EXPERT OPINION BY BILL MURPHY JR., FOUNDER OF UNDERSTANDABLY AND CONTRIBUTING EDITOR, INC. @BILLMURPHYJR (If you find this useful, I hope you’ll download my free e-books Jeff Bezos Regrets Nothing, Warren Buffett Predicts the Future, and How to Raise Successful Kids, 7th Edition) Let’s go straight to the insight Bezos and Buffett have articulated so often and add three short guidelines that make the whole thing more attractive and practical for a younger generation We’ll start with Bezos, if only for the reason that his name comes alphabetically before Buffett’s. Here’s what he had to say back in 1997 when he was asked how he got the idea to start Amazon.com: that web usage was growing at 2,300 percent a year … And I picked books … [after compiling] a list of like 20 different products Bezos has kept to this story almost verbatim for more than two decades Anything growing that fast, even if its baseline usage was tiny, it’s going to be big,” he said in 2018 I should come up with a business idea on the internet and then building an idea around that growth an indelible quote from Buffett — making the same point has its roots in a 19th-century textile company that Buffett bought in the early 1960s he held onto the failing textile business for two decades Finally, he gave up and closed down the mills. He’s talked about this lesson many times, but here’s an apt quote: You don’t get any extra points for the fact that something’s very hard to do So you might as well just step over one-foot bars instead of trying to jump over seven-foot bars like the burgeoning insurance industry that became Berkshire’s biggest concern Synthesize both of these perspectives and what do you get practical advice for people who are just starting out in the world It applies whether we’re talking about starting careers or choosing where to live or what passions to pursue For people with the potential to be successful in many different fields teach kids to orient themselves toward places and things that are growing Bigger is better; small now but growing big is likely even better until we realize that it’s the exact opposite of the advice that many young people follow Perhaps the most obvious and quantitative examples will come from higher education For example, consider the graduates of Columbia University’s film program, who, The Wall Street Journal recently reported take out federal loans totaling a median of $181,000 to compete for jobs that can pay as little as $30,000 Or graduates of the speech pathology program at Northwestern University half of whom borrowed $148,000 or more to compete for jobs with a median income of $60,000 These are talented people pursuing something they’re interested in But they’re reportedly doing so in an environment in which the economics suggest a lack of growth As one passionate film student put it in the article: “financially hobbled for life.” This is a good time to mention a few short caveats The first is that you don’t have to like or respect Bezos or Buffett as people in order to impart this advice to your kids even though both Bezos and Buffett would likely qualify their self-worth in part as a result of their net worth you don’t even have to think about monetary rewards for this advice to make sense “going toward growth” isn’t the only factor in success it would be silly to suggest that young children should think about things like market size or consumer trends or population growth That’s the time to explore and learn the things that you become passionate about not to worry about how they’ll fit into a life plan and they start to think about decisions that will have a more profound effect on their long-term success this doesn’t mean kids should ignore the things they’re interested in or passionate about (Even if that’s what Bezos largely suggests he did.) It’s more a matter of doing the hard work to figure out how they can best pursue their interests but do so in a context of growth rather than stagnation the film students in the Journal’s example might be better off looking for ways to harness their creativity in front of audiences and markets that are growing rather than shrinking The speech pathology students might focus on careers that help people overcome speech or developmental problems It’s about balancing passion and interests with economic realities how do we make this all a bit more practical Basically, this means things that you can understand and articulate. A big, broad example of this would be the census data released last week which shows how the United States is becoming a more ethnically diverse society and an older one — with people living longer but birth rates dropping This is what Bezos kept talking about in terms of the statistic that prompted him to start Amazon: 2,300 percent annual internet usage growth in the 1990s teach them to look for things that are actionable that means teaching kids to have the courage to cast aside growth insights that don’t match their personalities and concerns It’s less relevant to explore growth in medical careers if the health industry isn’t appealing to a young person exploring careers or to consider other parts of the country if someone is adamant for personal reasons about not wanting to leave the place where they grew up We don’t want kids to become slaves to any single consideration teach kids to think about the reflection that Bezos and Buffett (and frankly many other successful people) keep articulating: Your odds of success are better if you play and work in places where the forces of growth are with you And who wouldn’t want the odds to be more in their kids’ favor (Don’t forget the free e-books Jeff Bezos Regrets Nothing, Warren Buffett Predicts the Future, and How to Raise Successful Kids, 7th Edition.) The opinions expressed here by Inc.com columnists are their own Refreshed leadership advice from CEO Stephanie Mehta Learn More Indian Punchline The visit by the head of the Russian Direct Investment Fund and special representative of the Russian president for investment and economic cooperation Kirill Dmitriev to Washington on April 2-3, the first such visit by a senior Kremlin official since 2022, appears to have been a modest achievement whose productive outcome will be crucial in the rest of US-Russia relations If President Vladimir Putin’s choice fell on Dmitriev for such a hugely important pathbreaking mission An enduring settlement in Ukraine hinges on the stabilisation of the Russian-American ties which is deeply flawed due to the meagre content of it in substance Putin and US President Donald Trump share a conviction that the latter’s MAGA project offers a rare window of opportunity to couple Russia’s vast resources with the regeneration of the American economy in a new order where geopolitics will no longer be the pivot this involves a formidable challenge insofar as it is nothing short of the rollback of a century of adversarial mindset that began in the period 1918-1920 when the US and Japan sent thousands of troops from the Baltics to northern Russia to Siberia to Crimea—and despatched massive millions of financial aid and military supplies to the anti-communist White Russians—in an abortive attempt to strangle Bolshevism in its crib The Biden administration had already sanctioned Dmitriev after spotting him as “a known Putin ally.” But Trump sees that fatal flaw in Dmitriev’s DNA as actually qualifying him to be an excellent counterpart to his own special envoy Steve Witkoff the billionaire businessman and close friend of the US president Dmitriev is a former banker who studied at Stanford and Harvard and worked at McKinsey and Goldman and is familiar with the ways of Wall Street where he has old friends and associates White House appreciated Dmitriev’s role in the release of the American prisoner Marc Fogel in February in a deal negotiated by Witkoff “key members of the US administration” received Dmitriev Dmitriev’s posts in the social media have been in an upbeat tone the nascent US-Russian dialogue is on track The Russian reports mentioned that Dmitriev’s agenda included the possibility of resumption of direct flights between the two countries cooperation in the Arctic as well as in rare earths Trump left out Russia from the list of countries against which new tariffs were announced on “Liberation Day” (April 2) it transpires that American companies have applied for participation in the St Petersburg International Economic Forum (SPIEF) due to be held in St Putin personally interacts with the foreign participants in the SPIEF event Dmitriev took stock of all this probably when he told reporters in DC  that his meetings with administration officials constituted a step forward “I would say that today and yesterday we made three steps forward on a large number of issues,” Dmitriev pointed out He acknowledged that issues have been piling up for three years as there was virtually no communication between Russia and the US the process of resolution will take some time but it is definitely positive and constructive,” he said Russian Deputy Foreign Minister Sergei Ryabkov hinted on Friday that the forthcoming second round of discussions between the Russian and US delegations will focus on simplifying the visa process improving the mechanisms of mutual travel and resolving issues of financial transfers for diplomatic missions the elephant in the room is the West’s “sanctions from hell” against Russia Even an affectionate  tweak of sanctions for Russia’s exports of agricultural products and fertiliser meets with resistance from the European Union The Russian-American dialogue continues to face attempts at sabotage by Ukraine and the EU as well as certain groups within the US who simply do not want any normalisation with Russia, including  influential lawmakers such as Senator Lindsey Graham who is otherwise a staunch political supporter of Trump While in DC, Dmitriev remarked that “numerous forces interested in maintaining tension” stand in the way of restoring dialogue. He said they deliberately distort Russia’s position and try to disrupt any steps towards US-Russia cooperation, “sparing neither money nor resources for this.” (See a vicious attack on Dmitriev’s visit by the CNN here.)  The Ukrainian leadership sees the US-Russia negotiations as posing an existential threat Their belligerence and attempts to sabotage the negotiation process are directly linked to their main agenda of willy-nilly retaining the levers of power in Kiev it is appreciable that Washington and Moscow are still on the ball in regard of the full restoration of the functioning of diplomatic missions — although the negotiations on the Black Sea Initiative at the recent Riyadh meeting are on hold at the last summit of European leaders a week ago rare calls were heard for the EU to reach out to Russia for dialogue The Finnish President Alexander Stubb suggested that France or Britain should enter into negotiations with Putin Slovakia and Hungary have traditionally advocated such a pathway This kite-flying is an important enough signal that the matrix may no longer be seen in binary terms — as confrontation between the West and Russia — but creeping toward a modus operandi of  “every man for himself.” If Europe sees that sanctions continue to harm the EU itself it is possible that they will reconsider old positions the US remains a significant economic player in the transatlantic grid and the western system including the EU and the Trump administration is capable of exerting pressure on Brussels the question narrows down to how far Trump’s team shares the president’s vision of friendship and camaraderie with Putin at a personal level and a constructive engagement by the erstwhile rivals in a spirit of cooperation Secretary of State Marco Rubio  who harbours presidential ambitions Maybe their tone has softened a bit but there’s no sign yet of ‘new thinking’ All these are disturbing signs that a full-fledged Russian-American détente remains a long haul Above all, as if the Russia policy shift is not complicated enough, Trump has also got to grapple with the Iran question where a deadline is expected by October and a spectre of confrontation haunts both Washington and Iran unless a deal appears in the next 3-4 months.  But then, “Sweet are the uses of adversity, which, like the toad, ugly and venomous, wears yet a precious jewel in his head.” Adversity, as Shakespeare suggested, often conceals valuable lessons and opportunities for growth. Even in challenging times, there is wisdom to be gained and strength to be found.  The ‘known known’ is that Putin commands immense respect in Tehran. And the ‘known unknown’ is, how far Russia can help in a mediatory mission to wrap up a US-Iran deal. Put differently, the ‘unknown unknown’ is, will Trump seek Putin’s helping hand?  Anything is possible in Trump’s revolutionary mind. After all, the administration has stopped demanding the withdrawal of Russian troops from Syria. This tendentious item is missing from Washington’s new list of conditions for the authorities in Damascus.  Mail to me your questions, feedback and suggestions at indianpunchline@gmail.com Volume 11 - 2020 | https://doi.org/10.3389/fimmu.2020.01039 Aire is a transcriptional controller in medullary thymic epithelial cells (mTECs) modulating a set of peripheral tissue antigens (PTAs) and non-PTA mRNAs as well as miRNAs Even miRNAs exerting posttranscriptional control of mRNAs in mTECs the composition of miRNA-mRNA networks may differ networks exhibited greater miRNA diversity controlling mRNAs Variations in the number of 3'UTR binding sites of Aire-dependent mRNAs may represent a crucial factor that influence the miRNA interaction we analyzed through bioinformatics the length of 3'UTRs of a large set of Aire-dependent mRNAs The data were obtained from existing RNA-seq of mTECs of wild type or Aire-knockout (KO) mice We used computational algorithms as FASTQC STAR and HTSEQ for sequence alignment and counting reads 3USS for the alternative 3'UTRs and TAPAS for the alternative polyadenylation sites We identified 152 differentially expressed mRNAs between these samples comprising those that encode PTAs as well as transcription regulators most of these mRNAs featured an increase in the length of their 3'UTRs originating additional miRNA binding sites and new miRNA controllers Results from the in silico analysis were statistically significant and the predicted miRNA-mRNA interactions were thermodynamically stable Even with no in vivo or in vitro experiments they were adequate to show that lack of Aire in mTECs might favor the downregulation of PTA mRNAs and transcription regulators via miRNA control This could unbalance the overall transcriptional activity in mTECs and thus the self-representation The meaning of the Aire expression is relative to the immunological tolerance that occurs in the thymus, since the expression of this gene is preferentially in that organ. Other organs such as pancreas, adrenal cortex, testes, lymph nodes, fetal liver and appendix also express Aire, without however knowing its exact function in these organs (6, 16–19) The human AIRE gene maps on chromosome 21q22.3 and in the mouse on chromosome 10 position 39.72 cM and the primary structure of Aire gene suggests that the distribution of its domains encodes a protein that interacts with the chromatin and regulates transcriptional processes (2, 20–27) Of note, the DNA-topoisomerase (DNA-TOP2a) protein, which is a strong AIRE partner acts on the DNA topology by removing positive and negative supercoils when it causes transient DNA breaks. DNA breaks result in a local relaxation of chromatin that facilitates the initiation and post-initiation of gene transcription (6, 33) and attracts DNA repair elements such as DNA-PK and PARP1 (6) Evidence suggests that AIRE and its partners bind to chromatin super-enhancers, which include the transcription start sites of most Aire-dependent genes (37). This multimolecular complex enables the transcription elongation step of a myriad of genes including those encoding autoantigens (4, 6, 28) AIRE also associates to proteins involved with pre-mRNA processing (1), alternative splicing (38) and polyadenylation factors (HNRNPL/F, SFRS1, and CLP1) (39) It is plausible that this might be directly or indirectly related to the processing of Aire-dependent mRNAs in mTECs By means of classical gene knockout (KO) (40–42) or gene KO through Crispr-Cas9 (43) it was demonstrated that Aire affects the abundance of mRNA isoforms in mTECs. Previously we observed that miRNAs exert posttranscriptional effect on Aire-dependent mRNAs (44). However, this action is not uniform and a greater set of miRNAs exert their effect on mRNAs when Aire is down-regulated in mTECs (45) These findings prompted us to investigate whether Aire could affect isoforms of mRNAs involving the length of 3'UTR end as we have previously suspected (3) Whether this process exists among the Aire-dependent mRNAs this may be influencing the control of the PGE and consequently of the self-representation in the thymus the aim of this study was to evaluate whether Aire affects the length of the 3'UTR region of mRNAs it controls To test this we analyzed the existing RNA-seq data of mTECs isolated from Aire wild-type or Aire-knockout (KO) mice (41) identifying and quantifying those mRNAs differentially expressed between these two samples and that showed changes in the length of the 3'UTR region and thus in the miRNA binding sites We obtained the sequences from raw RNA-seq data deposited in the Gene Expression Omnibus GEO repository (https://www.ncbi.nlm.nih.gov/geo/) and available under accession number GSE65617 (41) which consisted of RNA-seq of CD45− EPCAM+ sorted as cTECs (Ly51+ UEA1−) Ly51− UEA+ CD80lo MHC-IIlo sorted as mTEClo Ly51− UEA+ CD80hi MHC-IIhi sorted as mTEChi The analyzed sequences were from mTEChi cells (Ly51− UEA+ CD80 hi MHC II hi) isolated of wild type (WT) or Aire knockout (KO) C57BL/6 mice Each biological replicate (n = 3 for each cell type) consisted of a pool of mTECs (FACS sorted) of four isogenic animals (two males and two females from the same litter) WT or KO Sequencing was performed on an Illumina HiSeq 2000 RNA-seq platform with paired-end libraries with fragments of 100 bp (2 × 100 bp) which outputs a BAM file containing both the sequence and its genomic coordinate Together with a General Transfer Format (GTF) the file containing all the genomic annotations were used to determine the total number of reads per transcript using the HTSeq Count program (https://htseq.readthedocs.io/en/release_0.11.1/) (27) Information about genome assembly and gene annotation corresponds to the mm10 reference genome that was submitted by the Genome Reference Consortium (mouse genome reference GRCm38) The complete description of this annotation as for example can be found available at (m.ensembl.org/Mus_musculus/Info/Annotation) For each sample analyzed, we obtained a list of genes with their respective counts, which were analyzed to determine the genes differentially expressed by using the DESEq2 package (https://bioconductor.org/) (47), which is carried out through the platform R (https://www.r-project.org/) A heat-map was constructed in order to hierarchically cluster samples and mRNAs based on Euclidean distance and the complete linkage method Benjamini-Hochberg FDR correction and fold change ≥ 1.5 were considered as differentially expressed We then classify the 3'UTR alternative sequences into two categories Aire-dependent or Aire-independent considering the differentially expressed genes between WT and Aire KO mTECs In addition, we use the TAPAS tool (https://github.com/arefeen/TAPAS) for the determination of the number of APA sites from RNA-seq data, which works with two or more APA sites or with APA sites located prior to the last exon of a RNA target sequence (50) This tool compares RNA-seq data of pairs of biological samples that in this study were samples from Aire WT or Aire KO mTECs The tool identify those differentially expressed APA sites as well as mRNAs that contain 3'UTRs with shortening/ lengthening events New interactions were those identified in this study but not predicted and deposited in any of these databases The new and predicted miRNA-mRNA interactions were validated using the RNA-Hybrid algorithm (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid) (51) It is a bioinformatics tool that analyzes the secondary structure and determines the most favorable hybridization site between a given miRNA and its target mRNA The methodology involves a dynamic programming algorithm that calculates the location for the most energy-efficient hybridization This methodology is based on the thermodynamic property of miRNA-mRNA interaction in which the RNA double strand is more stable the miRNA-mRNA binding is stronger when the energy is lower The interaction force is determined by the quantification of the minimum free energy (mfe) normalized by the length of the target mRNA we selected interactions with mfe ≤ −20 kcal / mol which are considered thermodynamically stable To better understand the functional annotation of Aire-dependent mRNAs other than those encoding PTAs but that featured alterations in the length of their 3′UTRs we use the DAVID (david.ncifcrf.gov/) considering a P-value EASE score ≤ 0.1 to examine the significance of gene-term enrichment with a modified Fisher's exact test The false discovery rate (FDR) under certain rate (e.g. ≤ 0.05) provided by DAVID is calculated on the basis of the multiple testing correction as Bonferroni and Benjamini methods Shortening or lengthening of 3'UTR in mRNAs Changes in 3'UTR of mRNAs expressed by Aire wild type or Aire KO mTECs were quantified using the Mus musculus reference genome Venn diagram showing the three categories of mTEC transcripts with short or long 3'UTRs: 642 transcripts were specific of Aire wild type 359 were specific of Aire KO and 500 were common to Aire wild type and Aire KO mTECs Heat-map showing the large-scale expression profiling of Aire-dependent transcripts when comparing Aire wild-type vs Unsupervised heat-maps and dendrograms were constructed using R platform downregulated mRNAs (Fold-change FC ≥ 1.5 Differentially expressed PTA and non-PTA mRNAs that featured alternative 3'UTRs Identification of Aire-dependent PTA mRNAs that featured increased (+) or reduced (–) 3'UTRs in Aire KO mTECs in comparison to the Mus musculus (mm10) reference genome miRNAs that hybridize with 3'UTRs of reference PTA mRNAs or with Aire-dependent PTA mRNAs expressed by Aire KO mTECs Supplementary Figure 1 show that when we compared the interaction between the seed region of miRNA 4661-5p or miRNA 6924-5p with the 3'UTR region of the GenBank reference mRNA Cxr6 with the result obtained with mTECs (this study) for Cxcr6 mRNA we observed that increased 3 'UTR sequence of this PTA mRNA does not unbalance the miRNA-mRNA interactions This increase favors energetically stable interactions presenting mfe of −25.6 kcal/mol (miR-4661-5p and Cxcr6) and−26 kcal/mol (miR-6924-5p and Cxcr6) With respect to the Rcvrn mRNA, Supplementary Figure 2 shows that the 3'UTR of this reference mRNA predicts interaction with the seed region of miRNA 4763-3p and miRNA 6754-5p which present mfe of −35 kcal/mol and −29.3 kcal/mol The increase of 3'UTR of mRNA Rcvrn observed in mTECs created new interactions involving miRNA 7076-5p and miRNA 7075-5p mfe −32.7 kcal/mol and −28.9 kcal/mol Supplementary Figure 3 shows that the reference Nos1 mRNA interacts with the seed region of miRNA-466k The reduction of Nos1 mRNA 3′UTR in mTECs created interaction with two new miRNAs Supplementary Figure 4 shows that the 3'UTR of the reference Ttc9 mRNA predicts interaction with the seed region of miRNA-3057-3p and miRNA-767 mfe of −31.5 kcal/mol and −27.7 kcal/mol The 3'UTR reduction of the Ttc9 mRNA observed in mTECs did not alter the interaction with miRNA 3057-30 but created two new possibilities of interaction involving miRNA-705 and miRNA-7008-5p The 41 mRNAs that encode PTAs and the featured 3'UTR alterations were assigned to 11 anatomic functional body systems as follows (in descending order): central nervous, glands, immune and lymphatic, reproductive, ocular, endocrine, digestive, renal and urinary, muscular, integumentary and respiratory (Figure 4) Distribution of the expressed mRNAs that encode peripheral tissue antigens into 11 anatomic/physiologic body's systems which guarantee their self-representation through promiscuous gene expression by mTECs We performed the functional enrichment of mRNAs that do not encode PTAs, but which are Aire-dependent and which have altered the size of the 3′UTR region (Table 2). The biological process most represented by these mRNAs is relative to the regulation of transcription (Figure 5) including the transcription factors (Tbx6 Functional enrichment of Aire-dependent non-peripheral tissue antigen (non-PTA) mRNAs which have altered the size of the 3'UTR Note that the most represented biological process in which these mRNAs were involved is the regulation of transcription Table 4 shows the positioning of APA sites throughout the nucleotide sequence of the Aire-dependent PTA mRNAs those most frequent and those APA sites not previously described but which were identified in the mTEC cell samples studied We observed that the number of APA sites does not change significantly between WT and Aire KO mTEC cells we observed new APA sites in PTA mRNAs expressed by this cell type Identification (nucleotide position) of alternative polyadenylation (APA) sites in Aire-dependent PTA mRNAs In this study, we analyzed two large groups of mRNAs that were expressed in medullary thymic epithelial cells (mTECs) isolated from autoimmune regulator (Aire) wild type (WT) or Aire-knockout (KO) mice (41) The first group of mRNAs encode peripheral tissue antigens (PTAs) and the other encode proteins involved with transcription control including transcription factors (TFs) The analysis was done to assess the hypothesis that Aire regulates the mRNA 3'UTR length the preferential region where are found the binding sites of miRNAs In previous studies, we have observed that Aire regulates a set of miRNAs in mTECs (52), which establish miRNA-mRNA network and regulate a set of downstream Aire-dependent mRNAs (45). Interestingly, reduction in Aire in these cells imbalance miRNA-mRNA interactions and a larger set of miRNAs now act on the target mRNAs (45) These observations raised questions about the factor (s) that lead to increased miRNA diversity acting on mRNAs (3, 44, 45) factors such as stoichiometry between miRNAs and mRNA targets or stability of base pairing may have influenced the miRNA interaction The stoichiometry between these RNA species is expected to vary during the cell cycle and does not explain the augmentation of miRNA diversity that interact with their mRNA targets The Gibbs free energy or minimum free energy (mfe) ≤ −20 is considered optimal for maintaining miRNA-mRNA pairing and this value is widely used as a cutoff in the search for stable miRNA interactions. Even so, different miRNAs could still play a role at different mfe values (51) a plausible explanation for the increase in the number of miRNAs that interact with target mRNAs would be the modification in the target these miRNAs would have to have more binding sites in the target 3'UTR region we analyzed the length of 3′UTRs of a large set of Aire-dependent mRNAs whose data were obtained from existing RNA-seq data of mTECs isolated of wild type or Aire-knockout (KO) mice we also analyze the variations of the 3′UTRs of mRNAs that encode transcription factors as these elements are at the basis of transcriptional gene expression control which may influence self-representation by mTECs The use of the 3USS software allowed us to identify the existence of alternative 3'UTR sequences when comparing the mTEC wild type cell transcript set with the Aire KO mTECs Both samples showed shortening or lengthening in this region and this demonstrated that Aire influences the generation of mRNA isoforms with respect to the 3'UTR region loss of tolerance may occur with consequences in the emergence of autoimmune reactions our results showed that in the absence of Aire increased by 7,729 nucleotides in its 3'UTR and the generation of two new miRNA binding sites (for miR-7076-5p and miR−7075-5p) As this PTA represents retinal pigment epithelium which are target during autoimmune reactivity this suggests a mechanism of autoimmunity of the eye structures the mRNA that encodes the PTA Syde-1 had an increase of 5,800 nucleotides in its 3'UTR with the appearance of a new miR-1187 binding site This PTA represents ovary and adrenal glands that are target organs for autoimmune reactivity in APECED syndrome This illustrates in what way changes in the 3'UTR of mRNAs could clinically affect aggressive autoimmunity Two other mRNAs that encode proteins involved in the biology of mTECs such as Crebbp (an AIRE partner) and Ago-2 (a key element in the RISC complex during the miRNA mechanism of action) also had an increase in their 3′UTRs and consequently the generation of new miRNA binding sites CREBBP being negatively regulated by miRNAs may destabilize the AIRE complex in the chromatin of mTECs with consequences for PGE control Ago-2 being negatively regulated might cause a general imbalance in the posttranscriptional control Aire gene/protein is the main transcriptional regulator of PTAs that acts in synergy with Forebrain embryonic zinc finger-like protein 2 (FEZF2) in mTECs (4–9). Although Aire is an upstream transcriptional controller, this gene / protein has a link to posttranscriptional control in mTECs (11, 45, 52) In addition to evaluating variations in the length of 3'UTR sequences of the Aire-dependent mRNAs we also examined whether these variations could affect miRNA-mRNA interactions which is a widely used method for different model-systems We show that Aire interferes with the 3'UTR length of Aire-dependent mRNAs and that its absence modify the composition of miRNA-mRNA interactions creating new miRNA sites on PTA mRNAs This might have influence in reduced PTA protein translation We evidence that absence of Aire not only interferes directly with transcription of PTAs but also with the structure of its respective mRNAs As previously shown (41, 43, 55) Aire also controls the expression of non-PTA mRNAs it was found that those mRNAs associated with transcription control including those encoding transcription factors were also under Aire control that affected the size of their respective 3'UTRs This certainly leads to an imbalance in the overall gene expression of mTECs with implications for self-representation Although FEZF2 plays its role as a TF in mTECs that works in synergy to AIRE we were not able to evidentiate its transcriptional expression in the samples analyzed It is well-known that the maturation of the 3′ ends of most eukaryotic mRNAs is dependent on cleavage and polyadenylation (C/P) and that this RNA species has several C/P sites. Processing of mRNAs by C/P, which involves endonucleolytic cleavage of the nascent RNA and then addition of the poly (A) tail is one of the processes of control of gene expression in eukaryotes (56–60) AIRE protein is also involved with these processes either interacting with proteins of pre-mRNA processing (1), alternative splicing (38) and polyadenylation factors (39) we investigated whether Aire could affect the number of APA sites of Aire-dependent mRNAs Our results show that rather reflecting the presence or absence of Aire the new APA sites in mRNAs is more associated to mTEC cell type This is still an open question to search for genes/proteins involved in this process these results point toward the existence of a controlling mechanism in mTECs in which Aire gene/protein affects the length of 3'UTR of Aire-dependent mRNAs the mRNA targets feature increased sequence were is found miRNA binding sites The miRNAs then exert posttranscriptional control over these mRNAs and might decrease protein translation that in the case of PTAs influence self-representation in the thymus The datasets generated for this study can be found in the GEO acc number GSE65617 EO: conceived the study and performed data analysis This work was funded by the following agencies Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq EHO was a PNPD post-doctoral fellow from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES This study was financed in part by the CAPES– Financial code 001 The 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 The reviewer PP declared a past co-authorship with one of the authors GP to the handling editor Tiballi (School of Dentistry of Ribeirão Preto The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2020.01039/full#supplementary-material Molecular interactions between miRNAs and mRNA targets that feature reduction or increase in their 3'UTRs and their respective thermodynamic minimal free energy (mfe) Comparison of the interaction between the seed region of miRNA 4661-5p or miRNA 6924-5p with the reference 3'UTR (GenBank NCBI) of mRNA Cxr6 with the respective interaction obtained with mTECs (this study) for Cxcr6 mRNA The increase of 3'UTR of mRNA Rcvrn observed in mTECs created new interactions for miRNA 7076-5p and miRNA 7075-5p The reduction of Nos1 mRNA 3'UTR in mTECs created interaction with two new miRNAs Aire's partners in the molecular control of immunological tolerance the master activator of self-antigen expression in the thymus Chapter 7 Thymus Transcriptome and Cell Biology CrossRef Full Text | Google Scholar and cell-cell interactions during induction of central tolerance Update on Aire and thymic negative selection The thymus as a mirror of the body's gene expression Chapter 9 Thymus Transcriptome and Cell Biology PubMed Abstract | CrossRef Full Text | Google Scholar PubMed Abstract | CrossRef Full Text | Google Scholar Fezf2 orchestrates a thymic program of self-antigen expression for immune tolerance The mechanisms of T cell selection in the thymus AIRE: from promiscuous molecular partnerships to promiscuous gene expression Promiscuous gene expression in the thymus: a matter of epigenetics Medullary thymic epithelial cells: deciphering the functional diversity beyond promiscuous gene expression Chapter 8 Thymus Transcriptome and Cell Biology CrossRef Full Text | Google Scholar CrossRef Full Text | Google Scholar The mouse Aire gene: comparative genomic sequencing Expression and function of the autoimmune regulator (Aire) gene in non-thymic tissue Expression of the autoimmune regulator gene and its relevance to the mechanisms of central and peripheral tolerance Aire-expressing ILC3-like cells in the lymph node display potent APC features The autoimmune regulator (AIRE) is a DNA-binding protein The biophysical and biochemical properties of the autoimmune regulator (AIRE) protein AIRE expands: new roles in immune tolerance and beyond CrossRef Full Text | Google Scholar caused by mutations in a novel gene featuring two PHD-type zinc-finger domains Mapping DNA-binding domains of the autoimmune regulator protein HTSeq a python framework to work with high-throughput sequencing data Husebye ES Autoimmune regulator and self-tolerance - molecular and clinical aspects PubMed Abstract | CrossRef Full Text | Google Scholar Autoimmune regulator is acetylated by transcription coactivator CBP/p300 AIRE acetylation and deacetylation: effect on protein stability and transactivation activity PubMed Abstract | CrossRef Full Text | Google Scholar The deacetylase Sirt1 is an essential regulator of Aire-mediated induction of central immunological tolerance Roles of eukaryotic topoisomerases in transcription AIRE recruits P-TEFb for transcriptional elongation of target genes in medullary thymic epithelial cells Aire unleashes stalled RNA polymerase to induce ectopic gene expression in thymic epithelial cells Brd4 bridges the transcriptional regulators to promote elongation of peripheral-tissue antigen transcripts in thymic stromal cells The transcriptional regulator Aire binds to and activates super-enhancers Extensive RNA editing and splicing increase immune self-representation diversity in medullary thymic epithelial cells An RNAi screen for Aire cofactors reveals a role for Hnrnpl in polymerase release and Aire-activated ectopic transcription Population and single-cell genomics reveal the Aire dependency and distribution of self-antigen expression in thymic epithelia Differential features of AIRE-induced and AIRE-independent promiscuous gene expression in thymic epithelial cells Promiscuous mRNA splicing under the control of AIRE in medullary thymic epithelial cells Aire disruption influences the medullary thymic epithelial cell transcriptome and interaction with thymocytes Aire-dependent peripheral tissue antigen mRNAs in mTEC cells feature networking refractoriness to microRNA interaction Aire downregulation is associated with changes in the posttranscriptional control of peripheral tissue antigens in medullary thymic epithelial cells Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 3USS: a web server for detecting alternative 3'UTRs from RNA-seq experiments TAPAS: tool for alternative polyadenylation site analysis Fast and effective prediction of microRNA/target duplexes Autoimmune regulator (Aire) controls the expression of microRNAs in medullary thymic epithelial cells Thymic crosstalk: An overview of the complex cellular interactions that control the establishment of T-cell tolerance Chapter 6 Thymus Transcriptome and Cell Biology CrossRef Full Text | Google Scholar Positive and negative selection of the T cell repertoire: what thymocytes see (and don't see) Aire knockdown in medullary thymic epithelial cells affects Aire protein deregulates cell adhesion genes and decreases thymocyte interaction Mechanism and regulation of mRNA polyadenylation Alternative cleavage and polyadenylation: the long and short of it Alternative cleavage and polyadenylation: extent Alternative polyadenylation in the regulation and dysregulation of gene expression Alternative cleavage and polyadenylation in health and disease 3' end mRNA processing: molecular mechanisms and implications for health and disease Processing and transcriptome expansion at the mRNA 3' end in health and disease: finding the right end Implications of polyadenylation in health and disease A 3'UTR polymorphism marks differential KLRG1 mRNA levels through disruption of a miR-584–5p binding site and associates with pemphigus foliaceus susceptibility Cooperative translational control of polymorphic BAFF by NF90 and miR-15a Duarte MJ and Passos GA (2020) Aire Gene Influences the Length of the 3′ UTR of mRNAs in Medullary Thymic Epithelial Cells Received: 03 November 2019; Accepted: 29 April 2020; Published: 28 May 2020 Copyright © 2020 Oliveira, Assis, Speck-Hernandez, Duarte and Passos. 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: Geraldo A. Passos, cGFzc29zQHVzcC5icg== 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 Luis Henrique’s name would have been met with derision Since joining Marseille in 2020 from the Brazilian outfit Três Passos the winger has by all accounts been a transfer flop and like a bad penny Les Phocéens could not seem to rid themselves of him He had returned in January from a year-and-a-half-long loan at former club Botafogo The Brazilian side had the option to make the deal permanent but didn’t exercise it as interest was fairly low following what was ultimately a disappointing stay back in his homeland Marseille likewise did not plan on keeping Henrique beyond January an intervention from then-manager Gennaro Gattuso who had been impressed by the winger filling in the gaps for a squad decimated by injuries and absences from the Africa Cup of Nations was enough to keep him at the club beyond the winter transfer window Gattuso did not last very long into the year and his interim replacement Jean-Louis Gasset was similarly intrigued by the winger Henrique became a useful option in the first team a player the coach believed could be played anywhere and do a bit of everything While all of these head coaches appreciated Henrique’s skillset none have quite understood him as much as Roberto De Zerbi the winger has grown from a player who had scored only once in his entire Marseille career before the 2024/25 campaign to one that has recorded six goals Henrique is on course for a career-best season with the winger also registering four assists De Zerbi has allowed Henrique to flourish and helped the player to have the confidence to take his chances The flexibility of the winger has been highly appreciated by the Italian coach who has fielded him as a midfielder and also as a wing-back in a three-at-the-back system that has been used in wins against Lens Henrique has come on leaps and bounds since the start of the season and should be understood as one of the key attacking threats within a team that is hoping to remain hot on Paris Saint-Germain’s trail for the entire season It cannot be understated how impressive this transformation has been for the young Brazilian GFFN | Nick Hartland who has been given the armband by head coach Roberto De Zerbi Luis Henrique joined Marseille from Brazilian outfit Três Passos in a €8m deal four years ago He failed to make an impression under the different tutelages of former Marseille bosses André Villas-Boas and Jorge Sampaoli Luis Henrique was then loaned out to Botafogo in 2022 with an option to buy The winger then returned to Marseille last season during the winter transfer window where he finally made his mark in a somewhat struggling team Inter Milan & Bayern Munich Go Head-To-Head For Marseille – Bayern Interest Stretches Back Several YearsSempreInter.comApril 4 2025 at 5:54 PM UTC·2 min readInter Milan and Bayern Munich are set to go head-to-head for Marseille’s Bayern Munich with the Bavarians having been keen for several years This according to Brazilian outlet Globoesporte, via FCInterNews They report that Bayern first showed an interest in signing the 23-year-old when he was a teenager at Tres Passos Inter Milan are reportedly working to sign Marseille winger Luis Henrique The Brazilian is a player who the Nerazzurri see as a potential backup option for Denzel Dumfries at wingback Inter are not the only club keen on Henrique’s signature Inter’s primary competition to sign Henrique could come from the Bundesliga Bayern Munich will be Inter’s opposition in the Champions League quarterfinals this month But the two European heavy-hitters are also ready to go head-to-head off the pitch Bayern see Luis Henrique as a target just as much as Inter do this is not the first time that the Bavarians have eyed up Henrique The Brazilian caught Bayern’s eye in a friendly match against his former club Tres Passos all the way back in 2016 Luis Henrique never totally fell off of Bayern’s radar And the Brazilian’s form for Marseille this season has resulted in the German giants reigniting their pursuit of Henrique The Magazine of the Science Fiction and Fantasy Field The shortlist for the 2025 Grand Prix de l’Imaginaire honoring the best SF/F work published in France in 2024 Winners will be announced on May 12, 2025 and awarded at a ceremony in Montpellier, to be held May 17. 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Suscribe x MercoPress, en Español Montevideo, May 5th 2025 - 11:58 UTC Chinese imports of meat and offal totaled 830,000 tons in September 63% higher than the volume recorded for the same month last year based on information from Beijing's customs office with an increase of 121.6% in the annual comparison because of the problems caused to the local supply by the African swine fever China has already imported 3.29 million tons of pork China’s beef imports in September increased by 18.9% or 180,000 tons Brazilian food companies BRF and Marfrig Global Foods received the approval of Beijing on October 23 to resume meat exports from the Dourados (MS) and Várzea Grande (MT) units that had been suspended amid concerns about the Covid-19 pandemic Shipments of chicken from the BRF unit were suspended in July and are expected to resume in the coming days Commenting for this story is now closed.If you have a Facebook account, become a fan and comment on our Facebook Page! - Photo: AuroraChina has suspended importations from the main slaughterhouse of cooperative Aurora in Chapecó Although no further reasons were communicated it is likely the ban is related to fears over Covid-19 The information about the suspension was shared last week by Brazil’s Ministry of Agriculture, Livestock and Food Supply (MAPA) after Chinese communication just before New Year The ministry said that China had “requested information on Covid-19 cases at the facility” and justified the suspension by “concerns” regarding this unit without giving further details According to a document available on the website of the General Administration of Customs of China (GACC) the suspended unit has the registration number SIF 3548 referring to a plant of the cooperative located in Chapecó The Brazilian Animal Protein Association (ABPA) supports Aurora and provides information to MAPA that might to be necessary to send to Chinese authorities in order to reverse the situation ABPA stated: “ABPA emphasises that this is a punctual and past situation All information and demonstrations of good practices from the cooperative – following strict sectoral and official protocols for Covid-19 – have been demonstrated in detail to Chinese authorities.” ABPA reiterated that there is no scientific proof of the risk of contamination of Covid-19 through food consumption China has already carried out similar suspensions against other Brazilian slaughterhouses China has suspended imports of 11 slaughterhouses and 1 fish unit from Brazil because of concerns over Covid-19 infections exports from BRF units in Dourados and Lajeado Monteiro Indústria de Pescados and meat processor Agra have already been reestablished Protecting Brazil from pig diseases 5 slaughterhouses are still suspended by China JBS Três Passos and Aurora Chapecó produce pork meat Brazil has confirmed a new milestone for pork meat exports International sales of pork totalled 1.021 million tonnes in 2020 a result 42.2% higher than that achieved the year before Your Ads Privacy ChoicesIMDb, an Amazon company© 1990-2025 by IMDb.com, Inc. COPS are hunting a paedophile suspected of raping and murdering his teenage stepdaughter while on parole for sex crimes. The body of Jordana Tamires Christ Watthier, 13, was found just hours after she vanished from her home in Bom Principio, Brazil, on Sunday. Cops said the girl's stepfather, 39-year-old Elias dos Santos Silvestre, took the teenager to a wooded area near a motorway at dawn before the rest of the family woke up. The marks left on the teen's body, which was reportedly found by firefighters, led police to believe the suspect raped the youngster and strangled her to death. Detectives also found an audio message sent by the suspect to a family member in which he confessed he had "done something foolish". Cops said the suspect, who worked as a bricklayer, has a criminal record for three other sex crimes - two involving minors - and a robbery. He had been sentenced to 12 years in prison and was on parole when the teenager was killed. Officers are now hunting Silvestre on suspicion of the brutal murder and rape of Jordana. Cops found the suspect's abandoned car with his car keys and mobile phone in Montenegro and believe he may have fled by bus. studying and dreamed of becoming an artist was buried on Tuesday in the municipality of Tres Passos The youngster lived in a quiet street of about 40 houses with her mum, older brother and younger sister, according to local news site GZH Neighbours described Jordana as "a very calm and dedicated girl" and said her murder has left the community "disgusted" Sie haben erfolgreich Ihre Einwilligung in die Nutzung von Transfermarkt mit Tracking und Cookies widerrufen Sie können sich jetzt zwischen dem Contentpass-Abo und der Nutzung mit personalisierter Werbung