79 eruption of Vesuvius Mathematical contribution to the theory of evolution On the reconstruction of the stature of prehistoric races Estimation of stature from long bones of American Whites and Negroes A re-evaluation of estimation of stature based on measurements of stature taken during life and of long bones after death Estimation of stature from long limb bones of American Whites and Negroes—Reply Stature in archeological samples from central Italy: Methodological issues and diachronic changes Reconstructing medical knowledge in ancient Pompeii from the hard evidence of bones and teeth I fuggiaschi di Ercolano: paleobiologia delle vittime dell’eruzione vesuviana del 79 dC Temporal patterns of nucleotide misincorporations and DNA fragmentation in ancient DNA Accurate sex identification of ancient human remains using DNA shotgun sequencing HaploGrep 2: Mitochondrial haplogroup classification in the era of high-throughput sequencing PhyloTree Build 17: Growing the human mitochondrial DNA tree Ancient Rome: A genetic crossroads of Europe and the Mediterranean Ancient DNA from hunter-gatherer and farmer groups from Northern Spain supports a random dispersion model for the neolithic expansion into Europe Origin and diet of the prehistoric hunter-gatherers on the mediterranean island of Favignana (Egadi Islands Phylogeography of mitochondrial DNA in western Europe Rare human mitochondrial HV lineages spread from the Near East and Caucasus during post-LGM and Neolithic expansions Fine dissection of human mitochondrial DNA haplogroup HV lineages reveals paleolithic signatures from European glacial refugia The genetic landscape of Serbian populations through mitochondrial DNA sequencing and non-recombining region of the Y chromosome microsatellites High resolution analysis and phylogenetic network construction using complete mtDNA sequences in Sardinian genetic isolates Excavating Y-chromosome haplotype strata in Anatolia The Levant versus the Horn of Africa: Evidence for bidirectional corridors of human migrations Low-pass DNA sequencing of 1200 Sardinians reconstructs European Y-chromosome phylogeny The peopling of the last Green Sahara revealed by high-coverage resequencing of trans-Saharan patrilineages Early Neolithic genomes from the eastern Fertile Crescent Late Upper Palaeolithic hunter-gatherers in the Central Mediterranean: New T archaeological and genetic data from the Late Epigravettian burial Oriente C (Favignana The first horse herders and the impact of early Bronze Age steppe expansions into Asia The spread of steppe and Iranian-related ancestry in the islands of the western Mediterranean Ancient genomes from North Africa evidence prehistoric migrations to the Maghreb from both the Levant and Europe Ancient genomes reveal social and genetic structure of Late Neolithic Switzerland The genetics of an early Neolithic pastoralist from the Zagros Massive migration from the steppe was a source for Indo-European languages in Europe Genome-wide patterns of selection in 230 ancient Eurasians Early farmers from across Europe directly descended from Neolithic Aegeans Upper Palaeolithic genomes reveal deep roots of modern Eurasians New insights into the Tyrolean Iceman’s origin and phenotype as inferred by whole-genome sequencing The demographic development of the first farmers in anatolia Ancient human genomes suggest three ancestral populations for present-day Europeans Genomic insights into the origin of farming in the ancient Near East Genetic origins of the Minoans and Mycenaeans Parallel palaeogenomic transects reveal complex genetic history of early European farmers Genetic history from the Middle Neolithic to present on the Mediterranean island of Sardinia The genetic prehistory of the Baltic Sea region Kinship-based social inequality in Bronze Age Europe Derived immune and ancestral pigmentation alleles in a 7,000-year-old Mesolithic European The Beaker phenomenon and the genomic transformation of northwest Europe Upper Palaeolithic Siberian genome reveals dual ancestry of Native Americans The origin and legacy of the Etruscans through a 2000-year archeogenomic time transect The genetic and cultural impact of the Steppe migration into Europe Ancient genomes reveal structural shifts after the arrival of Steppe-related ancestry in the Italian Peninsula Identification of Pathological Conditions in Human Skeletal Remains (Academic Press Ancient pathogen DNA in human teeth and petrous bones Improved metagenomic analysis with Kraken 2 Exploring alternative biomaterials for diagnosis of pulmonary tuberculosis in HIV-negative patients by use of the GeneXpert MTB/RIF assay Detection of Mycobacterium tuberculosis DNA on the oral mucosa of tuberculosis patients Molecular detection of Mycobacterium tuberculosis from buccal swabs among adults in Peru Screening methods for detection of ancient Mycobacterium tuberculosis complex fingerprints in next-generation sequencing data derived from skeletal samples Mitochondrial variability in the Mediterranean area: A complex stage for human migrations Ancient Roman mitochondrial genomes and isotopes reveal relationships and geographic origins at the local and pan-Mediterranean scales and communication in the Roman Empire: Three aspects of movement in history A case of healing spinal infection from Classical Rome Palaeopathology of human remains from the Roman Imperial Age DSP: A tool for probabilistic sex diagnosis using worldwide variability in hip bone measurement Bullettins et Mèmoires de la Société d’ Anthropologie de Paris 17 The interpretation of variations in the symphysial area Age estimation from the auricular surface of the ilium: Arevised method Illumina sequencing library preparation for highly multiplexed target capture and sequencing AdapterRemoval: Easy cleaning of next-generation sequencing reads Fast and accurate short read alignment with Burrows–Wheeler transform The sequence alignment/map format and SAMtools mapDamage2.0: Fast approximate Bayesian estimates of ancient DNA damage parameters ANGSD: Analysis of next generation sequencing data A revised timescale for human evolution based on ancient mitochondrial genomes Martiniano, R., De Sanctis, B., Hallast, P. & Durbin, R. Placing ancient DNA inot reference phylogenies. bioRxiv https://doi.org/10.1101/2020.12.19.423614 (2020) Sequencing Y chromosomes resolves discrepancy in time to common ancestor of males versus females Second-generation PLINK: Rising to the challenge of larger and richer datasets Principal components analysis corrects for stratification in genome-wide association studies Diverse variola virus (smallpox) strains were widespread in northern Europe in the Viking Age Download references We thank the Parco Archeologico di Pompei for the authorization to publish this paper (prot 2917 of 29.03.2021) in the journal "Scientific Reports" (prot Digital radiograph images were made by X-Ray Sas radiology center (Aradeo We would like to thank James Fallon for his assistance with the English revision of the manuscript Support for this project was also provided by PRIN MIUR (Italian Ministry for the Universities) 2009–11 (3 years) prot.2010EL8TXP National Scientific Coordinator and Principal Investigator OR: Biological and cultural heritage of the central-southern Italian population trough 30 thousand Years EPIC and by an in-kind contribution of the Laboratory of molecular Psychiatry at the University of California These authors contributed equally: Olga Rickards and Fabio Macciardi Centre of Molecular Anthropology for Ancient DNA Studies Laboratório de Biodiversidade e Evolução Molecular (LBEM) Department of Psychiatry and Human Behavior performed laboratory work and analysed the genetic data; F.M all the relevant information about the archaeological context and performed the anthropological analysis; G.S performed the genetic data interpretation; T.P performed the Y-chromosome data analyses and interpretation; G.S. wrote the manuscript with input from all co-authors The authors declare no competing interests Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1038/s41598-022-10899-1 Anyone you share the following link with will be able to read this content: a shareable link is not currently available for this article International Journal of Legal Medicine (2025) Sign up for the Nature Briefing newsletter — what matters in science Receive unfettered access to our digital content including our Examiner+ bonus content newsletter and get other perks like free tickets to local performing arts complimentary advertising for your favorite local charity Republican Robert Scorrano was elected Somers supervisor on Election Day who announced in January he would not be seeking a fifth two-year term The supervisor seat carries with it an annual salary of $109,233 I am proud to be your next town supervisor I look forward to serving our great town,” Scorrano stated Scorrano served on the Zoning Board of Appeals from 2015 to 2018 and is heavily involved in Somers Youth Sports as a coach he set his sights on a fast-paced career in the energy trading industry trading for such companies as Allegheny and NRG Energy His then shifted his career to insurance and financial services where he is currently Vice President Employee Benefits for one of the largest insurance brokers in the U.S Prior to that he worked for New York Life Insurance Company and Prudential Financial where he managed a team of successful financial advisors which he recruited is a former educator who also worked in the medical field After a long struggle with PTSD and addiction Keegan has been carrying forward his legacy ever since She’s been to the State of the Union in Washington and spoken on panels with presidential candidates She’s traveled the nation to tell Daniel’s story and deliver legislation that puts veterans first Keegan lost in her bid to unseat State Assemblyman Kevin Byrne and doing my best to convince a historically Republican community that it might be time to look at Somers through a different lens the race has ended with a far different outcome than I had hoped for,” Keegan stated “There are countless reasons for this disappointing ending let me say thank you to everyone who supported me and the amazing team I ran with I will forever hold all the support we have received during this year in my heart but the effort to help is not over by a long shot in the Town Board race for two available seats GOP councilmen Anthony Cirieco (3,579) and William Faulkner (3,246) were reelected beating Tom Newman (2,538) and Margaret DiLorenzo (2,436) Rick has more than 40 years’ experience covering local news in Westchester and Putnam counties, running the gamut from politics and crime to sports and human interest. He has been an editor at Examiner Media since 2012. Read more from Rick’s editor-author bio here. Read Rick’s work here: https://www.theexaminernews.com/author/pezzullo_rick-writer/ We'd love for you to support our work by joining as a free, partial access subscriber, or by registering as a full access member. Members get full access to all of our content, and receive a variety of bonus perks like free show tickets. Learn more here. Copyright © 2025 The Examiner News Regarding "Somers shooting survivor alerted grandparents to mom’s violent boyfriend, Fernando Jimenez," lohud.com 31 coverage of the tragedy that has struck the Town of Somers particularly the horrific level of detail with which the scene in the Raimondi family’s home was described was offensive and dismaying and exceeded the bounds of responsible journalism both the town and the school district went into action to provide support and coping strategies to the classmates staff and community members impacted by this senseless attack on our Somers family we enlisted the resources of the Regional Crisis Team from PNW BOCES to supplement our amazing school district counseling staff we updated the community with factual information provided to us by law enforcement and the family There was much misinformation posted on social media that made navigating the many emotions we all felt more difficult We aimed to counter the rumors and unreliable information by presenting only facts from official sources Our community is coping with heartbreaking news Our goal has always been to offer aid and support to students and staff who have been devastated by the loss of one wonderful young man and the injuries suffered by his younger brother and mother We also aim to help the Raimondi family in any way that we can Many individuals and organizations have reached out to help But this community and school district family face a long road ahead in healing from the pain and loss A description of the injuries and crime scene in such detail as was recounted in lohud.com’s coverage exploits the situation and makes our healing process more difficult It reignites and prolongs the pain and anguish so many in the community feel We understand and appreciate the important role that journalism — responsible journalism — plays in the healthy functioning of a community we hope that lohud.com’s reporters and editors will keep in mind the impact that their future reporting has on the people and community struggling to face this overwhelming tragedy Harry LeFevre is Interim Superintendent of the Somers Central School District Rob Scorrano is supervisor of the town of Somers POLICE SAY they are investigating a fatal accident that occurred yesterday The police said in a release that Scorrano was said to be working on the roof of the hotel which had sustained damage during the passage of Hurricane Beryl Police officers were called to the scene and found the employee motionless on his left side He was pronounced dead at the scene by a medical practitioner A post-mortem examination is expected to be conducted to determine the exact cause of death commented briefly on the incident in Parliament on Thursday The dates displayed for an article provide information on when various publication milestones were reached at the journal that has published the article activities on preceding journals at which the article was previously under consideration are not shown (for instance submission All content on this site: Copyright © 2025 Elsevier B.V. Metrics details Close proximities between organelles have been described for decades only recently a specific field dealing with organelle communication at membrane contact sites has gained wide acceptance attracting scientists from multiple areas of cell biology The diversity of approaches warrants a unified vocabulary for the field Such definitions would facilitate laying the foundations of this field streamlining communication and resolving semantic controversies written by a panel of experts in the field aims to provide this burgeoning area with guidelines for the experimental definition and analysis of contact sites It also includes suggestions on how to operationally and tractably measure and analyze them with the hope of ultimately facilitating knowledge production and dissemination within and outside the field of contact-site research intracellular membranes delimit organelles that have distinct biochemical functions While for decades the organelle field was governed by studies aimed at identifying the unique characteristics of each compartment the last years have seen a revolution in the field as more focus is being placed on the interactions between the organelles and their role in maintaining cellular homeostasis Together these two views delayed the appreciation of the importance of membrane tethering between two organelles it is rapidly becoming evident that organelles are highly interconnected and that there are multiple important functions for these physical associations at contact sites devoted to the investigation of the molecular mechanisms the physiological and pathological implications of contact sites While it is now obvious that such appositions are central to the structure and function of any eukaryotic cell and that they are becoming center-stage in cell biology research and experimental approaches to define and measure processes are vaguely defined leading to potential controversies and hampering development of knowledge we decided to offer a lexicon and a set of experimental guidelines to the field since these contacts do not occur between two organelles bound by membranes they might be physiologically very different and consequently we will also not discuss them here We now propose a set of unifying characteristics which we consider essential features of contact sites We suggest that an organelle juxtaposition can be defined as a contact site if it is characterized by the following We hence suggest that distance cannot be a sole measure and that simple juxta-positioning of organelles is not sufficient to be considered a contact site regardless of distance What does define a contact site in all cases reported to date is the presence of tethering forces that arise from protein–protein or protein–lipid interactions Fusion intermediates have been in the past referred to as “docking” events and this nomenclature Limited vesicular trafficking between apposed organelles may but would follow established mechanisms and terminology this requires that they be regulated and hence that dysregulation of contacts should impact cell function and contacts should therefore be selected for by evolutionary pressure We suggest that period of existence is therefore not a defining characteristic of contacts we recommend that the above four features all be experimentally characterized when a new type of contact is described Graphical representation of the four types of proteins that should reside in contact sites many proteins can have multiple roles at a contact site an additional function may yet be described For all other tethering pairs an additional function at the contact site has already been characterized It seems that for the major contact sites complete ablation is not viable but this has to be better studied to be confirmed any functional protein that is proposed to also be a tether can be tested by re-expression of a version with mutation of the functional domain even if partners on the opposing membrane exist a role in tethering has to be taken as untested since they could in theory define a unique lipidome for these areas this categorization is meant purely to aid in ordering the current knowledge and for ease of communication We propose fusion is inhibited at contacts by either: and the repelling force increases exponentially as the water molecules that hydrate the phospholipid heads are squeezed out fusion proteins are required to destabilize the bilayers enough to provoke lipid bilayer mixing Juxtaposed membranes at contact sites must therefore be devoid of fusogens The mechanisms that hinder the entry of fusogens into the contact sites have not been well-studied and would be interesting topics for future research Fusion occurs once lipid bilayers have been forced into a proximity of 1–2 nm whereas all reported contacts are not closer than 10 nm in distance Such spacing at contacts may be actively mediated by distancing proteins such as spacers Such dedicated spacers have not yet been described in detail it may very well be that spacing is simply a result of the large population of proteins resident at contacts that would need to be cleared before fusion can occur we suggest that newly identified contact sites are characterized for the presence of proteins that might inhibit fusion The characterization of potential spacers could contribute to our understanding of interorganellar proximity without fusion While the parallel alignment would lead to vesicle–membrane fusion These finding support a novel additional function for SNARE proteins in stabilizing contacts beyond their well-established role in membrane fusion and in the future maybe more of these examples will be found While tagging two organelles each with a different color is fast easy and amenable to adaptation to high-content approaches most contact sites are smaller than the optical diffraction limit: on the x–y axis resolution is limited to 250 nm; on the z-axis point spread functions of microscopes limit resolution to approx chemical fixation and single-plane confocal microscopy can alter contacts and offer a partial representation of interactions that occur in three dimensions visual quantification of proximity measured in confocal microscopy experiments must be accompanied by indexes of pixel-by-pixel overlap like Pearson’s and Manders’ coefficients while contact-site measurements based on confocal pseudo colocalization experiments are important they are more conclusive when (i) accompanied by a second approach that is endowed with a resolution power amenable to detect distances in the range of contact sites; (ii) performed on live cells using piezoelectric z-stepper or other similar approaches to acquire the whole cellular volume in very short times; (iii) accompanied by careful experiments of reconstitution of organelle shape and number to exclude possible artifacts caused by variability in these traits such approaches are clearly limited to the analysis of a subset of contacts whose components have already been identified This can be very powerful to study alterations that occur in response to genetic or environmental perturbations While this approach can indeed give a sense of membrane proximities it also suffers from a number of limitations: first reductions in PLA signals can be caused by changes in expression or localization of one of the PLA partners; second it requires that the PLA partners are unequivocally localized at the contact site; third it might not recognize changes in contact-site extent not accompanied by changes in proximity between the two proteins measured in the PLA believe that PLA can be a powerful tool to corroborate protein–protein interaction in situ especially in trans but we do not recommend PLA as the tool to measure contact extent If used it should be very carefully controlled fluorescence resonance energy transfer (FRET) and split fluorescence sensors can detect proximities between two membranes based on the effect of proximity on the fluorescence of the pair and can be used in live cells Such probes can be broadly divided in three classes: By changing the targeting sequence of the individual fluorescent proteins this approach can be adapted to measure any potential contact site FRET measurements require proficient experimenters and dedicated equipment thereby limiting the utilization of such probes this approach does not require any pre-existing knowledge about a contact site and enables discovery of novel contact sites these probes suffer from the fact that complementation of the two fragments is thermodynamically stable In such approaches the dynamics of contact sites cannot be studied and some contacts may become toxic under some conditions It is important to also remember that these approaches do not discriminate between close associations of organelles and tethered contacts and can cause synthetic expansion of the associations the intrinsically low fluorescence of these probes might restrict their application Brighter versions will be required for increased usage all above probes enable proximity measurements between two organellar membranes While this can be highly important for detecting the presence of a contact site it should be utilized carefully in measuring the effect of any single tether all contact sites described have more than a single tethering pair in them—hence loss of any one protein does not necessarily cause reduction in either the amount of contact or the distance between organelles increased distance between two organelles does not necessarily mean loss of contact and could also represent the formation of alternate contact areas by an alternate tethering mechanism as a compensation Super-resolution methods that can be used include structured illumination microscopy stimulated emission depletion (STED) microscopy and single molecule localization microscopy all such techniques require highly dedicated microscopes and technical expertise and despite the ongoing development of sub-diffraction microscopy techniques their application to contact-site analysis is still limited The latter two approaches can also both be used to pinpoint a protein specifically at a contact site and is therefore considered the best proof of it being a resident protein We therefore recommend that TEM be used to provide qualitative and quantitative features of contact sites but not to study occurrence and changes in the extent unless rigorous morphometric analyses are included ET often requires serial sectioning to reconstruct the 3D model of the entire structure and the combination of such laborious approaches could be technically challenging it should be considered that the 3D reconstructions created from ET tilt series of images are not complete representations This is due to the limited tilt range of the microscope holder in ET that leaves the 3D reconstruction with regions empty of information appearing as undefined cone shaped areas (so-called “missing wedge”) combined with advances in cryo-ET sample preparation such as the introduction of focused ion beam milling to thin samples to ideal thicknesses for imaging the potential for cryo-ET imaging is constantly increasing 3D-SEM resolving power is still limited and requires extended research time and computer power for processing the large amount of datasets produced The biochemical characterization of contact sites occurred contemporaneously with their discovery by EM protocols for isolating membrane contacts utilized subcellular fractionation followed by sucrose gradient centrifugation Any successful isolation of heterotypic contacts must consider that these have features of two organelles or maybe of neither purification of such contact regions can be challenging as they need to resist dilution and cell fractionation during the lengthy fractionation procedure potential protein modifications and interactions (such as phosphorylation or dimerization) might be reversed from thereon termed mitochondria-associated membranes became the first functional characterization of a contact-site function biochemical fractionation assays for contact sites are important tools for studying functional aspects of the contacts as well as identifying their unique lipid composition and resident proteins such protocols have not yet been developed for all contact sites can be used to study contact sites by labeling two opposing membranes and retrieving biotinylation activity only at the areas of interface Such approaches should be developed to appraise if changes in contact extent or composition affects cellular physiology Appropriate functional analyses need to be performed in combination with the imaging approaches detailed above to draw conclusions on whether contact sites are present and/or changed in the studied system Care should be taken not to confuse a lack of effect on any singular function as an indication that a protein is not a tether or a functional contact-site protein This is because deletion of a tether is often backed up by many other tethering molecules and its effect will therefore not necessarily be measurable if a function that it does not carry out is measured (for example deletion of a lipid-binding tether which probably functions in lipid transfer and measurement of the effect of Ca2+ transfer) it is necessary to generate separation-of-function alleles to inactivate just contact sites while maintaining the other function Many new and exciting questions remain to be answered in the field of contact sites—are there more contact sites that have not yet been described Are there different varieties of contact sites between similar organelles How is the distribution of signalling proteins determined between contact sites and non-junctional areas And when these proteins are multi-subunit complexes (such as the mitochondrial calcium uniporter What is the repertoire of functions carried out at contact sites How does the loss of each contact affect cellular physiology and organismal function How are contact sites regulated and co-regulated to maintain cellular homeostasis given the apparent inability to completely deconstruct contact sites what mechanisms exist that compensate malfunctions interest in these structures will only increase We anticipate that in the next decade the above questions will start to be addressed calling for extended guidelines that help define good practices in these additional areas of contact-site research An association between mitochondria and the endoplasmic reticulum in cells of the pseudobranch gland of a teleost Intracellular sites of lipid synthesis and the biogenesis of mitochondria A subfraction of the yeast endoplasmic reticulum associates with the plasma membrane and has a high capacity to synthesize lipids Ca2+ store depletion causes STIM1 to accumulate in ER regions closely associated with the plasma membrane Coordinated lipid transfer between the endoplasmic reticulum and the Golgi complex requires the VAP proteins and is essential for Golgi-mediated transport Piecemeal microautophagy of nucleus in Saccharomyces cerevisiae ER–plasma membrane contact sites contribute to autophagosome biogenesis by regulation of local PI3P synthesis ER-associated mitochondrial division links the distribution of mitochondria and mitochondrial DNA in yeast ER–mitochondria contacts couple mtDNA synthesis with mitochondrial division in human cells ER contact sites define the position and timing of endosome fission and everywhere: the importance of ER membrane contact sites Transport and retention mechanisms govern lipid droplet inheritance in Saccharomyces cerevisiae A tether is a tether is a tether: tethering at membrane contact sites Piecing together the patchwork of contact sites Stitching organelles: organization and function of specialized membrane contact sites in plants Mind the organelle gap—peroxisome contact sites in disease Organelle communication at membrane contact sites (MCS): from curiosity to center stage in cell biology and biomedical research Real time imaging reveals a peroxisomal reticulum in living cells Maturation of peroxisomes in differentiating human hepatoblastoma cells (HepG2): possible involvement of the peroxisome proliferator-activated receptor alpha (PPAR alpha) Peroxisomal aggregates forming large stacks in the lipid segment of the canine kidney Identification of seipin-linked factors that act as determinants of a lipid droplet subpopulation Lipid droplets are essential for efficient clearance of cytosolic inclusion bodies Num1 anchors mitochondria to the plasma membrane via two domains with different lipid binding specificities Short-range intracellular trafficking of small molecules across endoplasmic reticulum junctions Calcium signaling at ER membrane contact sites Lipid droplet biogenesis is spatially coordinated at ER–vacuole contacts under nutritional stress Structure and function of ER membrane contact sites with other organelles Repeated ER-endosome contacts promote endosome translocation and neurite outgrowth Membrane contacts between endosomes and ER provide sites for PTP1B–epidermal growth factor receptor interaction Autophagosomes form at ER–mitochondria contact sites Osh proteins regulate phosphoinositide metabolism at ER–plasma membrane contact sites Dynamic formation of ER–PM junctions presents a lipid phosphatase to regulate phosphoinositides Quasi-synaptic calcium signal transmission between endoplasmic reticulum and mitochondria Proteomic analysis of lipid raft-enriched membranes isolated from internal organelles Evidence for the involvement of lipid rafts localized at the ER–mitochondria associated membranes in autophagosome formation STIM proteins and the endoplasmic reticulum–plasma membrane junctions PI(4,5)P(2)-dependent and Ca(2+)-regulated ER-PM interactions mediated by the extended synaptotagmins Triggered Ca2+ influx is required for extended synaptotagmin 1-induced ER–plasma membrane tethering Palmitoylation determines the function of Vac8 at the yeast vacuole PI4P/phosphatidylserine countertransport at ORP5- and ORP8-mediated ER–plasma membrane contacts Gatta, A. T. et al. A new family of StART domain proteins at membrane contact sites has a role in ER–PM sterol transport. Elife 4, e07253 (2015). https://doi.org/10.7554/eLife.07253 Mitochondria and melanosomes establish physical contacts modulated by Mfn2 and involved in organelle biogenesis Systematic mapping of contact sites reveals tethers and a function for the peroxisome–mitochondria contact Mitofusin 2 tethers endoplasmic reticulum to mitochondria Kv2 potassium channels form endoplasmic reticulum/plasma membrane junctions via interaction with VAPA and VAPB a novel scaffold for endoplasmic reticulum membrane contact sites The yeast cell cortical protein Num1 integrates mitochondrial dynamics into cellular architecture Junctophilins: a novel family of junctional membrane complex proteins Mdm1/Snx13 is a novel ER–endolysosomal interorganelle tethering protein Vps39 interacts with Tom40 to establish one of two functionally distinct vacuole–mitochondria contact sites Bohnert, M. & Schuldiner, M. Stepping outside the comfort zone of membrane contact site research. Nat. Rev. Mol. Cell Biol. https://doi.org/10.1038/s41580-018-0022-1 (2018) Lam6 regulates the extent of contacts between organelles Homology of SMP domains to the TULIP superfamily of lipid-binding proteins provides a structural basis for lipid exchange between ER and mitochondria ER-mitochondria tethering by PDZD8 regulates Ca2+ dynamics in mammalian neurons A conserved membrane-binding domain targets proteins to organelle contact sites ER-to-plasma membrane tethering proteins regulate cell signaling and ER morphology Characterization of the yeast tricalbins: membrane-bound multi-C2-domain proteins that form complexes involved in membrane trafficking GM1-ganglioside accumulation at the mitochondria-associated ER membranes links ER stress to Ca(2+)-dependent mitochondrial apoptosis Detergent-resistant microdomains determine the localization of sigma-1 receptors to the endoplasmic reticulum–mitochondria junction What does S-palmitoylation do to membrane proteins Where the endoplasmic reticulum and the mitochondrion tie the knot: the mitochondria-associated membrane (MAM) Phosphoinositides in membrane contact sites The role of phosphatidylinositol-transfer proteins at membrane contact sites Cellular metabolism regulates contact sites between vacuoles and mitochondria p53 at the endoplasmic reticulum regulates apoptosis in a Ca2+-dependent manner In-depth proteomic analysis of mammalian mitochondria-associated membranes (MAM) A different kind of love—lipid droplet contact sites Synaptotagmin-1 binds to PIP(2)-containing membrane but not to SNAREs at physiological ionic strength The SNARE Sec22b has a non-fusogenic function in plasma membrane expansion A role for the ancient SNARE syntaxin 17 in regulating mitochondrial division Single-molecule measurements of dissociation rates and energy landscapes of binary trans snare complexes in parallel versus antiparallel orientation ORP5/ORP8 localize to endoplasmic reticulum-mitochondria contacts and are involved in mitochondrial function Direct observation of individual endogenous protein complexes in situ by proximity ligation Imaging interorganelle contacts and local calcium dynamics at the ER-mitochondrial interface Critical reappraisal confirms that Mitofusin 2 is an endoplasmic reticulum–mitochondria tether SPLICS: a split green fluorescent protein-based contact site sensor for narrow and wide heterotypic organelle juxtaposition Visualizing multiple inter-organelle contact sites using the organelle-targeted split-GFP system A novel fluorescent reporter detects plastic remodeling of mitochondria–ER contact sites Dimerization-dependent green and yellow fluorescent proteins An infrared reporter to detect spatiotemporal dynamics of protein–protein interactions Single-molecule analysis of diffusion and trapping of STIM1 and Orai1 at endoplasmic reticulum–plasma membrane junctions Physical interaction between peroxisomes and chloroplasts elucidated by in situ laser analysis Structural and functional features and 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endoplasmic reticulum and plasma membrane-bound ribosomes in erythropoietic cells A combined approach of quantitative interaction proteomics and live-cell imaging reveals a regulatory role for endoplasmic reticulum (ER) reticulon homology proteins in peroxisome biogenesis ACBD5 and VAPB mediate membrane associations between peroxisomes and the ER Peroxins Pex30 and Pex29 dynamically associate with reticulons to regulate peroxisome biogenesis from the endoplasmic reticulum Directed evolution of APEX2 for electron microscopy and proximity labeling Ascorbate peroxidase proximity labeling coupled with biochemical fractionation identifies promoters of endoplasmic reticulum-mitochondrial contacts Hung, V. et al. Proteomic mapping of cytosol-facing outer mitochondrial and ER membranes in living human cells by proximity biotinylation. Elife 6, e24463 (2017). https://doi.org/10.7554/eLife.24463 Han, Y. et al. Directed Evolution of Split APEX2 Peroxidase. ACS Chem. Biol. Preprint available at: https://pubs.acs.org/doi/10.1021/acschembio.8b00919 (2019) The ER stress sensor PERK coordinates ER–plasma membrane contact site formation through interaction with filamin-A and F-actin remodeling A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells Split-BioID: a proximity biotinylation assay for dimerization-dependent protein interactions A conserved endoplasmic reticulum membrane protein complex (EMC) facilitates phospholipid transfer from the ER to mitochondria An ER-mitochondria tethering complex revealed by a synthetic biology screen Ltc1 is an ER-localized sterol transporter and a component of ER–mitochondria and ER–vacuole contacts ER-mitochondria associations are regulated by the VAPB-PTPIP51 interaction and are disrupted by ALS/FTD-associated TDP-43 Download references Gilbert Omenn and Martha Darling Professorial Chair in Molecular Genetics The work on contact sites in the Schuldiner lab is supported by ERC CoG 646604 a Weizmann-EPFL collaborative grant and a Volkswagen Foundation grant Work in LS lab on contact sites was supported by ERC 282280 is funded by CIHR grant MOP 133541 and NSERC RGPIN-2015-04105 is supported by grants of the DFG (SFB 944 G.H.’s studies of contacts are supported by NIH (RO1-DK51526 and R33-ES025672) is funded by the NIH NIGMS grant R01GM120303 MADM acknowledges the support of Telethon (grant TGM11CB1) the Italian Association for Cancer Research (AIRC European Research Council Advanced Investigator grant no We thank Noam Schuldiner for the graphics in this paper These authors contributed equally: Benoît Kornmann Telethon Institute of Genetics and Medicine Department of Molecular Medicine and Medical Biotechnology Weill Institute for Cell and Molecular Biology and Department of Molecular Biology and Genetics Institute for Integrative Biology of the Cell (I2BC) and Maya Schuldiner wrote the initial version of the manuscript and György Hajnóczky contributed their expertise to the second version of the paper Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Reprints and permissions Download citation DOI: https://doi.org/10.1038/s41467-019-09253-3 Acta Neuropathologica Communications (2025) Nature Reviews Molecular Cell Biology (2025) Transportation Research Interdisciplinary PerspectivesCitation Excerpt :This demonstrates that most respondents are concerned about the EV price and public infrastructure which is in line with most of the previous studies (Berkeley et al. This underscores the significance of incentives and the need to minimize the ownership cost of EVs so that it is competitive with conventional equivalents on the US market (Liu et al. Journal of Cleaner ProductionCitation Excerpt :The probability of purchasing an EV even increases more through financial incentives than through technological improvements (Danielis et al. the introduction of cheaper EVs in the small and medium-sized vehicles which account for more than half of the Italian car market is advisable to boost EV sales (Giansoldati et al. Incentives can be offered by the government or by the company itself and can improve the chances of companies to implement profitable business models Metrics details Recent improvements in the analysis of ancient biomolecules from human remains and associated dental calculus have provided new insights into the prehistoric diet and genetic diversity of our species integrating metagenomic and proteomic analyses of dental calculus and human ancient DNA analysis of the petrous bones of two post-Last Glacial Maximum (LGM) individuals from San Teodoro cave (Italy) to reconstruct their lifestyle and the post-LGM resettlement of Europe Our analyses show genetic homogeneity in Sicily during the Palaeolithic representing a hitherto unknown Italian genetic lineage within the previously identified Villabruna cluster We argue that this lineage took refuge in Italy during the LGM followed by a subsequent spread to central-western Europe Analysis of dental calculus showed a diet rich in animal proteins which is also reflected on the oral microbiome composition Our results demonstrate the power of this approach in the study of prehistoric humans and will enable future research to reach a more holistic understanding of the population dynamics and ecology a more complete picture of the post-LGM population history of western Eurasia remains elusive as fossils from Southern Europe are still underrepresented in genomic studies a deeper knowledge of diet and nutrition becomes necessary to solve the complex co-evolution of oral microbiomes and their human hosts We used these analyses to investigate genetic ancestry and dispersal of southern European hunter-gatherers after the LGM and to reconstruct their oral microbiomes and dietary lifestyle Given the complexity of its archaeological records Southern Italy is one of the key geographic areas for understanding human and biological responses to postglacial climate evolution in Europe supporting the authenticity of the generated data which refers to a previous phase of the Epigravettian (Evolved Epigravettian) suggesting further substructure among individuals of the Villabruna cluster Map showing geographic locations (black crosses) and ancestry proportions (bar plots) of post-LGM hunter-gatherers, inferred using qpAdm (Supplementary Data 14) Individuals were modelled using four source groups representing major post-LGM lineages identified in this and previous studies Approximate geographic locations of source groups are indicated with coloured symbols Shown are estimated abundances of the top 20 most abundant genera (a) or species (b) across all ancient calculus samples (Supplementary Data 18–19) Overall percentage of deamidation for asparagine (N) and glutamine (Q) amino acids for the proteins found in the dental calculus samples: a San Teodoro 3; b San Teodoro 5 Numbers above each bar represent the number of peptides used for the analysis and the error bars represent standard deviation Then we interpret the identification of ovicaprid collagen as a possible evidence of ibex exploitation rapid progress has been made in the recovery of DNA from ancient human remains These data have revolutionised our understanding of human demographic history and the processes that shaped genetic diversity in the past metagenomic and palaeoproteomic data to characterise ancestry diet and microbial environment of two Upper Palaeolithic hunter-gatherers from Sicily our results suggest that geographic clines and isolation-by-distance played an important role in shaping European hunter-gatherer diversity we also find evidence for local transformations and possible migrations in regions including northern Iberia they could be evidence of a further arrival of human groups in Sicily from Calabria where they might have eaten ibex meat the possibility of occasional or habitual movements towards Southern Calabria by the Epigravettian hunters of San Teodoro Our results further confirm and illuminate the exploitation of marine and freshwater resources during Late Epigravettian showing the important benefits of the proteomic approach to identify species often absent in the archaeological records of the ancient sites while the sample size of two individuals warrants some caution in generalising our findings to a broader context they nevertheless demonstrate the value of integrating ancient genomic metagenomic and metaproteomic data in the study of prehistoric hunter-gatherer communities we could show that the individuals from San Teodoro were part of a likely genetically homogenous Sicilian hunter-gatherer metapopulation with a mode of subsistence predominantly relying on exploitation of meat and aquatic resources Applying it to a broader range of prehistoric hunter-gatherer communities in future studies is needed to reach a more comprehensive understanding of their population dynamics and ecology Two individuals from San Teodoro cave in Sicily: San Teodoro 3 (male) and 5 (female) were sampled for aDNA analysis and lifestyle evaluation For the human aDNA analysis the petrous bone was selected dental calculus was analysed by mass spectrometry-based proteomics and for the oral microbiome reconstruction by the metagenomic approach Each sample was then divided into two different tubes 22.8 mg (San Teodoro 3) and 9.9 mg (San Teodoro 5) for metagenomics analysis and about 7 mg each for proteomic analysis 50 mg have been instead used for protein extraction from petrous bones All the molecular work was performed in DNA clean laboratory facilities at the Lundbeck Foundation GeoGenetics Centre Globe Institute of the University of Copenhagen The Allentoft55 protocol was used for the aDNA extraction from both ancient matrices A starting amount of 150–400 mg of bone powder and 10–23 mg of dental calculus were added a pre-digestion buffer and incubated for 45 min at 37 °C in order to remove the surface contaminants Negative extraction controls were processed along with the samples After centrifugation at 2000 × g for 2 min the supernatants were discarded and a new digestion buffer was added then the samples were left for 24 h at 37 °C The DNA extraction was performed on the digestion buffer by Silica powder the supernatant was transferred into new tubes and 100 ml of silica suspension and 10× volume of binding buffer was added This solution was incubated at room temperature for 1 h the samples were centrifuged at 2000 × g for 2 min and the supernatant was discarded An additional 1 ml of binding buffer was added to the samples and the DNA was cleaned by ice-cold ethanol in a new tube the DNA was eluted in 90 μl Qiagen EB buffer DNA libraries for sequencing were prepared following a method proposed by Allentoft55 This method is divided into 4 steps: End-repair around 20 μl of DNA extraction was used and NEB End-repair (module E6050L) Mix was added according to the manufacturer’s instructions The solution was incubated at 12 °C for 20 min and 37 °C for 15 min Before the ligation a purification step by Qiagen MinElute spin columns was performed and the DNA was eluted in 17 μl EB buffer the mix was purified using Qiagen MinElute spin columns and the DNA was eluted in 20 μl Qiagen EB buffer 30 μl of NEB (module M0275L) reaction mix was added and incubated at 65 °C for 20 min and 80 °C for 20 min The library was then quantified using IS8 and IS7 primers and SYBER green solution The quantification results were used to assess the optimal number of PCR cycles required for DNA library indexing The indexing was performed by adding 1 μl of each primer (10 μM inPE forward primer and indexed reverse primer) and 2× Kapa (following the manufacturer’s temperature instruction) the amplified DNA was purified by Qiagen MinElute Kit and quantified using an Agilent Bioanalyzer 2100 The libraries were shotgun sequenced by the Illumina HiSeq 2500 and HiSeq 4000 platforms (81 bp single-read) at the Danish National High-Throughput DNA Sequencing Centre the library preparation followed the same steps previously described but dual-indexed libraries construction and amplification were used The mitochondrial contamination was evaluated for both samples by contamMix 1.015 that it reports a Bayesian-based estimate of the posterior probability of the contamination proportion Ancestry proportions of post-LGM hunter-gatherers were inferred using qpAdm The protein extraction was performed following the method proposed by Jersie-Christensen et al.73 To clarify the role of dental calculus in protein preservation we compared the deamidation patterns of proteins obtained from dental calculus and petrous bone of the same individuals The sample preparation of the bone fragments closely followed that of the dental calculus The main difference was the overnight demineralisation: for dental calculus 1 ml 15–20% acetic acid was added to about 7 mg of powder while about 50 mg of the bone fragments were demineralised in 300 ml of EDTA pH 8 After centrifugation for 10 min at 2000 × g the supernatant was removed reduction and alkylation buffer (2 M guanidine hydrochloride 10 mM tris(2-carboxyethyl) phosphine hydrochloride 20 mM chloroacetamide in 100 mM TRIS pH 8,5) was then added to the powder and the pH was adjusted to 7–9 The pellet was crushed by disposable sterile micro-pestles and then incubated either at 99 °C for 10 min (calculus) or at 80  °C for 2 h (bone) at 500 rpm The protein concentration was then measured by Bradford Assay The samples were then digested with rLysC (0.2 µg Sweden) incubating under agitation at 37 °C for 2–4 h the samples were diluted to a final concentration of 0.6 M GuHCl solution adding 25 mM Tris in 10% acetonitrile This was followed by digestion by trypsin (0.8 µg Sweden) and incubation overnight at 37 °C under agitation the samples were acidified (pH < 2) using 10% trifluoroacetic acid then the proteins were collected in home-made C18 StageTips and stored in the freezer until mass spectrometry analysis Dental calculus samples were eluted from the stage tips using 20 μL 40% ACN in water and then 10 μL 60% ACN while the bone samples by 30 μL 40% ACN in water both into a 96-well MS plate Samples were placed in a SpeedVacTM Concentrator (Thermo Fisher Scientific Denmark) vacuum centrifuge at 40 °C until approximately 3 μL of the solution was left and then 5 μL of 0.1% TFA Samples were then separated on a 15 cm column (75 μm inner diameter) in-house laser pulled and packed with 1.9 μm C18 beads (Dr Denmark) connected to a Q-Exactive HF (Thermo Scientific The column temperature was maintained at 40 °C using an integrated column oven The peptides were separated with increasing buffer B (80% ACN and 0.1% formic acid) held at 80% for 5 min before dropping back down to 5% in 5 min and held for 5 min 5% ACN was run in between each sample to hinder cross-contamination The Q-Exactive HF was operated in data-dependent top 12 mode (dental calculus) and top 10 mode (bones) Full scan mass spectra were recorded at a resolution of 120,000 at m/z 200 over the m/z range 350–1400 with a target value of 3e6 and a maximum injection time of 25 ms HCD-generated product ions were recorded with a maximum ion injection time set to 45 ms (dental calculus) and 118 ms (bones) with a target value set to 2e5 and recorded at a resolution of 30,000 (dental calculus) and of 60,000 (bones) The normalised collision energy was set at 28% and the isolation window was 1.2 m/z with the dynamic exclusion set to 20 s All identifications are based on 100% identity and common sources of misidentifications (for example leucine vs isoleucine and deamidated residues) were also checked Patterns of human admixture and shared genetic drift were evaluated by f-statistics using a reference panel of 170 individuals 6 other oral microbiome studies and 21 ancient human dental calculus genomes already published have been used for the metagenomic comparison The transformation method for compositional data was used to analyse the samples Detailed information of the statistical analyses carried out as described in the methods section All analyses can be reproduced by accessing the associated data linked in the Data Availability statement Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article Genome sequence of a 45,000-year-old modern human from western Siberia Meltwater pulse 1A from Antarctica as a trigger of the Bolling-Allerod warm interval Ancient biomolecules and evolutionary inference The effect of diet on the human gut microbiome: a metagenomic analysis in humanized gnotobiotic mice Eneolithic subsistence economy in Central Italy: first dietary reconstructions through stable isotopes The medieval population of Leopoli-Cencelle (Viterbo Latium): dietary reconstruction through stable isotope analysis from bone proteins Pathogens and host immunity in the ancient human oral cavity Multi-omic detection of Mycobacterium leprae in archaeological 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Protoc. pdb.prot5448 https://doi.org/10.1101/pdb.prot5448 (2010) AdapterRemoval: easy cleaning of next-generation sequencing reads Fast and accurate short read alignment with Burrows-Wheeler transform The Sequence Alignment/Map format and SAMtools mapDamage2.0: fast approximate Bayesian estimates of ancient DNA damage parameters ANGSD: analysis of next generation sequencing data PhyloTree Build 17: growing the human mitochondrial DNA tree Bayesian phylogenetics with BEAUti and the BEAST 1.7 Dating of the human-ape splitting by a molecular clock of mitochondrial DNA Improved calibration of the human mitochondrial clock using ancient genomes Robust relationship inference in genome-wide association studies Microbiome datasets are compositional: And this is not optional Wickham, H. ggplot2: Elegant Graphics for Data Analysis. https://doi.org/10.1111/j.1541-0420.2011.01616.x (2016) Quantitative metaproteomics of medieval dental calculus reveals individual oral health status Visualization of LC-MS/MS proteomics data in MaxQuant The PRIDE database and related tools and resources in 2019: improving support for quantification data Download references The Novo Nordisk Foundation Center for Protein Research (CPR) is funded in part by the Novo Nordisk Foundation (Grant number NNF14CC0001) This work was also supported by the Lundbeck Foundation the Novo Nordisk Foundation and the Wellcome Trust (grant no We would like to thank Benedetto Sala for his suggestions in order to identify wild animal species inferred by proteomic dental calculus data Mikkel Winther Pedersen & Martin Sikora Novo Nordisk Foundation Center for Protein Research Centro di Antropologia Molecolare per lo studio del DNA antico Cristina Martínez-Labarga & Olga Rickards Trace and Environmental DNA (TrEnD) Laboratory DANTE: Diet and Ancient Technology Laboratory initiated the project on Multi-omics analysis performed the human genetic extraction and library preparation performed the proteomic extraction on dental calculus with support and resources provided by J.V.O performed the genetic extraction from dental calculus and library preparation carried out the dental calculus metagenomic data analysis performed and discussed the comparison between the dental calculus metagenomic and the soils results carried out human genetic and proteomic dental calculus data analysis provided palaeobotanical input for plants consumption interpretation inferred by proteomic data provided input about the archaeological context and the radiocarbon date published in the present paper used for the contextualisation and interpretation of data obtained carried out the human bones morphological analysis provided supervision of data analysis and supervised the interpretation of the results and the formulation of the conclusions wrote the manuscript and all authors reviewed and approved it wrote the archaeological part of the manuscript wrote the anthropological part of the manuscript The data published fall into an overall project about the revision of the archaeological and anthropological collections from San Teodoro curated at the Museo e Istituto Fiorentino di Preistoria (Florence) designed by F.M. Communications Biology thanks Noemi Procopio and the other 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/s42003-022-04190-2 Metrics details A Corrigendum to this article was published on 10 September 2014 This article has been updated Juxtaposition between endoplasmic reticulum (ER) and mitochondria is a common structural feature providing the physical basis for intercommunication during Ca2+ signalling; yet the molecular mechanisms controlling this interaction are unknown a mitochondrial dynamin-related protein mutated in the inherited motor neuropathy Charcot–Marie–Tooth type IIa is enriched at the ER–mitochondria interface Ablation or silencing of mitofusin 2 in mouse embryonic fibroblasts and HeLa cells disrupts ER morphology and loosens ER–mitochondria interactions thereby reducing the efficiency of mitochondrial Ca2+ uptake in response to stimuli that generate inositol-1,4,5-trisphosphate An in vitro assay as well as genetic and biochemical evidences support a model in which mitofusin 2 on the ER bridges the two organelles by engaging in homotypic and heterotypic complexes with mitofusin 1 or 2 on the surface of mitochondria a juxtaposition required for efficient mitochondrial Ca2+ uptake Prices may be subject to local taxes which are calculated during checkout 605–610 (2008); doi:10.1038/nature07534 In Fig the representative image of a volume-rendered three-dimensional reconstruction of a z-stack of confocal images of endoplasmic-reticulum-targeted yellow fluorescent protein (ER-YFP) in a Mfn2−/− cell expressing MFN2IYFFT and that of a Mfn1−/− cell appear to be duplicated Organelle-specific initiation of cell death pathways Microdomains of intracellular Ca2+: molecular determinants and functional consequences Rapid changes of mitochondrial Ca2+ revealed by specifically targeted recombinant aequorin Microdomains with high Ca2+ close to IP3-sensitive channels that are sensed by neighboring mitochondria Close contacts with the endoplasmic reticulum as determinants of mitochondrial Ca2+ responses Decoding of cytosolic calcium oscillations in the mitochondria BAX and BAK regulation of endoplasmic reticulum Ca2+: a control point for apoptosis Phospholipid synthesis in a membrane fraction associated with mitochondria Interrelationships of endoplasmic reticulum and microtubules–a quadruple fluorescence labeling study PACS-2 controls endoplasmic reticulum-mitochondria communication and Bid-mediated apoptosis The dynamin-like protein DLP1 is essential for normal distribution and morphology of the endoplasmic reticulum and mitochondria in mammalian cells Drp-1-dependent division of the mitochondrial network blocks intraorganellar Ca2+ waves and protects against Ca2+-mediated apoptosis OPA1 requires mitofusin 1 to promote mitochondrial fusion OPA1 controls apoptotic cristae remodeling independently from mitochondrial fusion Mitofusins Mfn1 and Mfn2 coordinately regulate mitochondrial fusion and are essential for embryonic development Structural basis of mitochondrial tethering by mitofusin complexes Mitofusin 1 and 2 play distinct roles in mitochondrial fusion reactions via GTPase activity Mutations in the mitochondrial GTPase mitofusin 2 cause Charcot–Marie–Tooth neuropathy type 2A Brucella evades macrophage killing via VirB-dependent sustained interactions with the endoplasmic reticulum A role for Fis1 in both mitochondrial and peroxisomal fission in mammalian cells Dysregulation of HSG triggers vascular proliferative disorders Organellar relationships in the Golgi region of the pancreatic beta cell line visualized by high resolution electron tomography Measurement of co-localisation of objects in dual-colour confocal images Endoplasmic reticulum localized Bcl-2 prevents apoptosis when redistribution of cytochrome c is a late event Membrane topology and mitochondrial targeting of mitofusins ubiquitous mammalian homologs of the transmembrane GTPase Fzo Disruption of fusion results in mitochondrial heterogeneity and dysfunction Role of Bax and Bak in mitochondrial morphogenesis The Ca2+ concentration of the endoplasmic reticulum is a key determinant of ceramide-induced apoptosis: significance for the molecular mechanism of Bcl-2 action A class of membrane proteins shaping the tubular endoplasmic reticulum ER vesicles and mitochondria move and communicate at synapses Ca2+ shuttling between endoplasmic reticulum and mitochondria underlying Ca2+ oscillations Mitofusin 2 triggers vascular smooth muscle cell apoptosis via mitochondrial death pathway Nucleus-vacuole junctions in Saccharomyces cerevisiae are formed through the direct interaction of Vac8p with Nvj1p Organelle isolation: functional mitochondria from mouse liver Download references received a ‘Bolsa de Doutoramento’ of FCT Portugal is senior scientist of the Dulbecco-Telethon Institute and EMBO YIP United Mitochondrial Disease Foundation USA Muscular Distrophy Association USA and Swiss National Science Foundation 3100A0-118171 conceived and designed the experiments and wrote the manuscript Department of Cell Physiology and Metabolism This file contains Supplementary Figures 1-12 Supplementary Methods and Supplementary Notes 180° rotation along the y-axis of a 3D-reconstruction of a z-axis stack of a wt MEF expressing erYFP (green) and mtRFP (red) ER-mitochondria interaction in Mfn2-/- MEF 180° rotation along the y-axis of a 3D-reconstruction of a z-axis stack of a Mfn2-/- MEF expressing erYFP (green) and mtRFP (red) Electron tomography showing ER-mitochondria interaction in a wt MEF Rotation along the y and x-axis of a 3D-rendered reconstruction of representative area from an electron tomogram of a wt MEF Electron tomography showing ER-mitochondria interaction in a Mfn2-/- MEF Rotation along the y and x-axis of a 3D-rendered reconstruction of representative area from an electron tomogram of Mfn2-/- MEF Download citation Mitochondria are in close contact with the endoplasmic reticulum a juxtaposition that is important for cellular processes such as calcium homeostasis and lipid metabolism So far the identity of molecules mediating contact between these organelles has remained elusive Olga Martins de Brito and Luca Scorrano now show that a mitochondrial protein mitofusin-2 is enriched at the mitochondrion–endoplasmic reticulum interface and mediates tethering of both organelles endoplasmic reticulum morphology calcium transfer between the two organelles are disrupted Metrics details The Original Article was published on 01 December 2008 Three-dimensional reconstructions of endoplasmic reticulum in mouse embryonic fibroblasts of the indicated genotype co-transfected with ER-YFP and the specified plasmids The online version of the original article can be found at 10.1038/nature07534 Download citation Sorry, a shareable link is not currently available for this article. Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily. Menu.page-122585831{--colorD:#d9c2fe;--colorJ:#d9c2fe;--gradientTransparentJ:#d9c2fe00;--colorDC:#d9c2fe;--colorDA:#d9c2fe;--colorDF:#d9c2fe;--colorJD:#d9c2fe;--colorDJ:#d9c2fe;--colorJF:#d9c2fe;--colorJG:#d9c2fe;--colorDDC:#d9c2fe;--colorDTransparent:#d9c2fe;--colorJTransparent:#d9c2fe}MercyAncient Roman man lived in agony before Vesuvius eruption killed him — DNA analysis His death in the volcanic eruption may have been a relief Sebastian Condrea/Moment/Getty ImagesInside a dining room, the Roman lay back on his triclinium as the eruption of Mount Vesuvius began Every movement of the earth as the volcano came alive made him grimace in pain — indeed any sudden or strong movement could make him wince This ailing Roman was among them — a grisly Now, thanks to the latest genetic analysis tools, researchers are getting a glimpse into who this man was, how he lived, and what relationship he bore to the ancient people of Pompeii and modern Italians today It is thanks to this groundbreaking research that we now know how great his suffering must have been in the time before he died This image was taken in 1933 during one of the excavations of the Casa del Fabbro an assistant professor at the University of Copenhagen “Individual A looks very similar to other Imperial Roman Age genomes we have available Some of its genetic profile — the Y chromosome and the mitochondrial DNA — is very unusual for both the time and location,” Scorrano tells Inverse “Actually both his lineages were very rare What we believe it means is that he represents some Iron Age genetic diversity that was lost due to the homogenization of the Italic peninsula after the Roman Empire,” he says the individual’s Y chromosome line appears to be most similar to that of individuals in Sardinia Interestingly, the researchers also found evidence the man suffered from Pott’s disease — this is a kind of musculoskeletal tuberculosis which can cause deformation of the spine and intense pain on moving exacerbated by exertion or coughing and sometimes accompanied by sciatica limits the mobility of those affected,” Scorrano explains The Pompeiian man’s vertebra shows signs of tuberculosis-inflicted damage Why it matters — The study involves two skeletons discovered at the site inside the Casa del Fabbro — the House of the Craftsman (in this case Excavated at various times throughout the last century the house and its preserved occupants offer a window into life as a middle-class Roman citizen in Pompeii the study is also a rarity in terms of the depth of genetic analysis Scorrano and his colleagues managed to pull off — generally heat destroys DNA and other genetic material It is actually the same logic of why we put food in the fridge to slow down molecular reactions,” Scorrano says the Pompeiian man was found with another body — likely a 50-year-old woman — but the remains of this person could not be analyzed as there was too little material to work with “Because the bodies in Pompeii were enveloped in high-temperature ash this could very well have been the reason why one of the individuals did not yield sufficient DNA for us to analyze,” Scorrano says “I think the main surprise was that one of the individuals had a good DNA preservation We believe that the ash kept the individual in an anoxic environment but it is hard to be sure at this stage,” he adds “The preservation of ancient DNA is always a challenge,” says Scorrano’s co-author and colleague on the study, Serena Viva a postdoctoral researcher at the Università del Salento in Italy “especially the remains from individuals who died during the eruption and suffered a severe heat shock “Only thanks to the most innovative techniques was it possible to sequence the genome of the male individual,” she explains Serena Viva examines the Pompeiian skeleton for analysis “This disease was endemic in Imperial Roman times, but it is rare to find it in archaeological contexts because it only manifests skeletal changes in small percentages,” Viva says. The man’s spinal vertebrae show signs of wasting as a result of his disease, suggesting he had at least one collapsed vertebra in his backbone. But there is also the question of how this individual fit within the fabric of Roman society. His tuberculosis is a sign of the perils of urbanization in Roman Italy — as cities became more densely populated, diseases like tuberculosis spread more rapidly. In turn, Iron Age Italy — circa 1000 B.C.E. — appears to have experienced migration from the Middle East and other areas, evident in the diversification of the Italian genome from this period. Yet this man’s genes suggest a less cosmopolitan origin story within Italy itself — they also suggest he was not a slave. The Ancient Roman’s skeleton yielded curious DNA insights. What’s next — This study is a first — but Scorrano and his team hope it will not be the last. “The results in Pompeii show us clearly that there is still a lot to learn on the genetic diversity of the Roman period, but also about the populations before them, the so-called Pre-Roman Italic populations,” Scorrano says. “Very little is known about them, and even their relationship with the Romans and present-day Italians is uncertain.” In the paper, Scorrano and his co-authors say the work “confirms and demonstrates the possibility of applying paleogenomics methods to study human remains” from Pompeii. It could act as a blueprint for future studies to perform similar analyses and glean more clues to life in Pompeii before Mount Vesuvius’ historic eruption wiped it off the map. “Supported by the enormous amount of archaeological information that has been collected in the past century for the city of Pompeii, their paleogenetic analyses will help us to reconstruct the lifestyle of this fascinating population of the Imperial Roman period,” the authors write. This article was originally published on May 26, 2022 Please enable JS and disable any ad blocker new video loaded: House Tour | Palazzo Ducale Guarini transcriptBackbars0:00/1:54-1:54transcript Metrics details This study describes the preparation of ion-imprinted polymers (IIPs) for the selective removal of Hg(II) ions from aqueous media Polymeric sorbents were prepared using different synthesis approaches to understand the influence of diphenylcarbazone (DPC) bulk polymerization was first used to prepare two polymers demonstrated by Fourier Transform Infrared Spectroscopy promotes the formation of ternary complexes with mercury ions and 4-vinylpyridine induces an increase in binding performance as indicated by the Ka values (1.7 × 103±0.4 M−1 and 12.1 × 103±0.5 M−1 respectively) of IIP1 and IIP2 high affinity binding sites A third polymer (IIP3) was also synthesized using precipitation polymerization to evaluate the contribution of morphological characteristics on absorption performance compared with the addition of DPC Competitive studies revealed a stronger influence of IIP3 morphology on selectivity performance monodisperse microbeads were obtained only in this case the applicability of the polymers to real-world samples was demonstrated through batch experiments using drinking water spiked with 1 μg ml−1 of Hg(II) ions and the best removal efficiency of nearly 80% was obtained for IIP2 scanning electron microscopy (SEM) dynamic light scattering (DLS) and electrophoretic light scattering (ELS) Comparative selectivity studies were performed using heavy metals typically found in polluted water such as Co(II) to confirm the applicability of the prepared polymers to real-world water samples drinking water was chosen and analyzed before and after incubation with the prepared polymer particles and the extraction recovery was calculated 4VP and EGDMA were supplied from Sigma-Aldrich (Steinheim Hydrochloric acid (HCl) and α-α′-azoisobutyronitrile (AIBN) were purchased from Fluka (Steinheim Analytical grade acetonitrile and ethanol were obtained from J.T Elemental standard solutions of Pb(II) and Hg(II) were prepared by appropriate dilution of 1000 mg l−1 stocks purchased from Fluka Nitric Acid (67–69%) for trace metal analysis and Cu(II) and Co(II) standard solutions (1000 mg l−1) were supplied from Romil-SpA (Prato Buffer solutions were prepared with deionized water provided by a water purification system (Human Corporation UV–visible spectra were obtained with a Cary 100 Scan UV/vis spectrophotometer bulk polymerization was first used to obtain IIP1 EGDMA (0.936 mmol) and AIBN (0.037 mmol) were dissolved in 3 ml of acetonitrile and water (4/1 The polymerization was carried out at 65 °C for 24 h under magnetic stirring (400 r.p.m.) was synthesized in a similar manner to the above procedure with a slight modification DPC was dissolved in 3 ml of a porogen solution containing 4VP the resulting mixture was stirred for 5 h to form a stable complex between Hg(II) ions was prepared by precipitation polymerization similar to IIP1 but with a higher volume of porogen (13 ml) All polymers were washed several times with ethanol to remove any unreacted materials and subsequently washed with 2 M HCl to extract Hg(II) ions until Hg(II) in solution was no longer detected by ICP-AES analysis polymeric particles were washed with double distilled water to obtain a neutral pH The resulting fine powders were dried under vacuum in a desiccator prior to absorption studies The corresponding non-imprinted polymers were prepared using the same procedures but without the presence of the target ion FT-IR analysis was performed using dry polymers dispersed in a matrix of KBr followed by compression at 10 tons to form pellets DLS and ELS measurements were both carried out on diluted samples to establish the size and zeta potential of the polymer particles The hydrodynamic diameter of the dispersed beads was determined at 25 °C by measuring the autocorrelation function at a 90° scattering angle Three separate measurements were made to compute an average a measure of the distribution of molecular mass in a given polymer sample was evaluated for all prepared polymers and the results were compared size and shape of the polymer particles were examined by SEM The binding capacity of all polymers was evaluated by batch rebinding experiments by dissolving 5 mg of polymer particles in 1 ml of HgCl2 phosphate buffer solution (pH 8) spanning a concentration range from 0 to 600 mg l−1 The suspension was shaken for 18 h at room temperature and the resulting solution was analyzed by ICP-AES The amount of analyte adsorbed on the polymer (mg g−1) was calculated by where Ci and Ce represent the initial and equilibrium concentration (mg l−1) V is the volume of water solution (l) and m is the mass of polymer (g) Scatchard analysis was performed using the following equation: where Qe represents the equilibrium concentration of Hg(II) bound per gm polymer (mg g−1) and Bmax (μM g−1) is the apparent maximum number of binding sites Ka and Bmax of the polymer were determined from the slope and the intercept A simple Langmuir absorption isotherm was also used to evaluate the maximum absorption capacity Qmax (μmol g−1) of the polymers: where Qe and KL correspond to the amount of analyte ion adsorbed at equilibrium (μmol g−1) and the Langmuir constant (l μmol−1) Qmax is determined from the linear plot of 1/Qe against 1/Ce Ion recognition capacity of Hg-IIP materials are well-reflected by Hg(II) selectivity in the presence of other competing ions Cu(II) and Pb(II) was used for bath experiments The following equation was employed to evaluate the selectivity of the different prepared polymers: Ci and Cf represent the distribution coefficient (l mg−1) of an ion on the polymer the initial and the final concentration of solutions (mg l−1) The selectivity coefficient k of Hg(II) relative to the competing ion was calculated using the following equation: a relative selectivity coefficient k’ was defined as follows: Batch experiments were conducted to explore the application of prepared polymers to real-world samples the extraction capabilities of IIP2 and IIP3 in drinking water were evaluated Fifty milligrams of dried polymer were suspended in 100 ml of drinking water containing 1 μg ml−1 of Hg(II) ions and stirred for 1 h unextracted and extracted concentrations of Hg(II) ions were determined FT-IR spectra of IIP1 (a), IIP2 (b) and unleached IIP2 (c). The black circles underline the presence of DPC in IIP2 (b) compared with IIP1 (a) and their changes of peaks between unleached (b) and leached IIP2 (c). FT-IR, Fourier TransformInfrared Spectroscopy; IIP, ion-imprinted polymer. SEM images (20000 × magnification) of IIP1 (a) when the number of nucleated particles increases the average particle diameter decreases for a given degree of a monomer conversion These data confirmed that DLS analysis was suitable only for monodisperse spherical beads with a lower polydispersity index value whereas in other cases the SEM technique was considered the most reliable to obtain accurate morphological information the complete solubility of Hg(II) was verified at the concentrations used low adsorption capacities for all polymers tested were recorded at both high and low pH values it was decided to use a phosphate buffer solution with pH 8 to study the adsorption performance of polymers Scatchard plot of IIP1(▪) with equations y=1657.2x+0.0231 for high affinity sites (left line), y=1349.2x+0.4092 for low affinity sites (right line), and IIP2 (▴) with equations y=−12138x+4.4955 for high affinity sites (right line), y=9433.5x−0.0328 for low affinity sites (left line) (a); Langmuir isotherm of IIP3 (b) with equation y=0.562x−0.004. IIP, ion-imprinted polymer. Comparison of IIPs absorption performance with the corresponding non-imprinted polymers and imprinting factor (IF) evaluation IIP2 and IIP3 using a mixture solution of Pb(II) To verify the feasibility of the application of these polymers the removal of ions from drinking water was tested which showed the best absorption performance were incubated with drinking water samples spiked with 1 μg ml−1 of Hg(II) ions Despite the high selectivity performance showed by IIP3 the ligand rather than the monodispersity of the polymer particles had a more significant role in the removal of mercury ions IIP2 demonstrated the best removal efficiency (78.8% suggesting a good anti-interference ability in environmental water samples selective Hg(II) imprinted polymers were prepared using different synthesis approaches on absorption performance was demonstrated Absorption studies on IIP1 and IIP2 in the absence and presence of DPC confirmed a strong impact of this compound on the binding behavior of polymers against Hg(II) ions with a significant difference between Ka values of high affinity binding sites of IIP1 (1.7±0.4 M−1) and IIP2 (12.1±0.5 M−1) an increase of absorption capacity compared with IIP1 was also observed for IIP3 demonstrating that the morphological characteristics of the polymer influenced the presence of homogenous binding sites SEM images of IIP3 showed the presence of monodisperse spherical microbeads with a low propensity to aggregate whereas the presence of some aggregates was observed for other polymers Selectivity studies revealed that morphological characteristics of polymers were more effective than the ligand inclusion for the selectivity of Hg(II) imprinted polymers batch experiments conducted using drinking water spiked with 1 μg ml−1of Hg(II) ions showed a greater incidence of ligand on mercury ion extraction with a removal efficiency near 80% for IIP2 after 1 h of treatment Cadmium and lead contamination in tap water samples from Tokat in Handbook of Ecotoxicology 2nd edn (eds Hoffman Synthesis and characterization of Hg(II)-ion-imprinted polymer: Kinetic and isotherm studies A review of permissible limits of drinking water Liquid–liquid extraction of mercury (II) from hydrochloric acid solutions by Aliquat 336 Adsorption by liquid-liquid extraction of Hg(II) from aqueous solutions using the 2-butyl-imidazolium Di-(2-ethylhexyl) phosphate as ionic liquid Polymer-supported ionic liquid solid phase extraction for trace inorganic and organic mercury determination in water samples by flow injection-cold vapor atomic absorption spectrometry Flotation separation of mercury(II) from environmental water samples using thiosemicarbazide derivatives as chelating agents and oleic acid as surfactant Achieving very low mercury levels in refinery wastewater by membrane filtration mechanism and application of solid-phase extraction using a dithiocarbamate resin- for the sampling and determination of mercury species in humic-rich natural waters Selective adsorption of mercury (II) on chitosan derivatives from hydrochloric acid Relationships between the renal handling of DMPS and DMSA and the renal handling of mercury Silica gel-immobilized-dithioacetal derivatives as potential solid phase extractors for mercury(II) Noncovalent imprinted microspheres: preparation evaluation and selectivity of DBU template Experimental and computational studies on non-covalent imprinted microspheres as recognition system for nicotinamide Synthesis of nicotinamide-based molecularly imprinted microspheres and in vitro controlled release studies Molecularly imprinted polymer for solid phase extraction of nicotinamide in pork liver samples Molecularly imprinted polymer for solid-phase extraction of 1-methyladenosine from human urine Synthesis of molecularly imprinted polymers for amino acid derivates by using different functional monomers Developments in the synthesis of a water compatible molecularly imprinted polymer as artificial receptor for detection of 3-nitro-L-tyrosine in neurological diseases Molecularly imprinted polymers: present and future prospective A molecularly imprinted polymer as artificial receptor for the detection of indole-3-carbinol Selective recognition of arsenic by tailoring ion-imprinted polymer for ICP-MS quantification Highly selective determination of inorganic mercury(II) after preconcentration with Hg(II)-imprinted diazoaminobenzene–vinylpyridine copolymers Development of a highly selective voltammetric sensor for nanomolar detection of mercury ions using glassy carbon electrode modified with a novel ion imprinted polymeric nanobeads and multi-wall carbon nanotubes Ion-imprinted beads for molecular recognition based mercury removal from human serum Synthesis and characterisation of nano structure lead (II) ion-imprinted polymer as a new sorbent for selective extraction and preconcentration of ultra trace amounts of lead ions from vegetables Synthesis and characterization of a high selective mercury(II)-imprinted polymer using novel aminothiol monomer Molecularly imprinted fluorescent polymers as chemosensors for the detection of mercury ions in aqueous media Synthesis by precipitation polymerisation of molecularly imprinted polymer microspheres for the selective extraction of carbamazepine and oxcarbazepine from human urine Selective separation of mercury (II) using a synthetic resin containing amine and mercaptan as chelating groups An ion-imprinted polymer for the selective extraction of mercury(II) ions in aqueous media Download references This work was supported by Ministero dell’Istruzione dell’Università e della Ricerca PON 2HE (grant number PONa3_00334) and PRIN NANOMED (grant number 2010FPTBSH) Institute for Microelectronics and Microsystems The authors declare no conflict of interest Download citation Metrics details They identified MTCH2 as a mitochondrial protein that interacts with Bid and whose ablation dramatically affects mitochondrial translocation of this BH3-only protein MTCH2 shares homology with members of the mitochondrial carrier family but it is located on the outer membrane of the organelle; and it was recently reported to be associated with increased body mass index this study not only unveils how BID is targeted to mitochondria during apoptosis but also opens interesting avenues to investigate the relationship between mitochondria The importance of tBID in the Fas death pathway is well established however the molecular mechanism of tBID recruitment on mitochondria is still unknown and this has been an area of intense investigation in the last years Two main models have been put forward to explain the affinity of BID for mitochondria: one postulates that BID travels to mitochondria as a consequence of its affinity for specific lipids or of its specific lipidation; the other involves the existence of one or more specific receptors on the mitochondrial surface that interact with tBID to assist its insertion in the mitochondrial membrane conclusive evidence for their role in this process is often lacking This evidence raised the hypothesis that MTCH2/MIMP could be involved in the mitochondrial apoptotic program but its role was not clear The recruitment of tBID on mitochondria is mediated by the novel target protein MTCH2/MIMP The diagram depicts the sequence of events that occur in type II cells following an extrinsic death stimulus Pro-caspase 8 binds to cardiolipin (yellow) on mitochondria where it undergoes self-proteolytic activation to cleave BID The active tBID is then recruited on mitochondria by MTCH2/MIMP This in turn leads to oligomerization of BAX/BAK and cytochrome c release Zaltsman and coworkers analyzed whether loss of MTCH2/MIMP that abolished in vitro recruitment of tBID on mitochondria could have significant effects on hepatocellular apoptosis in vivo They generated MTCH2/MIMP liver-specific knockout mice and assessed ther sensitivity to Fas The liver-specific knockout animals show less liver injury and are more resistant to death than heterozygotes In order to investigate the molecular mechanism of these effects they analyzed the activation of caspases and the recruitment of tBID to mitochondria The results clearly showed that in mice lacking MTCH2/MIMP in liver caspase 8 was cleaved but the recruitment of tBID to mitochondria failed This causes less activation of caspase 3 and consequently the hepatocites are less prone to apoptosis after Fas stimulation substantiating a model in which lipids and proteins cooperate to target BID one can envision a role for cardiolipin in targeting and/or assembly of MTCH2 in the outer membrane in the case of MTCH2 it is not clear if the protein like BAD fulfils multiple functions in independent pathways or if its role in apoptosis is key also for the regulation of body weight it might be interesting to address if MTCH2 participates in the regulation of body weight by impacting on mitochondrial function Studies capitalizing on the use of the conditional knockout animals generated by Zaltsman et al will for sure help address these questions and place this protein in the broad context of integrated metabolism MTCH2/MIMP is a major facilitator of tBID recruitment to mitochondria Cardiolipin provides an essential activating platform for caspase-8 on mitochondria Mitochondrial rhomboid PARL regulates cytochrome c release during apoptosis via OPA1-dependent cristae remodeling A distinct pathway remodels mitochondrial cristae and mobilizes cytochrome c during apoptosis Posttranslational N-myristoylation of BID as a molecular switch for targeting mitochondria and apoptosis Cardiolipin provides specificity for targeting of tBid to mitochondria and lipids cooperate to form supramolecular openings in the outer mitochondrial membrane Mitochondrial outer membrane proteins assist Bid in Bax-mediated lipidic pore formation Mitochondrial targeting of tBid/Bax: a role for the TOM complex Mitochondrial carrier homolog 2 is a target of tBID in cells signaled to die by tumor necrosis factor alpha Bid-deficient mice are resistant to Fas-induced hepatocellular apoptosis BAD and glucokinase reside in a mitochondrial complex that integrates glycolysis and apoptosis Download references LS is a Senior Telethon Scientist of the Dulbecco-Telethon Institute and supported by The Italian Association for Cancer Research (AIRC) Download citation Cellular and Molecular Life Sciences (2012) Volume 11 - 2018 | https://doi.org/10.3389/fnmol.2018.00351 Epilepsies are a group of common neurological diseases exerting a strong burden on patients and society often lacking clear etiology and effective therapeutical strategies Early intervention during the development of epilepsy (epileptogenesis) is of great medical interest though hampered by poorly characterized epileptogenetic processes Using the intrahippocampal kainic acid mouse model of temporal lobe epilepsy we investigated the functional role of the endogenous opioid enkephalin during epileptogenesis We addressed three sequential questions: (1) How does enkephalin affect seizure threshold and how is it regulated during epileptogenesis (2) Does enkephalin influence detrimental effects during epileptogenesis (3) How is enkephalin linked to mitochondrial function during epileptogenesis? the expression of enkephalin is not regulated in a seizure dependent manner and enkephalin’s proconvulsive effects suggested it as a potential driving force in epileptogenesis enkephalin deficiency aggravated progressive granule cell dispersion in kainic acid induced epileptogenesis Based on reported beneficial effects of enkephalin on mitochondrial function in hypoxic/ischemic states we hypothesized that enkephalin may be involved in the adaptation of mitochondrial respiration during epileptogenesis we observed dynamic improvement of hippocampal mitochondrial respiration after kainic acid-injections in wild-type wild-type mice displayed higher efficiency in the use of mitochondrial capacity as compared to enkephalin-deficient mice Our data demonstrate a Janus-headed role of enkephalin in epileptogenesis but in subsequent stages it contributes to neuronal survival through improved mitochondrial respiration thus potentially representing a molecular link between the association of stress and seizures/epilepsy Furthermore, DOPr activation has been implicated in neuroprotection under conditions of hypoxia (Mayfield and D’Alecy, 1994a,b), ischemia (Gao et al., 2012), and excitotoxicity (Zhang et al., 2000). This effect might be mediated by mitochondrial alterations (Zhu et al., 2011) Changes of mitochondrial function have been described to occur after seizures and during epileptogenesis. Oxidative stress and mitochondrial dysfunction appear to be important factors in the pathogenesis of epilepsy (Folbergrova and Kunz, 2012; Rahman, 2015). Mitochondria are also intimately involved in pathways leading to neuronal cell death observed in experimental and human epilepsy (Blümcke et al., 1999) Epileptic patients often exhibit reduced mitochondrial Complex I (CI) activity and defects in CI are potent generators of epileptogenesis (Kunz et al., 2004; Rahman, 2015). In line with this, induction of seizures in rats causes secondary mitochondrial dysfunction, affecting primarily CI (Kudin et al., 2002; Folbergrova et al., 2010) We investigated the regulation of Enk during epileptogenesis and studied its effect on seizure threshold we probed for differential neuropathological and neurochemical outcomes in prepro-Met-Enk-deficient (Enk-/-) mice in the kainic acid (KA) model Hypothesizing Enk to play a role in mitochondrial dysfunction in epilepsy we compared wild-type (WT) and Enk-/- mice at different time intervals after KA injection applying high-resolution respirometry Young adult, male C57BL/6 mice (WT) and prepro-Met-enkephalin deficient (Enk-/-) mice (König et al., 1996) were used in all experiments. Quantitative real-time PCR (qPCR) applied on cortical and hippocampal samples revealed no differences in opioid receptor mRNA-expression between WT and Enk-/- mice (Supplementary Figures S2A–F) Mice were kept at 23°C with a 12/12 h light/dark cycle and free access to standard laboratory rodent chow and water All procedures involving animals were approved by the Austrian Animal Experimentation Ethics Board in compliance with the European convention for the protection of vertebrate animals used for experimental and other scientific purposes ETS no.: 123 Every effort was taken to minimize the number of animals used For pentylenetetrazole (PTZ) tail vein infusions pH 7.4) was injected until generalized clonic seizures were displayed At that point the mice were killed immediately by neck-dislocation The infused volume of PTZ was used to calculate the seizure threshold (mg PTZ/kg mouse) The DOPr agonist (+)-4-[(aR)-a-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC80 dissolved in 1 molar equivalent HCl) and the DOPr antagonist 17-(Cyclopropylmethyl)-6,7-dehydro-4,5α-epoxy-3,14-dihydroxy-6,7-2′,3′-indolomorphinan (Naltrindole dissolved in water) were purchased from Tocris 30 min before testing (2 mg/kg or 1 mg/kg diluted in saline Four–seven animals per interval after KA (2 N = 3) per genotype were used for histological studies Animals were killed by an overdose of thiopental (150 mg/kg) and brains were fixed by transcardial perfusion with paraformaldehyde (4% in 50 mM PBS Immunohistochemistry and Nissl staining were performed on 30 μm free-floating coronal sections covering the entire dorsal hippocampus Cell counts and measurement of granule cell layer area were performed on sections of the dorsal hippocampus covering the range from 1.4 to 2.4 mm caudal to bregma (Paxinos and Franklin, 2001) Mean cell numbers of each brain were taken for stereological and statistical analysis Cell numbers of non-principal neurons were assessed for CA1 principal neurons were counted in CA1 over a length of 250 μm and in CA3a and CA3c over a length of 125 μm covering the whole width of the layer Granule cell dispersion was measured as the area of the entire granule cell layer from photomicrographs (100× magnification In situ hybridizations were performed on frozen-sections (20 μm) obtained from snap-frozen brains in vicinity to the injection site as described elsewhere (Wittmann et al., 2005) 4–7 WT animals per interval were injected with KA (1 time point 0) and used for in situ hybridization studies Single stranded DNA oligonucleotides (5 pmoles) complementary to prepro-dynorphin (5′-GTTCTCCTGGGACCGCGTCACCACCTTGAACTGACGCCGCAG-3′) prepro-neuropeptide Y (5′-GAGGGTCAGTCCACACAGCCCCATTCGCTTGTTACCTAGCAT-3′) and prepro-enkephalin (5′-TCCCTCATCTGCATCCTTCTTCATGAAGCCGCCATACCTCTTGGC-3′) mRNAs were labeled with 35S-dATPs (Hartmann Analytic Germany; 1000 Ci/mmol) using terminal deoxynucleotidyltransferase (Roche Hybridization was performed at 52°C for 18 h Data analysis was performed with ImageJ (NIH1) Relative optical densities (ROD) were calculated from the gray values obtained from autoradiographs of the upper and lower granular cell layer of the dentate gyrus (DG) Means were calculated for values of both layers of the DG and background obtained over the corpus callosum was subtracted Dorsal hippocampi obtained from adult WT and Enk-/- mice (N = 4 per condition) were snap-frozen in liquid nitrogen at different time intervals after KA injections and stored at -80°C for quantitative real-time PCR (qPCR) Total RNA was isolated using RNeasy Micro kit (Qiagen) according to the manufacturer’s instructions 1 μg of total RNA was reverse transcribed to cDNA using the GoScript Reverse Transcription Mix (Promega qPCR based on the SYBR Green chemistry (Promega) was carried out using the CFX384 Real-time System (Bio-Rad Of the four biological replicates for each condition three technical replicates were performed Beta-actin (Actb) was used for normalization of mRNA expression level of the target genes: Ndufs3 fw 5′-CTGACTTGACGGCAGTGGAT-3′ rv 5′-CATACCAATTGGCCGCGATG-3′; Relnfw 5′-CAAGCCACTGGACCTCACTC-3′ rv 5′-CGCTGTTGCAACTGTCTGTC-3′; Sdhb fw 5′GTCTACCGCTGCCACACC-3′ rv 5′-AGGTCGCCATCATCTTCTTG-3′; Atp6 fw 5′-CCTTCCACAAGGAACTCCAA-3′ Rv 5′-GGTAGCTGTTGGTGGGCTAA-3′; Actb fw 5′-CTGGCTCCTAGCACCATGAAGAT-3′ rv 5′-GGTGGACAGTGAGGCCAGGAT-3′ Relative quantification was performed using the comparative cycle threshold (Ct) method after determining the Ct values for the reference and target gene in each sample according to the 2-ΔΔCt method The expression data were averaged across the technical replicates before comparing between biological replicates An optimized protocol was applied with succinate at a concentration of 50 mM to prevent potential inhibitory effects of 2 mM malate on Complex II Tissue-mass specific oxygen fluxes were corrected for residual oxygen consumption measured after inhibition of the mitochondrial electron transfer system fluxes of all respiratory states were divided by ET-capacity to obtain flux control ratios Terminology was applied according to http://www.mitoeagle.org/index.php/MitoEAGLE_preprint_2018-02-08 (see also (Lemieux et al., 2017) United States) was used for quantification of protein contents of the homogenized samples according to the manufacturer’s protocol One-way ANOVA was calculated to compare seizure threshold data Repeated measures 1-way ANOVA was applied to compare differences in granule cell dispersion across the rostro-caudal axis regular 2-way ANOVAs were used to compare neuropeptide mRNA-levels during epileptogenesis and for cell dispersion across time-points respiration and qPCRs for reelin and ETS components and cell count data were evaluated by 2-way ANOVA between genotypes and across different time intervals (derived from populations injected separately) Post hoc analyses were performed using Tukey’s post hoc test Repeated measures 2-way ANOVA was performed to assess differences across mitochondrial states between untreated controls of the tested genotypes Bonferroni corrected post hoc test was applied To elucidate the role of Enk on seizure threshold and its regulation during epileptogenesis we applied DOPr pharmacology in combination with pentylenetetrazole-induced seizure threshold and neurochemical methods in the intrahippocampal KA model The reported proconvulsive actions of Enk are supposed to depend on the activation of DOPr. The threshold for pentylenetetrazole (PTZ)-induced seizures was not altered by pretreatment with the specific DOPr agonist SNC80 (2 mg/kg; 30 min before PTZ) in WT mice. By contrast, pretreatment with the specific DOPr antagonist naltrindole (1 mg/kg; 30 min before PTZ) increased seizure threshold by about 25% (Figure 1A) This confirms DOPr mediated proconvulsant effects and suggests that exogenous agonists cannot further enhance the effects of endogenous Enk in WT mice Seizure threshold modulation via the Enk-DOPr system and Enk mRNA- and protein-levels during epileptogenesis (A) Wild-type (WT) mice treated with the DOPr-antagonist naltrindole (Nalt; 1 mg/kg; 30 min before PTZ) exhibited significantly elevated seizure thresholds as compared to untreated WT mice and similar seizure thresholds like untreated Enk-KO mice the DOPr specific agonist SNC80 (2 mg/kg; 30 min before PTZ) did not influence seizure threshold in WT mice but decreased it in Enk-KO mice Nalt treatment of Enk-KO mice did not further increase seizure threshold 1-way ANOVA with Tukey’s multiple comparison tests was calculated: F(5,23) = 18.54 and enkephalin (Enk) in the dentate gyrus (ROD – relative optical density) during epileptogenesis are depicted Post hoc significances calculated by two-way ANOVA are omitted for sake of clarity – please refer to main text for statistical information (C) mRNA- levels are shown for representative autoradiographs obtained after in situ hybridizations for Met-Enk mRNA close to the injection site of KA (1.8–2.0 mm caudal to bregma Middle and right columns depict images obtained from KA injected hippocampi after immunohistochemistry for pro-Enk (pEnk) or mature Enk Saline-injected controls (3 weeks after injection) and 21 days time intervals after KA-injection are depicted ∗∗Indicates a p-value < 0.01 ∗∗∗p-value < 0.001 Naïve Enk-/- mice displayed a threshold for PTZ-induced seizures comparable to those of WT mice treated with naltrindole (Figure 1A). This phenotype was fully reversed upon pre-treatment of Enk-/- mice with SNC80 (2 mg/kg; 30 min pre PTZ). By contrast, pre-treatment with naltrindole (1 mg/kg; 30 min pre PTZ) did not influence the seizure threshold of Enk-/- mice (Figure 1A) The two-way ANOVA was calculated comparing mRNA-expression of the different neuropeptides across the studied time intervals: Finteraction(14,55) = 7.483 Post hoc tests (Tukey’s multiple comparison tests) were applied to compare mRNA-levels across time intervals for individual neuropeptides suggesting accumulating pro- and mature peptides Based on the observed anticonvulsive effects of pharmacological antagonism of DOPr and the prominent continuous up-regulation of Enk mRNA and potentially strong release of Enk early after KA injection we hypothesized that Enk might be a driving force of epileptogenesis morphological and neurochemical alterations in KA injected WT and Enk-/- mice at different time intervals significant alterations of reelin mRNA were observed in Enk-/- mice only Enk-deficiency aggravates granule cell dispersion during epileptogenesis (A) Granule cell layer dispersion is depicted from Nissl-stained sections of the dentate gyrus of mice 21 days after KA injection WT and KO mice displayed increasing granule cell dispersion (area under the curve across the dorsal hippocampus) after KA-injection Statistical significance was reached for KO mice; 2 way ANOVA: Finteraction(4,33) = 0.4551 (C) Area covered by the granule cell layer was analyzed from Nissl stained sections granule cells of the dentate gyrus across the dorsal hippocampus were significantly more dispersed in KO mice; 1-way repeated measures ANOVA; F(1.4,7.2) = 81.26 Relative expression of reelin mRNA was measured in the ipsi (D) and contralateral (E) hippocampus A decrease of reelin mRNA was observed ipsi- (D) and for KO contralaterally (E) during early epileptogenesis which was restored to baseline levels in WT mice 3 weeks after KA injection In KO mice reelin mRNA was significantly overexpressed at this time-interval 2-way ANOVAs were calculated to assess statistical significances time and group statistics: F(2,12) = 30.42 F(1,12) = 28.85 (P < 0.001 for all) for (E) and F(2,12) = 1.25 (P = 0.321) Principal neurons in area CA1 and CA3 and non-principal neurons in area CA1 were reduced by 80–100% in the ipsilateral hemisphere. Non-principal neurons in area CA3 were slightly less affected. Cell counts of area CA3 subfields are given in Supplementary Figures S2H–K. No marked neuronal loss was observed contralaterally (Figures 3A–E and Supplementary Figures S2G–K) There were no differences between genotypes Enkephalin-deficiency does not alter hippocampal cell loss during epileptogenesis (A) Representative Nissl-stained brain sections close to the injection site after saline or KA-infusion in WT or Enk-/- (KO) mice are depicted Quantification of cell numbers in hippocampal subfields obtained from Nissl stained sections is shown in (B–E) cell loss was significant and cell numbers remained stable between 2 and 21 days after KA-infusion Interaction/time/group effects in 2-way ANOVAs were F(12,94) = 89.02/F(4,94) = 298.6/F(3,94) = 984.4 (B) F(12,90) = 12.72/F(4,90) = 84.54/F(3,90) = 147.70 (C) F(12,81) = 26.62/F(4,81) = 77.38/F(3,81) = 342.1 (D) F(12,86) = 3.95/F(4,86) = 18.97/F(3,86) = 29.79 (E) To investigate, whether highly vulnerable cell types are differentially affected by the lack of Enk, we quantified cell numbers of somatostatinergic interneurons during epileptogenesis. These cells are known to be highly vulnerable in epilepsy (Magloczky and Freund, 1993; Buckmaster et al., 2002). However, we did not observe differences across genotypes (Supplementary Figure S3) The lack of beneficial effects paralleled by aggravated morphological alterations in Enk-/- mice suggests that Enk induces not only a reduction in seizure threshold Reports on the interaction of Enk/DOPr with mitochondrial function and the strong involvement of mitochondrial malfunction in epilepsy stimulated us to investigate this aspect in more detail applying high-resolution respirometry ET-capacity was unchanged 2 days after injection but increased by 25% 3 weeks after injection No such effects were observed in Enk-/- mice Dynamic regulation of mitochondrial respiration during epileptogenesis in WT Traces of experimental runs in duplicates (red and green traces) are depicted for WT 2 days (A) WT 21 days (C) and KO 21 days (D) after KA injection Data are presented as oxygen flux in pmol/(mg∗s) where mg corresponds to wet tissue weight in (A–D) and to normalized protein in (E,F) Electron transfer capacity (NSE) (E,F) and OXPHOS capacity (NSP) (E,F) per mg protein ipsi- (E,G respectively) are shown with respect to the site of KA-injection Interaction/time/genotype statistics were as follows: F(2,34) = 2.98 ∗ and # indicate a p-value < 0.05 Alterations in oxidative phosphorylation (OXPHOS) capacity (NSP) per mg protein (Figures 4G,H) followed a very similar pattern as ET-capacity NADH-pathway (N) capacity was significantly higher (by about 30%) in WT controls than in Enk-/- ipsilaterally, but dropped to similar levels 2 days after KA injection, before it recovered almost to baseline level 21 days after injection (Figure 5A). Contralaterally, an up-regulation of N-respiration by about 40% was apparent in WT 21 days after injection (Figure 5B) No such dynamics were observed in Enk-/- mice Respiration pathways are differentially affected during epileptogenesis (A–D) Ipsilateral absolute depression in early epileptogenesis and contralateral absolute respirational increase in WT is absent in Enk-/- (KO) mice and SE-(C,D) respiration per mg protein of ipsilateral and contralateral dorsal hippocampi Interaction/time/genotype statistics: F(2,34) = 3.08 (E–J) Flux control ratios (FCRs) in WT but not in Enk-/- (KO) mice change across epileptogenesis NP FCR (E,F) remains similar across epileptogenesis and genotypes SE FCR (G,H) is increased in epileptogenesis in WT Apparent electron transfer-excess capacity decreases in WT in late epileptogenesis ipsilaterally (I) Interaction/time/genotype statistics: F(2,34) = 0.124 Succinate-pathway (S) capacity was increased 21 days after injection in WT both ipsi- (Figure 5C) and contralaterally (Figure 5D) by about 20 and >30% Statistically significant higher respiration as compared to Enk-/- was observed only contralaterally In line with the reduced respiratory activity in Enk-/- mice (Supplementary Figure S4G), overall Complex IV (CIV) activity appeared lower in Enk-/- (Supplementary Figures S4I,J) without reaching post hoc significance for individual time-points Except for ATP6 (which was also upregulated in KO about four–fivefold) the tested respiratory complex subunits in Enk-/- mice were upregulated to smaller extend (about two–threefold) 2 days post injection Transcription dropped to about one–twofold in both phenotypes 3 weeks after KA for NDUFs4 and SDHb ATP6 remained elevated (two–threefold) in KO animals Based on observations of involvement of pharmacological modulation of DOPr in seizure threshold we aimed to elucidate the role of DOPr’s preferential endogenous ligand We report a dual role of the DOPr/Enk system; proconvulsive properties on one hand enhancement of mitochondrial function on the other Both of these neuropeptides are regulated during epileptogenesis in a seizures-dependent manner Met-Enk mRNA was upregulated continuously during the 1st days after KA infusion it might act as a driving force in epileptogenesis that endogenous Enk is sufficient to fully activate DOPr in C57BL/6J and its continuous up-regulation during epileptogenesis suggest Enk as one driving force for those ill-defined molecular events transforming a non-epileptic brain into an epileptic one we applied the KA-model to Enk-deficient mice expecting to observe mitigated neuropathology (including reduced cell loss) due to reduced seizure frequency and severity Increased granule cell dispersion associated with overexpression of reelin mRNA but unchanged reelin immunoreactivity points to a functional deficit clarifying the underlying mechanisms is beyond the scope of this study Instead we were curious, how Enk confers protective effects on hippocampal morphological integrity and reelin regulation. A prominent role of mitochondrial dysfunction in temporal lobe epilepsy is well accepted [for reviews see (Kudin et al., 2002; Rowley and Patel, 2013)]. Based on reported links of the Enk/DOPr system with mitochondrial function (Zhu et al., 2009, 2011) we suspected mitochondrial parameters to be involved we observed a pronounced reduction of the ET-capacity 2 days after KA-injection which returned to control-levels 3 weeks after KA ipsilaterally The patterns were similar for most of the other respiratory states (N- These dynamic changes in respiration were not observed in Enk-/--animals Three weeks after KA-injection mitochondrial respiration contralaterally were significantly higher in WT-mice (about 30–50%) than in KO-mice (NP- The ratio of respiration of mitochondria in the coupled versus uncoupled state is an indicator of the apparent ET-excess capacity, which was significantly reduced in WT- but not in KO-mice ipsilaterally 3 weeks after KA (Figure 5I) This suggests alterations that made WT-mice use OXPHOS more effectively In other disease models cross-hemispheric adaptations to stressors have been described in more detail; Weilnau et al. (2018) for example report cross-hemispheric preconditioning effects following unilateral intrastriatal infusion of 6-hydroxy-dopamine in mice via upregulation of preconditioning-relevant proteins. Contra-lateral insult-related changes in metabolite levels were reported by Ruan et al. (2017) in a stroke model in rats Our findings support and expand these results by demonstrating contralateral upregulation of OXPHOS mRNA-levels and function in the intrahippocampal KA model of unilateral insult The Enk/DOPr system and its pivotal role in adaptations to one of the most important triggers of seizures and potential risk factor for epilepsies and its prominent effects on energy metabolism represents an attractive novel molecular target to reduce seizure-induced neuronal damage and potentially disease-modifying therapy JB performed most of the experimental work and contributed to the writing of the manuscript CB performed qPCR and related data analysis for mitochondrial mRNAs and AA performed some of the surgeries and parts of histochemical analysis and discussed the respirometry experiments This work was supported by the Austrian Science Fund (FWF; W1206-B05; P-30430 to CS) funded by the Land Tirol within the program K-Regio of Standortagentur Tirol (to EG) and the AFM-trampoline grant Project No.16662 (to CB) The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest We thank Andreas Zimmer for providing the enkephalin knockout mice Serena Quarta for qPCRs on opioid receptors and Inge Kapeller and Christina Schwarzer for excellent technical support The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnmol.2018.00351/full#supplementary-material FIGURE S1 | (Related to Figures 1–3) Enkephalin (Enk) protein- and mRNA-levels during epileptogenesis are depicted Images in (A) represent contralateral hippocampi stained for pro-Enk (pEnk) or mature Enk (Enk) after saline or kainic acid (KA) injection at different time intervals mRNA-levels in the dentate gyrus of WT mice for dynorphin (B) and Enk (D) during epileptogenesis are depicted as relative optical densities measured from film autoradiographs obtained by radioactive in situ hybridization FIGURE S2 | (Related to Figure 2) mRNA-levels of opioid receptors in WT and Enk-/- mice (KO) and additional data on cell numbers are depicted respectively) in the hippocampus and cortex of young adult male mice (A–F) Nissl-stained hippocampal sections 1 week (1w) and 2 weeks (2w) after KA injection (G) show the onset of granule cell dispersion Cell counts of area CA3 subfields are given in (H–K) (A–F) Cortex and hippocampi from naive adult WT and Enk-/- mice (N = 4) were analyzed for expression of mu and kappa opioid receptors (Taqman primer sets Mm01188089_m1; Mm01180757_m1; Mm01230885_m1; respectively; Thermo Fisher) by qPCR Reactions were performed in a MicroAmp Fast Optical 96-Well Reaction Plate (Applied Biosystems) using the 7500 Fast Real-Time PCR System (Applied Biosystems) Samples were run in duplicates using 50 ng of total RNA equivalents (cDNA) Positive and negative controls were included in all experiments Threshold cycle (CT) values were recorded as a measure of initial template concentration Relative levels of RNA were calculated by the ΔΔCT method using SDHa as a reference standard gene The fold-difference expression was calculated relative to a calibrator sample by 2-ΔΔCT FIGURE S3 | (Related to Figure 3) Numbers of somatostatin immuno-positive neurons were analyzed from WT and Enk-/- mice (KO) in the hippocampal subfields at different time-intervals after KA Almost complete cell loss was observed ipsilaterally (A,C,E) Somatostatin positive neurons were mostly conserved contralaterally (B,D,F) No genotype specific differences in cell numbers were detected applying 2way ANOVA on distinct hippocampal areas time effects were significant in all hippocampal areas (F = 157.1; F = 17.6; F = 119 from top to bottom Cell numbers represent area under the curve (AUC) across six hippocampal sections from 1.4 to 2.4 mm caudal to bregma FIGURE S4 | (Related to Figures 4, 5) mRNA levels of selected subunits of respiratory complexes of WT and enkephalin deficient mice (KO) normalized to beta-actin and basal levels of untreated animals are depicted: NDUFs3 (A,D) Data from (A–C) have been obtained from the hippocampus of the hemisphere of injection data from (D–F) from the contralateral hippocampus Absolute oxygen fluxes (G) of naïve WT and KO mice differed in a 2way repeated measures ANOVA [Finteraction(4,64) = 3.51 No changes were observed for Complex IV respiration across genotypes and time intervals after KA (I,J) Representative traces of respiration protocols for naïve controls are depicted for WT (K) and KO (L) and ∗∗∗P < 0.001 Prevalence and incidence of drug-resistant mesial temporal lobe epilepsy in the united states Stress is associated with an increased risk of recurrent seizures in adults Mitochondrial dysfunction in neurodegenerative disorders Striatal pre-enkephalin overexpression improves huntington’s disease symptoms in the r6/2 mouse model of 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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: Erich Gnaiger, ZXJpY2guZ25haWdlckBvcm9ib3Jvcy5hdA== Christoph Schwarzer, c2Nod2FyemVyLmNocmlzdG9waEBpLW1lZC5hYy5hdA== †Present address: Johannes Burtscher Laboratory of Molecular and Chemical Biology of Neurodegeneration École Polytechnique Fédérale de Lausanne 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 Get important news about your town as it happens Get the top stories from across our network Are you sure you want to unsubscribe from daily updates An iconic statue has returned in a more durable form in Northern Westchester Somers Town Supervisor Robert Scorrano pictured with the new Old Bet sculpture.  The statue in front of The Elephant Hotel in Somers known as Old Bet has returned to its post after being taken down in July 2023 The beloved landmark had been a fixture in Somers since 1827 when a gilt wood replica of an elephant was installed on iron scrollwork on top of a granite shaft in front of the hotel which was built between 1820 and 1825 and is now used as Town Hall The old statue was taken down after cracks were discovered in it the town commissioned a new sculpture created by Luigi Badia and funded by the Wittmann family Earlier Report - Here's When Beloved, Refurbished Elephant Landmark Will Be Returning To Somers The new statue is made of bronze and is expected to last for hundreds of years according to Somers Town Supervisor Robert Scorrano It is the fourth Old Bet sculpture to be installed in Somers The elephant was purchased by Hachaliah Bailey who built the hotel and was known for touring with Old Bet and other wild animals introducing the traveling menagerie as popular entertainment in the region The hotel was designated a National Historic Landmark in 2005 for its role in the development of the American circus The eruption of Mount Vesuvius buried the Roman city of Pompeii in ash in 79 CE Anthropologists recently sequenced ancient DNA from one of the victims providing a glimpse into the family background of a Roman citizen The results also suggest that he suffered from a tuberculosis infection in his lower spine the study found DNA from the bacterium that causes tuberculosis suggesting that the infection had traveled through the bloodstream from his lungs to his lower spine A team led by anthropologist Gabriele Scorrano of the University of Rome sequenced the genome of the victim Although the ancient man’s genome didn’t yield much new information about life in Pompeii it proves that bones from Pompeii may still contain enough DNA to sequence—and that could be exciting news Even partial genomes from several more Pompeiians could shed some light on the demographics of a cosmopolitan Roman city, where historical documents tell us that people came from all over the Roman Empire (willingly or not) But sequencing ancient DNA from skeletons at Pompeii has been a challenge because high temperatures—like the ones in the pyroclastic flow of superheated volcanic gas and debris that killed everyone in the city—tend to cause chemical changes in bone and damage the DNA inside Previous studies have managed to sequence only a few short stretches of mitochondrial DNA (which is stored in the meme-famous “powerhouse of the cell” and passed directly from mother to child) Scorrano and his colleagues say advances in technology make it possible to get DNA from sources that would have been unusable a few years ago that the volcanic ash and rock that buried Pompeii may also have shielded the remains from things like oxygen sequencing an ancient genome from Pompeii has worked once Scorrano suspected the man might have spinal tuberculosis based on the condition of his fourth lumbar vertebra (L4) An infection had eaten away a hole in the upper front part of the bone and the surrounding bone was badly pitted and eroded Scorrano and his colleagues found genetic material from Mycobacterium tuberculosis and it suggests some details about what the man’s life may have been like before Mount Vesuvius erupted mainly due to the crowded conditions of most Roman cities but occasionally bacteria from the lungs can travel through the bloodstream to other parts of the body and in the long bones of your arms and legs dense networks of blood vessels supply blood to the bone marrow those blood vessels may also carry bacteria into the bone archaeologists may be able to sequence DNA from those bacteria and discover that you had spinal tuberculosis until a volcano buried your city in ash and pumice Archaeologists found the man’s remains in a relatively modest house in southeast Pompeii huddled beside a couch in one corner of the dining room The two probably died instantly when the wave of hot gas and volcanic ash swept over the city and they would have been buried in a dense blanket of ash soon afterward archaeologists found a set of cabinetmaker’s tools; it’s reasonable to speculate that the man may have been the cabinetmaker in question but his skeleton hasn’t yet offered enough information to say for sure Scorrano and his colleagues took small samples of bone from both victims but the woman’s DNA was too badly degraded to offer any useful information we know that she was at least 50 years old and probably female But Scorrano and his colleagues managed to sequence the man’s entire genome as well as search the sample for DNA for bacteria and viruses his father’s ancestors probably came to Italy from what’s now Turkey during the first expansion of Neolithic farmers into Europe about 7,000 years before Vesuvius erupted What the man’s genome doesn’t tell us is whether he was born and raised in Pompeii or belonged to the 5 percent of Imperial Romans who migrated within Italy The answer would shed some interesting light on his story The traces of bacterial DNA still embedded in his bone Infection would have caused painful inflammation in his lower back and it also would have seriously weakened the bones supporting and protecting his lower spine Archaeologists couldn’t find his third lumbar vertebra (L3 but because the infection had eaten away so much of L4’s upper half it’s hard to imagine that L3 wasn’t also affected That means the soft disc between the bones was probably involved which is painful and sometimes debilitating A collapsed disc can put pressure on the nerves that connect the spine with other parts of the body If the disc between this man’s L3 and L4 collapsed or numbness in his legs due to the compressed nerve Meanwhile, the badly weakened bone, without a disc to support and cushion it, probably would have eventually collapsed if the man had lived much longer. That could have left his legs paralyzed, which is what happened to a young Nasca boy who died in Peru around 700 CE Scientific Reports, 2022 DOI: 10.1038/s41598-022-10899-1;  (About DOIs) Somers, NY HamletHub invites you to contribute stories, events, and more to keep your neighbors informed and connected. Don’t miss what’s happening in your community.Subscribe to receive a daily digest of the people, places and things that make our community great. Copyright ©2025 HamletHub™ 2023 –The home of the Lupi is now Avicor Stadium Selvapiana Campobasso Football Club and Montreal-based commercial construction giant Avicor today announced an historic partnership which includes naming rights to the former Stadio Selvapiana the 25,000-seat home stadium of the rossoblu.  The partnership materialized as a result of the relationship between Campobasso Football Club President Matt Rizzetta and Avicor Founder and CEO Aldo Vicenzo Aldo Vicenzo is a proud immigrant from Sepino a small town in the Province of Campobasso who rose to build one of Canada’s largest and most significant commercial construction empires.  Avicor and Campobasso Football Club are intent on adding the names Molinari and Scorrano to the stadium as a tribute to former owner Antonio Molinari who brought the team to Serie B and defeated European football powers such as Juventus; and legendary team captain and Molise hero Michele Scorrano The addition of the names Molinari and Scorrano to the stadium are subject to municipal approvals by the City of Campobasso Until approvals are received from the municipality the stadium name will remain Avicor Stadium Selvapiana.  “On behalf the entire Campobasso Football family across the world I would like to thank Aldo Vicenzo and the Avicor leadership team for their generous support of our football club and our social mission” said Matt Rizzetta parent company of Campobasso Football Club “Aldo Vicenzo is a true success story and represents the humility and ambition of the people of Molise and the values of Campobasso Football Club we will be able to make critical infrastructure upgrades and most importantly we will be able to ensure the legendary names Molinari and Scorrano will live forever on the crown jewel of the Region of Molise Campobasso is fresh off a promotion to Serie D following a championship season in Eccellenza which saw the Lupi win 28 out of 30 games and score the most goals of any team in Italy.  “I have been following the Campobasso Football journey with great interest and admiration since the new American ownership team took over the club,” said Aldo Vicenzo “Beyond building a winning football culture they are building a social platform for the Molisani community as well as for the many immigrants such as myself who worked hard to achieve our dreams I am proud to play a role in the mission of Campobasso Football Club through our support of the stadium and to give back to the community that has given me so much in my life”.  Campobasso Football Club becomes one of the few football clubs in Italy to have a naming rights agreement for its stadium Sassuolo and Atalanta to have a partnership of this nature Avicor Stadium Selvapiana will be inaugurated in August 2023 with a friendly match and fan event More details will follow in the coming weeks and months.  Campobasso is the premier football team of Campobasso and the hundreds of thousands of Molisani across the world You are using an outdated browser. Please upgrade your browser to improve your experience Deputy Chief of Mission of the Indian Embassy was also present at the award ceremony," Patnaik wrote on Twitter on Saturday.Pattnaik was awarded the Padma Shri by the union government in 2014.In 2017 the Odisha-based sand artist won the jury prize gold medal at the Xth World Sand Sculptures Championship in Moscow Vietnamese artist showcases gold powder Buddha painting at UN Vesak Day Inauguration Finance Minister Sitharaman meets ADB President Italian counterpart; highlights India's DPI success Rajnath Singh holds bilateral talks with Japanese counterpart Gen Nakatani thanks him for efforts to deepen defence cooperation Gen Nakatani expresses condolences on Pahalgam attack during India-Japan Defence Ministerial Meeting Defence Minister Rajnath Singh to hold bilateral talks with his Japanese counterpart tomorrow Angolan President Joao Manuel Lourenco pays tribute to Mahatma Gandhi at Rajghat New UK-India cultural agreement boost creative industries copyrights © aninews.in | All rights Reserved