Despite significant progress in the structural and functional characterization of the human genome, understanding of the mechanisms underlying the genetic basis of human phenotypic uniqueness remains limited. Here, I report that transposable element-derived sequences, most notably LTR7/HERV-H, LTR5_Hs, and L1HS, harbor 99.8% of the candidate human-specific regulatory loci (HSRL) with putative transcription factor-binding sites in the genome of human embryonic stem cells (hESC). A total of 4,094 candidate HSRL display selective and site-specific binding of critical regulators , and are preferentially located within the matrix of transcriptionally active DNA segments that are hypermethylated in hESC. hESC-specific NANOG-binding sites are enriched near the protein-coding genes regulating brain size, pluripotency long noncoding RNAs, hESC enhancers, and 5-hydroxymethylcytosine-harboring regions immediately adjacent to binding sites. Sequences of only 4.3% of hESC-specific NANOG-binding sites are present in Neanderthals’ genome, suggesting that a majority of these regulatory elements emerged in Modern Humans. Comparisons of estimated creation rates of novel TF-binding sites revealed that there was 49.7-fold acceleration of creation rates of NANOG-binding sites in genomes of Chimpanzees compared with the mouse genomes and further 5.7-fold acceleration in genomes of Modern Humans compared with the Chimpanzees genomes. Preliminary estimates suggest that emergence of one novel NANOG-binding site detectable in hESC required 466 years of evolution. Pathway analysis of coding genes that have hESC-specific NANOG-binding sites within gene bodies or near gene boundaries revealed their association with physiological development and functions of nervous and cardiovascular systems, embryonic development, behavior, as well as development of a diverse spectrum of pathological conditions such as cancer, diseases of cardiovascular and reproductive systems, metabolic diseases, multiple neurological and psychological disorders. A proximity placement model is proposed explaining how a 33–47% excess of NANOG, CTCF, and POU5F1 proteins immobilized on a DNA scaffold may play a functional role at distal regulatory elements. Identification of the genomic regulatory sequences contributing to the development of species-specific phenotypic features during primate evolution remains a significant challenge with important fundamental and translational implications. Determination of the molecular mechanisms underlying the genetic basis of human phenotypic uniqueness represents a particularly challenging task. Despite remarkable progress in understanding the structural–functional diversity, transcriptional and regulatory complexities, and biological significance of nonprotein-coding genomic regions, protein-coding genes remain the main focus of the experimental and theoretical analyses of human-specific genetic traits. Accelerated evolution of a selected set of protein-coding genes involved in various aspects of nervous system development and biology has been demonstrated . IncreasThe increase in the content and quality of genome and transcriptome sequence databases has facilitated comparative genomics studies focused on identification of primate-specific genes in the human genome. Taken together, these reports have identified a few hundred candidate primate-specific genes for follow-up functional studies, demonstrated insertions of interspersed repeats in exons of 92% of the identified primate-specific genes, documented the placement of sequences derived from transposable elements (TEs) in 53% of primate orphan genes, and highlighted the involvement of nonduplicated pericentromeric and subtelomeric regions of the human genome in the genesis of primate-specific genetic elements . Early pRepetitive and repeat-derived DNA sequences, including TEs, may account for up to two-thirds of the human genome . TEs conIn human cells, retrotransposon activity is believed to be suppressed to restrict the potentially harmful effects of mutations on functional genome integrity and maintenance of genomic stability. Human embryonic stem cells (hESCs) seem markedly different in this regard. Several studies demonstrated that hESCs express mRNAs from both human-specific (L1HS) and older L1 subfamilies of retrotransposons , and actHere, I report the identification and initial characterization of 4,094 candidate human-specific regulatory loci, 3,803 of which harbor putative TF-binding sites in hESC. Candidate human-specific regulatory elements are preferentially located in hESC within the matrix of hypermethylated DNA segments and appear engaged in selective and site-specific binding of the critical regulatory proteins NANOG (Nanog homeobox), POU class 5 homeobox 1 (POU5F1) (OCT4), CTCF, and Lamin B1. Sequences of hESC-specific TF-binding sites do not intersect with any chains in the mouse, rat, chimp, gorilla, orangutan, gibbon, rhesus, and marmoset genomes. This analysis reveals exclusive-to-primates DNA editing mechanisms involving DNA methylation at selected cytosine residues within LTR7 sequences coupled with methyl-cytosine deamination events that are overrepresented in the human genome and appear involved in the creation of regulatory sites with novel protein-binding specificities.http://genome.ucsc.edu/cgi-bin/hgLiftOver, last accessed May 20, 2015). Extraction of BLASTZ alignments by the LiftOver algorithm for a human query generates a LiftOver output “Deleted in new,” which indicates that a human sequence does not intersect with any chains in a given nonhuman genome. This indicates the absence of the query sequence in the subject genome and was used to infer the presence or absence of the human sequence in the nonhuman reference genome. Human-specific regulatory sequences were manually curated to validate their identities and genomic features using a BLAST  algorithm and the latest releases of the corresponding reference genome databases as of April/May 2013.Solely publicly available data sets and resources were used for this analysis as well as methodological approaches and a computational pipeline validated for primate-specific gene discovery . The anahttp://genome.ucsc.edu/ ; http://neomorph.salk.edu/human_methylome, last accessed May 20, 2015). The histone modification and TF chromatin immunoprecipitation sequence (ChIP-Seq) data sets for visualization and analysis were obtained from the UCSC data repository site . Consensus TF-binding site motif logos were previously reported . Direct access to the specific Genome Browser tracks utilized for analyses and visualization: http://genome.ucsc.edu/cgi-bin/hgTracks? db=hg18&position=chr10%3A69713986-69714099&hgsid= 393865029_yg7UixUE4a4awjjTahns4KTPkIl1 .The assessment of conservation of 826 human-specific NANOG-binding sites in individual genomes of three Neanderthals and five Modern Humans was carried out by direct comparisons of corresponding sequences retrieved from individual genomes and the human genome reference database . Any modifications or new elements of statistical analyses are described in the corresponding sections of the Results. Statistical significance of the Pearson correlation coefficients was determined using GraphPad Prism version 6.00 software. The significance of the differences in the numbers of events between the groups was calculated using two-sided Fisher’s exact and Chi-square test, and the significance of the overlap between the events was determined using the hypergeometric distribution test  identified 29,130; 14,003; and 29,018 sequences of 200 bp in length centered at NANOG-, OCT4-, and CTCF-binding sites, respectively, that do not intersect with any chains in the mouse and rat genomes . Overall, these sequences constitute 33% of all TF-binding events detected in hESCs for NANOG and CTCF and 47% of all OCT4-binding sites . Notably, these sets of human-specific regulatory sequences represent 0.9%, 8%, and 0.7% of all binding sites and 3%, 17%, and 2% of primate-specific binding sites for the NANOG, OCT4, and CTCF TFs, respectively . Each overlapping event was tabulated and the numbers of overlaps of each TF’s binding event with specific repetitive elements were calculated. Notably, 99.8%  of human-specific TF-binding sites were found embedded within repetitive elements, which is significantly higher than the proportion expected by chance . Follow-up analyses indicated that the “99% rule” is not limited to the NANOG-, OCT4-, and CTCF-binding sites and seems to have broad relevance. All human-specific binding events identified for five different regulatory proteins  mapped within repeat-derived sequences in the reference human genome database  Genome Browser . LINE- and LTR-embedded human-specific TF-binding sites constitute 64%, 5%, and 18% of all human-specific binding events for NANOG, OCT4, and CTCF, respectively. LINE and LTR elements appear to show equal contribution to the creation of human-specific TF-binding sites for NANOG, OCT4, and CTCF . Interestingly, a majority of the LINE- and LTR-embedded human-specific TF-binding events in hESCs are represented by the retrotransposons L1PA2 (196 events), L1HS (64), LTR7 (109), and LTR5_HS (81) that are transcriptionally active in human cells . Notably, human-specific TF-binding sequences are often embedded within full-length 6-kb L1PA2 and L1HS sequences. For example, 95% of L1PA2-embedded NANOG-binding sites are located within the full-length L1PA2 sequences. Similarly, full-length 6-kb L1HS sequences with less than 1% divergence constitute 77% of all L1HS sequences with human-specific TF-binding sites in hESCs.In total, 532, 116, and 104 of the candidate human-specific binding sites for NANOG, OCT4, and CTCF, respectively, were found located within long interspersed nuclear element (LINE) and long terminal repeat (LTR) sequences, suggesting that retrotransposons play a significant role in the creation of human-specific regulatory sequences  suggests that the function of a large number of CTCF-binding events is associated with the pluripotent state. Interestingly, loss of CTCF occupancy in differentiated cells appears limited to the human- and primate-specific binding events , indicating that pluripotency-associated CTCF activity may be mediated by primate-specific binding sites. Recent experimental evidence supports the idea of specialized CTCF-dependent regulatory mechanisms that are critical for the self-renewal and pluripotency properties of hESCs. One of the most notable features of the candidate human-specific regulatory loci reported in this study is that many of the TF-binding sites active in hESCs are not bound by TFs in differentiated cells. This observation is consistent with the documented essential role of NANOG and OCT4 in the maintenance of the pluripotent state and the restriction of their functions to ESCs. Therefore, a majority of candidate human-specific regulatory sequences identified in this study should be defined as candidate human-specific regulatory loci harboring putative TF-binding sites in hESC. Significantly diminished CTCF-binding in differentiated human cells compared with hESCs  are occupied by NANOG, which is significantly more than expected by chance (P = 0.00086). In contrast, only seven full-length L1HS-embedded human-specific TF-binding sites are occupied by OCT4, five of which overlap with NANOG-binding sites. Similarly, only three full-length L1HS-embedded human-specific TF-binding sites are occupied by CTCF, which is marginally significantly less than expected by chance. These conclusions remain statistically valid after accounting for five overlapping sites.As noted above, NANOG-binding sites in hESCs seem particularly interesting in this context because nearly two-thirds of human-specific NANOG-binding events are embedded within LINE and LTR retrotransposons in contrast to only 5% and 18% binding sites for OCT4 and CTCF, respectively. Consistent with this line of arguments, 34 of the 39 (87%) TF-binding loci embedded within transcriptionally active full-length L1HS retrotransposons  of the apparent structural differences between hESCs and differentiated cells.supplementary fig. S2, Supplementary Material online), 2) consistent transcriptional activity  Frequent location within lamina-associated domains (LADs)  activity . An examactivity . Therefoactivity . A signiactivity . In contactivity . These dactivity . Similaractivity , which iactivity .Fig. 2.Transcriptional activity is another notable feature of candidate human-specific genomic regulatory loci embedded within LINEs and LTRs. Analysis of corresponding RNA sequences in the Expression Tracks of the UCSC Genome Browser revealed that expression is not limited to established human cell lines, such as GM12878, K562, and H1-hESC. Active transcription from selected genomic loci containing human-specific TF-binding sites embedded within LINE and LTR sequences is apparent in the human brain, heart, breast, and lymph nodes. NANOG-binding sites embedded within LTR7 appear to be particularly active in H1-hESCs . RNA molsupplementary data set S7, Supplementary Material online). Human-specific LADs constitute more than 21% of all LADs in the human genome, indicating that genome-wide interaction profiles with nuclear lamina and three-dimensional folding patterns of human chromosomes are markedly distinct. The average length of human-specific LADs is 361 kb, which is 2.7-fold shorter than the average length of all LADs in the human genome , and 25–40% of all LADs of nearly half of all human chromosomes are represented by the human-specific LADs . Unexpectedly, a significant correlation between the chromosomal distributions of 290 human-specific LADs and either 29,018 primate-specific CTCF-binding sites  or 29,130 primate-specific NANOG-binding sites  was observed. In contrast, no significant correlation was found between the chromosomal distributions of 290 human-specific LADs and 14,003 primate-specific OCT4-binding sites (data not shown). These data suggest that the observed correlations cannot be explained solely by the chromosome size effects and may indicate that mechanisms of creation and retention of novel regulatory elements in the human genome are associated with common structural features. A survey of the genomic landscape in the vicinity of human-specific TF-binding sites revealed their apparent colocalization with DNA methylation sites, suggesting that genomic regions of DNA methylation may be relevant to these processes.Evidence of the frequent placement of the putative human-specific TF-binding sites within LADs suggests that at least some LADs in the human genome may constitute a component of the human-specific genomic regulatory network. To test this hypothesis, human-specific LADs were searched, which were defined as human LAD sequences which are 1) conserved in the mouse and human genomes and 2) located outside of the LAD boundaries in the mouse genome of the four distinct types of murine cells, namely mouse ESCs, neural precursor cells, astrocytes, and mouse embryonic fibroblasts . Using tP < 0.0001) were found to intersect with continuous DNA segments termed partially methylated domains (PMDs), which are hypermethylated in hESCs and hypomethylated in differentiated human IMR90 cells  of human-specific TF-binding sites with PMDs identified in normal human tissue. Notably, among seven different tissue-specific PMDs and highly methylated domains (HMDs), only neuronal-specific domains (N-HMDs) were found to be enriched for genes located near human-specific NANOG-binding sites . These data are consistent with the hypothesis that human-specific regulatory loci are preferentially placed within differentially methylated genomic regions, because N-HMD designates domains defined by sequencing of bisulfite-treated DNA (MethylC-seq) as PMDs (hypomethylated domains) in IMR90 fetal lung fibroblasts and placentas but as HMDs in SH-SY5Y neuroblastoma cells . The initial analysis was concentrated on the genome-wide assessment of the fully conserved 33- and 24-nt LTR7 sncRNA-encoding loci, defined as the full-length sequences with 100% identity, no gaps, and no mismatches. Both sequences were classified as primate-specific, because 1) there are no full-length 33- and 24-nt LTR7 sequences in either the mouse or rat genomes, and 2) chromosomal positions for 89% of the genomic loci encoding 33-nt LTR7 sncRNAs are conserved in primate genomes, with most significant similarities noted between the human genome and those of chimpanzee and gorilla . Reflecting their common origin, chromosomal distributions of 33- and 24-nt sequences in the human genome manifest highly correlated patterns . However, the number of loci encoding 24-nt LTR7 sncRNAs is 8.6-fold higher than that for 33-nt transcripts in the human genome . Additional analysis of this phenomenon is presented in the supplementary material, Supplementary Material online.Consistent with the recent reports of the increased LTR7 transcription in hESC , transcrsupplementary fig. S4, Supplementary Material online). Colocalization events were computed using data visualization tools in the AnnoJ browser , which are designed based on a genome-wide single-base resolution human methylome map . Of note, colocalization of sncRNA expression with noncanonical methylation sites at CHH or CGH (where H is any base except G) sequences appears to be significantly enriched . Intriguingly, methylation at noncanonical methylated (m)CH sites increases most rapidly during the primary phase of synaptogenesis in the developing postnatal human brain, and a rapid mCH level increase occurs primarily in neurons  is prone to mutate to T through spontaneous deamination . The netsupplementary fig. S4, Supplementary Material online). Strikingly, these analyses identified 21,906 genomic loci in the human genome encoding 12- to 24-nt-long single-site C to T mutants of the 24-nt LTR7 sncRNA-encoding sequence . Notably, C to T mutations at different positions within the 24-nt LTR7 sequence are generated and/or retained in the human genome with markedly different efficiency: Mutants at the C3, C4, C5, C9, and C10 sites are represented by 2,893–4,146 loci, whereas mutants at the C1, C2, C7, C8, and C11 positions are represented by 426–998 loci. The single-site C to T mutants seem to create primate-specific, predominantly highly conserved sequences because chromosomal positions of more than 90% of the examined mutant loci are conserved in the genomes of human, chimpanzee, gorilla, orangutan, and gibbon . Interestingly, the generation and/or retention of mutants harboring the second C to T mutation within the same 24-nt LTR7 sequence was negligible, as relatively few loci containing double C to T mutants were found in the human genome.The 24-nt LTR7 sncRNA sequence contains 11 potential noncanonical methylation sites, indicating that a spontaneous mC deamination mechanism may be relevant for generation of the C to T mutations within 24-nt LTR7 sequences. This hypothesis was tested by performing a systematic search for single-site C to T mutant sequences of differing lengths in the human genome . The UA9 motif logo  was previously associated with TF-binding events of multiple regulatory proteins exclusively in H1-hESCs, including NANOG, BCL11A, HDAC2, ESR1, GATA2, RXRA, TCF12, and a subset of HDAC2-associated POU5F1-binding sites .Of note, multiple sequence alignment analysis of the human ESC-specific NANOG-binding sites embedded within the LTR7-derived sequences identified motif logos that appear to closely resemble previously reported consensus binding sequences of several TFs, including NANOG, POU5F1 (OCT4), GATA1, and FOXA .Collectively, these observations suggest that mC to T mutations outside the canonical mCG sites may play an important role in continuous editing of TE-derived sequences in primate genomes. In the human genome, this mutation mechanism appears to be linked to the 25,756 DNA segments  containing LTR7-derived 24-nt sequences .An elite set of 24 genes regulating brain size and behavior in humans has been identified, which also shows markedly accelerated rates of protein evolution within the lineage leading from ancestral primates to humans . This sevolution . Thus, ivolution . Becausevolution , the strFor each gene in both gene sets, the number of human-specific TF-binding sites with genomic coordinates showing relative proximity to protein-coding gene boundaries defined in the hg19 reference human genome database was calculated. The quantitative limits of proximity were defined based on the metrics placing human-specific TF-binding sites closer to putative target genes than experimentally defined distances to the nearest targets of 50% of the regulatory proteins analyzed in hESCs . For eacsupplementary fig. S6, Supplementary Material online). On average, 4.00 and 3.98 human-specific TF-binding sites were located in close proximity to the rapidly evolving brain-specific and housekeeping genes, respectively. For genes in both the brain-specific and housekeeping sets, the evolutionary processes appear to favor the retention and/or creation of human-specific NANOG-binding sites over CTCF-binding sites . These data indicate that the overall patterns of the placement and retention of human-specific TF-binding sites are similar with respect to the genomic coordinates of either brain-specific or housekeeping genes.The housekeeping genes and brain-specific genes showed similar association patterns with human-specific TF-binding sites, as measured by the number of binding events located within the boundaries of putative target gene-specific regulatory regions . In the brain-specific gene set, a highly significant positive correlation  was observed; that is, genes that are located in close proximity to more human-specific TF-binding sites manifest higher rates of protein evolution . In striking contrast, in the housekeeping gene set, a highly significant negative correlation  was observed; that is, genes that are located in close proximity to more human-specific TF-binding sites manifest lower rates of protein evolution . Notably, the most highly significant correlation was observed in the brain-specific gene set between the evolutionary rates of proteins regulating brain size in humans and the numbers of human-specific NANOG-binding sites in close proximity to these genes . These data suggest that human ESC-specific NANOG-binding sites are located in close proximity to the rapidly evolving genes regulating the size of the human brain. In contrast, novel NANOG-binding sites appear to be preferentially located in close proximity to housekeeping genes with low rates of protein evolution. These associations are not necessarily due to the positive selective pressure. An alternative possibility is that rapidly evolving genes might be less constrained in humans and hence more tolerant to the transcriptional regulatory effect of nearby human-specific NANOG-binding sites, provided that this regulatory influence is important for neuronal gene expression. Therefore, this mechanism may be similar to the known depletion of TEs in promoters caused by the negative impact of TE insertions in genomic regulatory regions; TEs are preferentially found outside of promoter regions not because of selection for insertion in intergenic regions but rather owing to negative selection against insertion in promoters. It will be of interest to determine whether the association with rapidly evolving genes is a function of the new TF-binding sites or of TEs.Intriguingly, this analysis revealed that brain-specific and housekeeping genes manifest remarkably different correlation profiles between the numbers of human-specific TF-binding sites and rates of protein evolution of individual genes , which is significantly higher than the number expected by chance (P << 0.0001). Human-specific NANOG-binding sites comprised 88% of all human-specific TF-binding events associated with pluripotency lncRNAs , whereas human-specific CTCF-binding sites were detected in only 3 of 26 instances. Conducting similar analyses for 34 lineage-specification lncRNAs .Many candidate human-specific TF-binding sites manifest hESC-specific profiles of regulatory protein binding and transcriptional activities . Dispari lncRNAs , we obsesupplementary fig. S6, Supplementary Material online). Human-specific NANOG-binding sites appear enriched near pluripotency lncRNAs . Extended analysis of these associations is presented in the supplementary material, Supplementary Material online.The spectrum of human-specific binding events associated with lineage-specification lncRNAs appears significantly different from that of pluripotency lncRNAs . In contrast to TF-binding sites, only seven human-specific EDEs were found, which represents 0.09% of all EDEs in the human genome and is a marked reduction compared with the number of human-specific TF-binding events  play important roles in human embryonic development and are distinguished in hESCs by their unique chromatin signatures and biological activities . Next, hThe results of the present analysis suggested that one of the potential mechanisms of the bioactivity of human-specific NANOG-binding sites may be associated with the functions of promoter-distal regulatory elements. To test this hypothesis, the colocalization pattern of human-specific NANOG-binding sites and distal regulatory elements was assessed utilizing a comprehensive database of hESC enhancers . A totalsupplementary fig. S7, Supplementary Material online). In contrast, no enrichment of 5hmC nucleotides near human-specific CTCF-binding sites within 1-kb windows was observed. Only 15% of CTCF-binding sites were located near 5hmC, which is significantly lower than the number of human-specific NANOG-binding sites (P < 0.0001). Similarly, no significant enrichment of 5hmC nucleotides was observed near human-specific POU5F1-binding sites. Only 14.9% of POU5F1-binding sites were located within 1 kb of 5hmC, which is significantly lower than the number of colocalization events documented for human-specific NANOG-binding sites (P < 0.0001). These data indicate that a large number of human-specific NANOG-binding sites harbor 5hmC within the canonical CpG context immediately adjacent to TF-binding sequence motifs, because in hESCs nearly all (99.89%) 5hmCs exist in the canonical CpG context , which is most enriched in regions immediately adjacent to sequence motifs of TF-binding sites . Using a context . Additiosupplementary fig. S7, Supplementary Material online). Interestingly, human- and primate-specific NANOG-binding sequences were found to be located near hESC enhancers, whereas NANOG-binding sites common with rodents were located next to the transcription start site of protein-coding genes .Collectively, these observations suggest that candidate human-specific TF-binding sites are most likely to play a functional role in hESCs at promoter-distal regulatory elements. This hypothesis was confirmed by building regulatory maps of selected genomic regions depicting all NANOG-binding sites, hESC enhancer/coding gene regulatory pairs, and 5hmC distributions near regulatory loci . In total, 81 instances of colocalization of at least one copy of this three-letter 5hmC symbol in sequences containing multiple 5hmC nucleotides within 1-kb windows centered at 826 human-specific NANOG-binding sites were observed . This number is likely an underestimate because only high-abundance 5hmC sequences are detectable at the reported sequencing depth and significantly greater sequencing is required to obtain high-confidence resolution of low-abundance 5hmC sequences at the single-base level (P < 0.0001). Similarly to CTCF-binding sites and in sharp contrast to human-specific NANOG-binding sequences, only 20 events displaying this three-letter symbol of 5hmC sequences were observed within 1-kb windows near 2,386 human-specific POU5F1-binding sites, and NANOG protein was bound to 13 of these sites (P < 0.0001).Analysis of 5hmC sequences located near human-specific NANOG-binding sites revealed common patterns created by the frequent usage of particular sequences, examples of which are depicted in figure 5se level . In contTaken together, these data reveal association of human ESC-specific NANOG-binding sites with functional hESC enhancers and suggest that the common patterns of 5hmC sequences identified here may contain a code to signal recruitment of NANOG protein to specific genomic loci.http://www.ingenuity.com/, last accessed May 20, 2015). The Ingenuity Pathway Analysis of coding genes that have human-specific NANOG-binding sites within gene bodies or near gene boundaries identified a core set of 135 genes that appear to be interconnected in multiple networks associated with nervous system development and functions, embryonic development, behavior, and cardiovascular system development and functions . One of the notable features of this set of genes is their apparent association with development of a broad spectrum of common human disorders, including cancer, cardiovascular diseases, reproductive system diseases, metabolic diseases, multiple neurological and psychological disorders, hereditary and developmental diseases, and many other disease states . Taken together, these data argue that human ESC-specific NANOG-binding sites may contribute to regulation of coding genes with important physiological roles in development of nervous and cardiovascular systems during embryogenesis.To test whether coding genes located in close proximity to human ESC-specific NANOG-binding sites have pathophysiological associations or important developmental roles, the Ingenuity Pathway Analysis software was utilized .One of the limitations of the analyses conducted so far is that they were performed using the reference genome databases and not the individual genomes. To address this limitation, the assessment of conservation of 826 human ESC-specific NANOG-binding sites in individual genomes of three Neanderthals and five Modern Humans was carried-out by direct comparisons of corresponding sequences retrieved from individual genomes and the human genome reference database , 35.7% in Han (Chinese), 39.2% in Yoruba (West Africa), 43.3% in French (Western Europe), and 43.6% in San (Southern Africa). The sequence homology analysis identifies 152 sequences (18.4%) that are conserved in all five individual human genomes, whereas 58.1% of human-specific NANOG-binding sites are conserved in at least one individual genome of Modern Humans . In striThe reported numbers of NANOG-binding sites in zebrafish, mouse, and human genomes are 14,010, 16,667, and 88,351, respectively , suggestSimilar results were obtained during the analysis of human-specific and primate-specific CTCF-binding sites indicating that creation rates of novel CTCF-binding sites were 4.2-fold higher in humans compared with Chimpanzees . AssuminAnalysis of rates of creation of novel primate-specific and human-specific TF-binding sites indicates that there was a marked acceleration of novel TF-binding site creation in genomes of Chimpanzee and Modern Humans compared with rodents, which may have been associated with the increased activity of primate-specific retrotransposons during evolution. Consistent with this hypothesis, analysis of sequence conservation of 130 genomic loci encoding LTR7 RNAs in individual genomes of three Neanderthals and five modern humans reveals that 1) no LTR7 RNA-encoding sequences are present in Neanderthals’ genomes and 2) overall evolutionary conservation patterns of 130 LTR7 RNA-encoding loci and 826 human ESC-specific NANOG-binding sites are strikingly similar . In contSeveral additional lines of experimental evidence suggest that likely candidate retrotransposons with markedly accelerated activity may be found among HERVs, in particular, LTR7/HERVH family. The LTR7 subfamily is rapidly demethylated and upregulated in the blastocyst of human embryos and remains highly expressed in human ES cells . In humaIn human ESC, 128 LTR7/HERVH loci with markedly increased transcription were identified . These gAnalysis of estimated creation rates of novel TF-binding sites during evolution indicates that there was a marked acceleration of TF-binding site creation in primates’ genomes compared with rodent. This preliminary analysis reveals that estimated creation rates of novel NANOG-binding sites appear increased approximately 283-fold in the ESC genomes of Modern Humans compared with the Mouse ESC genome. During the most recent evolutionary period this phenomenon seems linked to the increased activity of HERVs, in particular, LTR7/HERVH family.At the nanoscale level, genomic DNA molecules are highly dynamic. DNA double helix continually acquires a transitory protein-less state due to highly dynamic nature of interactions with nucleosome core and linker histones. Methylation-associated DNA editing (MADE) alters the conventional double helix by replacing Watson–Crick base pairs with less energetically efficient noncanonical base pairing, creating nucleotide mismatches and nucleotide evictions at selected sites . Recent experiments have shown that structural changes of DNA sequences resembling MADE markedly enhance the responsiveness of DNA to invading DNA and noncoding RNA molecules  and these properties of “edited” DNA double helix can be exploited to engineer next-generation nanosensors, DNAzymes, and DNA motors . InvadinEnergetic biases introduced by MADE change kinetics of strand displacement reactions to favor creation of a thermodynamically-stable PSDS with single-stranded DNA exposed for interactions with regulatory proteins for extended time periods. Importantly, more than 90% of TF-binding sites, including binding sites for NANOG, POU5F1, and CTCF, are represented by the single-stranded DNA sequences . TherefoAccording to this model, sequence-specific interactions of LTR7 non-coding RNAs with DNA would induce thermodynamically-stable PSDSs and transition to triple-stranded DNA/RNA hybrid complexes with exposed single-stranded DNA chains. Binding of single-strand-specific TFs, such as NANOG, POU5F1, and CTCF, to the exposed single-stranded DNA sequences would lead to stabilization of a PSDS and maintenance of the open chromatin architecture in the vicinity of selected genomic loci. TEs are primary targets of MADE, because DNA methylation plays crucial roles in the inactivation of transposons to protect mammalian genomes from their harmful mutational activity . Accordisupplementary figs. S9–S11, Supplementary Material online). Consistent with this model, statistically significant placement enrichment of human-specific NANOG-binding sites in close proximity to the genomic coordinates of protein-coding genes regulating brain size that are rapidly evolving in primates, pluripotency lncRNAs, functional hESC enhancers, and EDEs was observed.The proximity placement enrichment model does not require that all primate- and human-specific TF-binding sites function in a manner similar to the classic promoter, enhancer, repressor, or insulator elements. This indicates that 33–47% of the excess NANOG and/or POU5F1 proteins  that areTEs contributed to the creation of thousands new species-specific TF-binding sites in mammals . Computasupplementary fig. S5, Supplementary Material online). These data provide a relatively short list of defined genetic loci, including candidate human-specific regulatory elements and their putative genomic targets, which may be functionally linked and should be useful for design of follow-up mechanistic experiments.Phylogenetic conservation can be considered as strong evidence for an important functional role of genetic regulatory elements. However, as species-specific regulatory events are by definition not conserved, it is difficult to differentiate the biological significance of such events from mere background noise without conducting detailed functional experiments. However, a total of 84,163 identified herein primate-specific sequences harboring putative TF-binding sites are present in the reference genome databases of humans and chimpanzees, suggesting that they were created before the Modern Humans/Chimpanzees split 13 Ma and conserved during divergent evolution of Modern Humans and Chimpanzees. Genome-wide proximity placement analyses of 826 human ESC-specific NANOG-binding sites revealed apparently nonrandom placement and significant enrichment near distinct genomic elements, including PMDs, N-HMDs, functional hESC enhancers, enhancer/coding gene regulatory pairs, asymmetrical 5hmC sites, pluripotency lncRNAs, EDEs, and protein-coding genes regulating brain size in humans that show rapid evolution among primates. Multiple sequence alignment analysis identified motif logos of the TE-embedded NANOG-binding sites, which closely resemble previously reported consensus binding sequences of the POU5F1 (OCT4) and NANOG TFs . These results suggest that genomic regions that are hypermethylated in hESCs (PMD domains) may provide a permissive chromatin environment for emergence of human-specific regulatory sites. PMD domains are significantly enriched in genes that are overexpressed in hESCs compared with differentiated cells, indicating that the chromatin state within these regions is permissive for high transcriptional activity in hESCs  and passive retrotransposition, DNA methylation of TE-derived sequences, and mC deamination and hydroxylation may facilitate the emergence of human-specific regulatory networks. The placement and/or retention of candidate human-specific NANOG-binding sites within regulatory networks seems to favor close proximity to the genomic coordinates of protein-coding genes regulating brain size that are rapidly evolving in primates, pluripotency lncRNAs, functional hESC enhancers, and a subset of EDEs .


Aire) mutation-induced autoimmune-polyendocrinopathy-candidiasis ectodermal dystrophy. However, the mechanisms and clinical implications of the aging-induced increased release of autoreactive T cells are not clear, especially since not all elderly individuals develop specific autoimmune diseases.It is well known that age-related involution (shrinkage) of the thymus  results in decreased output of naïve T cells . The insMost aged individuals possess low-grade, but above baseline, sustained pro-inflammatory factors, such as interleukin-6 and -1, tumor necrosis factor alpha, and C-reactive protein. This chronic inflammatory state, associated with the aging process termed “inflammaging”, has been implicated in the poor prognosis of virtually every age-related disease. Studies have shown that inflammaging can be attributed to senescent cell-induced “senescence-associated secretory phenotype” , and the+FoxP3+ T regulatory cells (Tregs). Firstly, using a FoxN1 conditional knockout (cKO) mouse model of accelerated thymic involution [Rag2-GFP reporter mouse , it was demonstrated that RTEs derived from the involuted thymus adopt an autoreactive phenotype. Generally, RTEs are not in a highly active state because they have not yet encountered their cognate antigens. The increased activation of CD4+ and CD8+ RTEs from the involuted thymus in the absence of infection suggests that these RTEs are autoreactive T cells that are responding to peripheral self-antigens. To confirm this, an IRBP  immunization model was used to amplify and detect autoreactive T cell clones within a polyclonal T cell repertoire. Mice with an involuted thymus showed a significant expansion of activated IRBP specific T cells, while they were mostly undetectable in littermate controls with a normal thymus.The study  focuses volution  crossed + and FoxN1-cKO background into the kidney capsule of young adult OT-II TCR-transgenic mice. RIP-mOVA is a transgene, which expresses chicken ovalbumin as a neo-self-antigen in an Aire-dependent manner in the thymic medulla where negative selection takes place. The OT-II TCR transgenic mice have CD4+ thymocytes that specifically recognize RIP-mOVA. Once these OT-II TCR transgenic thymocytes encounter RIP-mOVA+ medullary thymic epithelial cells (mTECs), they are signaled to undergo exaggerated negative selection. However, when bone marrow progenitor cells from OT-II TCR transgenic hosts were seeded into the grafted RIP-mOVA+/FoxN1-cKO thymus in the kidney capsule, the exaggerated negative selection was not observed. Furthermore, mTECs from FoxN1-cKO mice were found to have diminished expression of Aire, suggesting that impaired clonal deletion likely results from defects related to Aire-dependent promiscuous gene expression of tissue-specific self-antigens. This finding confirmed that negative selection is impaired in the involuted thymus.It is well-known that most autoreactive T clones are eliminated through thymocyte negative selection. Therefore, the question arises as to why so many autoreactive T clones are released from the involuted thymus. Negative selection in the involuted thymus was then examined using a triple genetically engineered mouse model. This was done by transplanting a newborn mouse thymic lobe from a RIP-mOVARag gene knockout mice, which do not have endogenous T and B cells. The age-related Treg accumulation and enhanced survival via diminished expression of the pro-apoptotic Bim gene in the Tregs could be reversed by the young microenvironment, while the inflammatory cell infiltration into non-lymphoid tissues could not. These data imply that changes in peripheral aged Tregs are non-intrinsic, while inflammatory infiltration is driven by intrinsic defects related to perturbed negative selection.In a healthy individual, the escape of a few autoreactive T cells from negative selection is not necessarily an issue because thymus-derived Tregs are able to suppress the autoimmune response . The stuTogether, these findings demonstrate that age-related thymic involution is involved not only in immunosenescence associated with the insufficient output of naïve T cells, but also in the emergence of inflammaging via the increased release of autoreactive T cells. Therefore, therapeutically targeting thymic involution should present a promising strategy for attenuating chronic inflammation, thereby reducing a major risk factor associated with morbidity and mortality in virtually every chronic age-related disease.


Ninety-four percent of mammalian protein-coding exons exceed 51 nucleotides (nt) in length. The paucity of micro-exons (≤ 51 nt) suggests that their recognition and correct processing by the splicing machinery present greater challenges than for longer exons. Yet, because thousands of human genes harbor processed micro-exons, specialized mechanisms may be in place to promote their splicing. Here, we survey deep genomic data sets to define 13,085 micro-exons and to study their splicing mechanisms and molecular functions. More than 60% of annotated human micro-exons exhibit a high level of sequence conservation, an indicator of functionality. While most human micro-exons require splicing-enhancing genomic features to be processed, the splicing of hundreds of micro-exons is enhanced by the adjacent binding of splice factors in the introns of pre-messenger RNAs. Notably, splicing of a significant number of micro-exons was found to be facilitated by the binding of RBFOX proteins, which promote their inclusion in the brain, muscle, and heart. Our analyses suggest that accurate regulation of micro-exon inclusion by RBFOX proteins and PTBP1 plays an important role in the maintenance of tissue-specific protein–protein interactions. Most vertebrate pre-mRNA are divided into short exonic sequences separated by longer intronic stretches that are removed during mRNA maturation. Although alternative splicing of exons allows multiple protein isoforms to be produced from the same gene, many isoforms appear to lack functional roles, owing to their low evolutionary conservation and expression levels . ConsequExons exhibit widely diverse characteristics and functions. They differ greatly in their nucleotide composition , inclusiIn contrast to ordinary exons, ultra-short exons are relatively uncommon and often have unknown functional roles. Several observations lend support to the hypothesis that micro-exons (exons of length ≤ 51 bp) are difficult for the splicing machinery to process and, consequently, that there is significant selective pressure on exon lengths to remain longer than 51 nt. Here, we study exons 51 nt or shorter because of their increased tendency to be skipped in mature transcripts, possibly because molecular crowding between large multimeric complexes cause steric hindrance that inhibits spliceosome assembly . Indeed,KDM1A is regulated by PTBP1 (polypyrimidine tract binding protein) in a brain-specific manner  to show that thousands of micro-exons are highly conserved across vertebrates and mammals at the sequence and exon inclusion levels, respectively. Analysis of 7949 brain-expressed micro-exons revealed that constitutively spliced (CS) micro-exons possess strong genomic signatures predicted to facilitate splicing, including stronger splice-site motifs, shorter flanking introns, and a higher density of exonic splicing enhancers compared to longer exons. In contrast, alternative spliced micro-exons are flanked by highly conserved intronic flanks that harbor intronic splicing enhancers including RNA motifs bound by RBFOX and PTBP1 proteins. We found that micro-exons regulated by RBFOX proteins are characterized by weaker splice sites, longer flanking introns and lower exonic splicing enhancer densities than other micro-exons. These observations indicate that RBFOX proteins can facilitate the splicing of micro-exons. We also found that PTBP1 likely regulates the inclusion of micro-exons, possibly by repressing the inclusion of micro-exons that are enhanced by RBFOX proteins and other splicing factors. Our analysis is the first, to our knowledge, to provide examples of how alternatively spliced (AS) micro-exons can impact cellular functions either by affecting post-transcriptional regulation or by regulating protein–protein interactions through changes in protein tertiary structure.Here, we provide a comprehensive characterization of micro-exons at the DNA conservation, RNA-splicing, and protein tertiary structure levels. We analyzed more than 57 billion reads from 901 human and mouse RNA-seq libraries including 25 postmortem human brain samples across development , 345 samTLN1 that is conserved in mammals . However, > 20% of micro-exons shorter than 3 nt mapped exactly to multiple positions within intronic sequences (Methods). Although it may be possible to use intron conservation to predict AS micro-exons that are shorter than 6 nt, they were discarded from further analysis because we expected 6- and 9-nt micro-exons, which we were able to detect, to possess similar splicing mechanisms as 3-nt micro-exons.A total of 12,835 Ensembl-annotated internal micro-exons were identified in protein-coding genes . To compCACNA1A gene  micro-exons discovered, several among our set of 310  lie within genes previously associated to human diseases or other genetic traits. After discarding 30 exons that were previously annotated in three additional databases , 64 novel micro-exons were contained in genes linked to at least one disease in the Online Mendelian Inheritance in Man database . Among mA1A gene . This enA1A gene . Accordi2 fold differences of 0.55–4.00) compared to TopHat2 and STAR . OLego’s performance was similar to ATMap’s (median log2 fold differences of 0.48–0.92). We therefore used ATMap to quantify the usage of novel and previously annotated micro-exons across all 901 RNA-seq samples (Methods).Nearly half (136 or 43.9%) of our novel micro-exons were 6–21 nt in length compared to only 12.3% (1575) of Ensembl-annotated micro-exons. This prompted us to investigate the ability of algorithms to detect and accurately map RNA-seq reads onto micro-exons. Mapping RNA-seq reads directly onto the genome is computationally difficult owing to a large search space for small exons. We expected reads spanning micro-exons (and thus three or more exons) to further complicate the mapping procedure. We therefore compared the ability of several RNA-seq aligners, including STAR , TopHat2We quantified the splicing inclusion ratios of micro-exons across all RNA-seq samples assigning PSI (percent spliced-in) values for each micro-exon and sample. Given our initial assumption that micro-exons tend to be skipped, we observed a surprisingly large proportion of micro-exons (∼81%) that were constitutively spliced across all tissues  . HoweverCPEB4, encoding a key RNA-binding protein that controls cytoplasmic polyadenylation     . Further U test)  score (5 U test) . Notably U test)  was signMethods) . DiffereWe also observed an increased thymine content 5–20 bp upstream of CS micro-exons compared to other classes of exons . When we calculated the median nucleotide content, we found a higher proportion of thymine immediately upstream of CS micro-exons (but not of AS micro-exons) immediately (5–20 bp) upstream of the start site. This increase in thymine content does not extend further into the upstream intron . Consequently, we expect that the polypyrimidine tracts are located downstream from the branch point for CS micro-exons. In contrast, AS micro-exons showed lower proportions of thymine content immediately upstream, but appeared to possess higher thymine contents > 20 nt into the upstream intron, and overall. These observations are consistent with the notion that different splicing mechanisms contribute to CS and AS micro-exon regulation.Because higher thymine and cytosine content can strengthen polypyrimidine tracts and enhance splicing, we searched for thymine-rich motifs within a compendium of RNA-binding motifs  that areRhesus macaque, cattle, dog, and mouse (Methods). Splicing motifs are generally 4–10 nt in length (< 1.0). In the 5′ intronic flank of AS micro-exons, CS micro-exons, and all AS exons, several pyrimidine-rich motifs were found to be overrepresented  is a well-characterized motif that is bound by RBFOX splicing factors . We therNext we hypothesized that AS micro-exons are regulated by RBFOX binding events in their intronic flanks, which possibly act by enhancing splicing. Indeed, we found an unexpectedly higher density of experimentally determined RBFOX binding events near AS micro-exons, most prominently in their 3′ flanks. We also found that this density is even higher for the 145 AS micro-exons that are brain specific. To do this, we first obtained CLIP-seq replicate data sets for all three RBFOX family members  from mouse brains . We thenThe higher density of RBFOX binding events in the 3′ intronic flanks compared to the 5′ intronic flanks of AS micro-exons encouraged us to investigate the putative role of RBFOX proteins in enhancing micro-exon splicing. This is because binding of RBFOX proteins downstream from alternatively spliced exons is known to enhance exon inclusion . FurtherTo further understand the connection between RBFOX proteins and micro-exon splicing, we analyzed 521 AS exons that were either predicted to be enhanced (410 micro-exons) or repressed (111 micro-exons) in the brain by the proximal binding of RBFOX . We founKDM1A was repressed by the polypyrimidine tract binding protein 1 (PTBP1): Knocking down PTBP1 resulted in a higher inclusion of the 12-nt micro-exon. Similarly to the RBFOX CLIP-seq study, our analysis of this PTBP1 CLIP-seq data from HeLa cells revealed a higher density of binding near AS micro-exons . Furthermore, the signal enrichment immediately upstream of AS micro-exons is consistent with binding to the polypyrimidine tract. To further investigate the regulation of micro-exons by PTBP1, we obtained a RNA-seq data set consisting of wild-type and Ptbp1 knockdown mouse N2A neuroblastoma cell line samples  of which a vast majority  increased in inclusion following Ptbp1 knockdown. PTBP1, unlike RBFOX proteins, may therefore regulate micro-exons by repressing their inclusion either directly by binding to the polypyrimidine tract or indirectly by regulating other splice factors.Owing to several pyrimidine-rich motifs conserved upstream of AS micro-exons, we also investigated the possibility that micro-exons were regulated by the binding of splice factors to their polypyrimidine tracts. Previously,  samples . After m samples . We thenP-value < 6.7 × 10−5; hypergeometric test). This suggests that PTBP1 may act coordinately with RBFOX proteins and perhaps other splicing factors to accurately regulate micro-exon inclusion. When we compared the intron length, splice-site strength, and exonic splicing enhancer density of RBFOX- and PTBP1-regulated micro-exons, we found a general trend of “weakened” splicing features compared to other micro-exons  and possibly those of AS exons . The density of exonic splicing enhancers was also significantly lower for PTBP1-regulated micro-exons . The binding of factors to these intronic splicing enhancers alone may therefore compensate for the difficulty in processing very short exons.We next wished to study the genomic features and inclusion of micro-exons regulated by RBFOX proteins and PTBP1. To this end, we projected the 171 micro-exons enhanced by RBFOX and the 129 micro-exons repressed by PTBP1 from the mouse genome to the human genome that resulted in 159 and 113 human RBFOX- and PTBP1-regulated micro-exons, respectively. Interestingly, if we assume that all 1480 AS micro-exons may be targeted by RBFOX proteins and by PTBP1, the number of micro-exons predicted to be both enhanced by RBFOX proteins and repressed by PTBP1 (25) is significantly larger than expected by chance  encoded peptides that were preferentially situated within intrinsically disordered regions of proteins  compared to CS micro-exons (46.4%–49.0%) and longer exons  . RemarkaTo explore whether AS micro-exons could alter protein–protein interactions, we searched for AS micro-exons that encode residues from domains known to interact with other proteins. Among genes with conserved AS micro-exons with coding potential, we identified a paralogous gene family consisting of three amyloid binding proteins: APBB1, APBB2, and APBB3. All three APBB  genes possess 6-nt micro-exons encoding two amino acids located within the first of two phosphotyrosine-binding (PTB) domains. Searching cDNA databases revealed that the micro-exons within APBB are also present and alternatively spliced in fishes, in chicken, and in diverse mammals . These mTo investigate the functional impact of these micro-exons on protein structure, we mapped the two amino acid residues encoded by the micro-exon onto the known 3D structure of APBB2 . This shThe splicing process requires a large number of steps, each of which depends on multiple proteins and genome-encoded signals . PreviouIt has become increasingly clear over recent years that splicing is tightly linked to human diseases , and it Based on previous studies , we expeDespite the presence of compensatory features for a large class of 6469 CS micro-exons, we observed that the 1480 AS micro-exons generally did not possess such splicing-enhancing features. Instead, they were characterized by highly conserved intronic flanks that harbor conserved intronic splicing enhancers. We identified RNA-binding proteins, RBFOX proteins, and PTBP1, which act as preferential regulators of micro-exons. Indeed, we confirmed that RBFOX proteins and PTBP1 bind with higher affinity near AS micro-exons compared to other exons. In addition to regulating inclusion ratios of micro-exons, RBFOX proteins appear to also enhance the splicing efficiency of micro-exons. We observed that micro-exons that are predicted to be targets of RBFOX proteins or PTBP1 possess weaker splicing features than other AS or CS micro-exons. This observation, coupled with the finding that most micro-exons appear to require compensatory genomic features to maintain splicing, suggests that the splicing-enhancing activity of RBFOX proteins alone is able to guarantee efficient splicing. Our results that micro-exons targeted by PTBP1 also possessed weak splicing signals were somewhat surprising. This is because PTBP1 generally functions as a splicing repressor, possibly through competition with U2AF2 to bind the polypyrimidine tracts of micro-exons. However, we found that 25 of the 113 PTBP1 targets were also predicted to be enhanced by RBFOX proteins. Given that the set of exons predicted to be targets of RBFOX proteins is likely a subset of all RBFOX targets , it is pAPBB gene family and all four members of the tensin (TNS) gene family possess alternatively spliced micro-exons mapping to their beta-turn loops. The alternative inclusion or exclusion of these micro-exons lengthens or shortens the beta-turn loop, and is therefore expected to alter protein–protein interactions: APBB with amyloid-beta protein and TNS with beta integrin. In light of these findings, we propose that alternate use of micro-exons often alters local binding-site specificities, whereas the inclusion of longer AS exons may more substantially alter global protein structure. Although we show that several AS micro-exons are likely to regulate protein–protein interactions, we believe it is premature to conclude that this is their canonical function. In fact, it is likely that micro-exons also function through other mechanisms. For example, a 13-nt micro-exon that is alternatively included near the 3′ end of Arl6 transcripts was found to be required for vision in mammals  and micro-exon splicing in the context of both splicing regulation and brain-related disorders.http://www.ebi.ac.uk/ena/]; accession number ERA022994); mouse transcriptome data were obtained from This study uses transcriptome data in the form of 76-bp paired-end RNA-seq reads from 345 postmortem prefrontal cortices, 74 muscle samples, 47 nerve samples from human, and up to 10 samples for other tissues within the GTEx , 76-bp pTo identify novel micro-exons from RNA-seq data, 307 brain samples, 74 muscle, and 47 nerve samples from GTEx were mapped onto Ensembl (release 70)  cDNA traRL and RR, representing the number of reads supporting the left and right junction, respectively, were then computed by counting the number of reads that span each junction. According to these quantities, the number of reads supporting each micro-exon, totR, was then computed using the following equation:Rtot, the percent spliced-in statistic was computed for each micro-exon: tot/(Rtot+Rskipped)R, where skippedR represented the number of reads supporting an exon skipping event.An in-house pipeline was developed in order to quantify micro-exon usage. A transcriptome augmented by alternatively spliced micro-exons was created as follows: (1) All micro-exons were identified from an annotation file, (2) for each transcript, we constructed a version with the micro-exon(s) included and another without, and (3) in cases where there were multiple micro-exons in the same gene, we constructed transcripts representing all possible combinations of micro-exon inclusion/exclusion for those that are 100 nt within one another. Step (3) was important for quantifying alternatively spliced micro-exons located in tandem . RNA-seq data sets were then mapped to this augmented transcriptome using BWA , allowing at most two mismatches per read. The quantities To compare our in-house pipeline to STAR , TopHat2Brain-specific micro-exons were defined to be those that have a median PSI of at least 25 in GTEx brain samples, and an 80 percentile of at most 10 PSI in other samples, excluding samples from pituitary gland (due to their relatedness to brain). A total of 145 brain-specific micro-exons were identified using these thresholds, but with 120–200 using different cut-offs.To identify a set of control exons to allow comparison with micro-exons analyzed in this study, 100 samples from the GTEx brain were chosen randomly and analyzed. Spliced reads were recovered from each of the samples and the PSI of each internal exon was computed in the same way as for internal micro-exons. Exons with median PSI at least 10% and at most 90% were classified as alternatively spliced, while exons with PSI higher than 90% were classified as constitutively spliced.We explored several PSI criteria for defining AS: 5%, 10%, 15% as the lower bound and 85%, 90%, 95% as the upper bound. Our first observation was that the qualitative interpretation of our results was independent of the criteria used. Our second observation, however, was that exons (both long and short) with median PSI 5%–10% in our samples were less conserved  than exons with higher median PSI. This motivated us to use 10% PSI as a lower bound.S denote a set containing vectors of size 300, each representing the phastCons score of one exon at each of the 300-nt positions, (2) draw randomly with replacement |S| vectors from this set, (3) compute the average conservation profile Si, (4) repeat this process 1000 times, obtaining {Si: 1 ≤ i ≤ 1000}. The lower and upper values of the confidence interval for each position 1 ≤ k ≤ 300 correspond to the 5- and 95-quantiles of {Si(k): 1 ≤ i ≤ 1000}, respectively.To characterize the sequence conservation of micro-exons and their intronic flanks, micro-exons were centered within 300-nt windows and vertebrate phastCons scores  were retrieved for each position. phastCons confidence intervals for different classes of exons were then computed by bootstrapping as such: (1) Let To compute the percent identity between human brain-expressed exons and orthologs in dog, mouse, platypus, and chicken, the internal human exons and their intronic flanks were aligned to corresponding orthologous sequences. The percent identity between human and the other species was then computed for 100 nt of intronic sequences flanking the 5′ and 3′ ends of each exon. Additionally, the percent identity of 12 nt from the exon (6 nt from both the 5′ end and 3′ end) was also computed to remove biases from differing exon lengths.k-mers, all regions consisting of human internal exons plus 500 nt intronic flanking sequences (250 nt from each flanking intron) were collected. These regions were projected onto the genomes of rhesus monkey, cattle, mouse, and dog using liftOver, and were then aligned using MUSCLE  and mapped using BWA  to the mTo identify motifs that were enriched upstream of CS micro-exons, the binding affinity of 131 human RNA-binding proteins (RBP) were computed using a sliding window of 7 bp, and were based on position weight matrices (7 nt) derived from experimental data . We testAll 8891 protein sequences containing a brain-expressed internal exon studied here were collected and disordered regions were predicted using DISOPRED . The dishttp://www.pymol.org/). Structural superposition of PTB domains was performed using Dali (To identify micro-exons overlapping protein domains, HMMSCAN  was useding Dali .


Hypertension is an independent predictor of acute kidney injury (AKI) in non-cardiac surgery patients. There are a few published studies which report AKI following non-suprainguinal vascular procedures, but these studies have not investigated predictors of AKI, including anti-hypertensive medications and other comorbidities, in the hypertensive population alone. We sought to identify independent predictors of post-operative AKI in non-suprainguinal vascular surgery patients with a pre-operative history of hypertension. We performed univariate  and multivariate (binary logistic regression) statistical analysis of prospectively collected data from 243 adult hypertensive patients who underwent non-suprainguinal vascular surgery  at a tertiary hospital between 2008 and 2011 in an attempt to identify possible associations between comorbidity, acute pre-operative antihypertensive medication administration, and post-operative AKI (a post-operative increase in serum creatinine of ≥ 25 % above the pre-operative measurement) in these patients. The incidence of post-operative AKI in this study was 5.3 % . Acute pre-operative β-blocker administration was independently associated with post-operative AKI in non-suprainguinal vascular surgery patients with a pre-operative history of hypertension . The acute pre-operative administration of β-blockers should be carefully considered in non-suprainguinal vascular surgery patients with a pre-operative history of hypertension, in lieu of an increased risk of potentially poor post-operative renal outcomes.  Acute kidney injury (AKI) is a significant cause of in-hospital morbidity and mortality in hospitalized patients  prospective database of adult patients who underwent elective vascular surgery at a tertiary hospital located in Durban, South Africa between 2008 and 2011 . We conpatients who did not undergo non-suprainguinal vascular surgery , patients with missing peri-operative serum creatinine measurements, patients with pre-operative renal dysfunction, as defined in the study by Abelha and colleagues  as a prpatients without a history of hypertension Figure 1. Data elements collected from patient medical records included demographic information (age and gender), comorbid conditions , acute pre-operative anti-hypertensive medication use , and peri-operative laboratory test results (pre- and post-operative serum creatinine measurements). The definitions of comorbid conditions used in this study were adopted from the study of Lee and colleagues . In thiUnivariate statistical methods used to analyse data included chi-squared and Fisher's Exact testing, where appropriate. For the multivariate statistical analysis, a binary logistic regression model was used to identify independent predictors of post-operative AKI in non-suprainguinal vascular surgery patients with a pre-operative history of hypertension. The clinical variables entered into the logistic regression equation were purposefully selected, with variables attaining a significance level of p <0.1 at the univariate analysis level being included in the regression analysis. Results for the univariate data analysis are presented as frequencies and percentages, whilst results for the binary logistic regression analyses are presented as odds ratios (OR) with 95 % confidence intervals (95 % CI). Any p-value < 0.05 was considered to be a statistically significant result. All statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS) version 21 .The process by which patients were selected for inclusion in this study is shown in Figure 1When stratified by post-operative AKI status, no statistically significant univariate associations were observed between patient demographic factors, comorbidities and post-operative AKI Table 1. The poiFive of every 100 hypertensive patients undergoing non-suprainguinal vascular surgery in this study suffered post-operative AKI. The incidence of post-operative AKI in our study was lower than that reported for two other studies of non-suprainguinal vascular surgery, which reported the incidence of post-operative AKI to be 12 % and 12.7 % , we didWe found acute pre-operative βB administration to be associated with a 3-fold increased risk of developing post-operative AKI in this study. In another study, we had reported that acute pre-operative βB administration was associated with a higher risk of peri-operative troponin leak and peri-operative major adverse cardiovascular events in vascular surgery patients with a pre-operative history of hypertension . In thaIn conclusion, acute pre-operative β-blockade was associated with a higher risk of post-operative AKI in non-suprainguinal vascular surgery patients with a pre-operative history of hypertension. Although this research requires validation in a larger cohort, our findings suggest that the acute pre-operative administration of βB should be carefully considered in non-suprainguinal vascular surgery patients who have a pre-operative history of hypertension.This study was funded by a South African Medical Research Council self-initiated research grant awarded to Bruce M. Biccard. This work forms a component of the doctoral studies of Yoshan Moodley, who is the recipient of a doctoral scholarship awarded by the South African National Research Foundation (NRF). Yoshan Moodley was also supported by the Columbia University-Southern African Fogarty AIDS International Training and Research Program (AITRP), Implementation Science Scholarship Program funded by PEPFAR through the Fogarty International Center, National Institutes of Health (grant # D43TW000231).The authors declare that they have no conflict of interest.


Multiple sclerosis (MS) is a debilitating chronic inflammatory disease of the nervous system that affects approximately 2.3 million individuals worldwide, with higher prevalence in females, and a strong genetic component. While over 200 MS susceptibility loci have been identified in GWAS, the underlying mechanisms whereby they contribute to disease susceptibility remains ill-defined. Forward genetics approaches using conventional laboratory mouse strains are useful in identifying and functionally dissecting genes controlling disease-relevant phenotypes, but are hindered by the limited genetic diversity represented in such strains. To address this, we have combined the powerful chromosome substitution (consomic) strain approach with the genetic diversity of a wild-derived inbred mouse strain. Using experimental allergic encephalomyelitis (EAE), a mouse model of MS, we evaluated genetic control of disease course among a panel of 26 consomic strains of mice inheriting chromosomes from the wild-derived PWD strain on the C57BL/6J background, which models the genetic diversity seen in human populations. Nineteen linkages on 18 chromosomes were found to harbor loci controlling EAE. Of these 19 linkages, six were male-specific, four were female-specific, and nine were non-sex-specific, consistent with a differential genetic control of disease course between males and females. An MS-GWAS candidate-driven bioinformatic analysis using orthologous genes linked to EAE course identified sex-specific and non-sex-specific gene networks underlying disease pathogenesis. An analysis of sex hormone regulation of genes within these networks identified several key molecules, prominently including the MAP kinase family, known hormone-dependent regulators of sex differences in EAE course. Importantly, our results provide the framework by which consomic mouse strains with overall genome-wide genetic diversity, approximating that seen in humans, can be used as a rapid and powerful tool for modeling the genetic architecture of MS. Moreover, our data represent the first step towards mechanistic dissection of genetic control of sexual dimorphism in CNS autoimmunity. MS prevalence is ~3 fold higher in females, although the disease course in men typically presents as a more rapid severe progressive disease , incidence , days affected (DA), cumulative disease score (CDS), severity index (SI), and peak score .(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.


Oncolytic viruses represent a diverse class of replication competent viruses that curtail tumor growth. These viruses, through their natural ability or through genetic modifications, can selectively replicate within tumor cells and induce cell death while leaving normal cells intact. Apart from the direct oncolytic activity, these viruses mediate tumor cell death via the induction of innate and adaptive immune responses. The field of oncolytic viruses has seen substantial advancement with the progression of numerous oncolytic viruses in various phases of clinical trials. Tumors employ a plethora of mechanisms to establish growth and subsequently metastasize. These include evasion of immune surveillance by inducing up-regulation of checkpoint proteins which function to abrogate T cell effector functions. Currently, antibodies blocking checkpoint proteins such as anti-cytotoxic T-lymphocyte antigen-4 (CTLA-4) and anti-programmed cell death-1 (PD-1) have been approved to treat cancer and shown to impart durable clinical responses. These antibodies typically need pre-existing active immune tumor microenvironment to establish durable clinical outcomes and not every patient responds to these therapies. This review provides an overview of published pre-clinical studies demonstrating superior therapeutic efficacy of combining oncolytic viruses with checkpoint blockade compared to monotherapies. These studies provide compelling evidence that oncolytic therapy can be potentiated by coupling it with checkpoint therapies. Oncolytic viruses are biological agents which are increasingly being explored to treat cancer. These typically represent two classes of viruses, one with the intrinsic property to preferentially replicate in tumor cells, while leaving normal cells alone. Viruses represented in this class exploit defective anti-viral pathways in cancer cells or use proteins specifically expressed in cancer cells as virus entry receptors to establish infection. The other class represents genetically engineered viruses which encode genes that enhance the preferential tropism and replication of viruses within tumor cells. Oncolytic viruses are an attractive class of tumor therapy as these viruses selectively replicate within tumor cells; thereby, offering better specificity and safety profile. The safety and efficacy of a plethora of oncolytic viruses have been established in numerous pre-clinical studies which has advanced the progression of oncolytic viruses into clinical development . The fieOncolytic viruses typically encode for genes that induce oncolysis and immunogenic killing of tumor cells . There aVarious strategies to further promote onco-immunotherapy have been evaluated for anti-tumor efficacy. For example, oncolytic viruses encoding transgenes such as granulocyte macrophage colony-stimulating factor (GM-CSF), interferon (IFN)-β, interleukin (IL)-2, IL-15, IL-12, heat shock proteins have been engineered. These viruses expressing immune stimulatory genes have shown to boost immune priming via release of tumor antigens and by the expression of viral molecular patterns, to facilitate a robust adaptive T cell response .regs) [Cancer cells typically acquire genetic mutations and undergo epigenetic changes to circumvent growth suppressors and apoptotic machinery to promote proliferation and metastasis . Tumors regs) ,14 and mregs) , and actregs) ,17. Negaregs) . There aregs)  and cytoregs) , T cell regs)  and lympregs) ,23. Thesregs) ,25,26. Hregs) . Given tregs [Two widely studied inhibitory immune molecules, CTLA-4 and PD-1, share some similar features but differ in the mechanism of the action to limit T cell activation . T cellsregs .regs [In vitro studies have shown that ITSM motif can recruit tyrosine protein phosphatases, SHP1 and SHP2 [Similar to CTLA-4, PD-1 is a transmembrane protein absent on resting naïve and memory T cells. It is expressed on activated T cells upon TCR engagement and has two known ligands. PD-1 modulates T cell suppression by binding to its ligands, PD-L1  and PD-Lregs . Both CTregs . Throughregs . PD-1 abregs . PD-1 liregs . CTLA-4 regs . In contand SHP2  which arIn various pre-clinical studies, the efficacy of antibodies blocking CTLA-4 or antibodies against PD-1 and PD-L1, which target the PD-1/PD-L1 axis, has been demonstrated ,47,48. TWe are only beginning to appreciate the mechanisms of action these antibodies employed to effectively control tumors. In the past few years, data from several clinical studies have provided insight into the characteristics of patients who respond and perhaps, more importantly, who do not respond to therapies ,56. FromThe combination of oncolytic with immune checkpoint modulators has several advantages. First, by combining oncolytic therapy with checkpoint blockade, multiple immune pathways inducing immune tolerance during cancer progression can be thwarted. Second, oncolytic viruses infection itself could induce the up-regulation of CTLA-4  or PD-L1et al. was the first to provide pre-clinical data to support clinical exploration of the use of checkpoint antibodies with oncolytic Newcastle disease virus (NDV). The authors have used NDV, a negative strand RNA virus, that has the natural propensity to infect and replicate in tumors that have defects in type I IFN signaling [regs ratio. Through depletion studies, the authors further showed that the therapeutic efficacy was largely driven by the CD8 T cells, natural killer (NK) cells, and type I and II IFN indicating interplay between innate and the adaptive arms of the immune system to dictate therapy. The combination therapy offered better protection against tumor challenge and induced a robust memory response [versus T cell exhaustion.The study published by Zamarin ignaling . Anti-tuignaling . The autresponse . Additioet al. provided the first pre-clinical data to support the use of oncolytic vaccinia virus in combination with anti-CTLA-4 antibody for the treatment of melanoma [tk gene deletion and encoding granulocyte macrophage colony stimulating factor (GM-CSF) [Rojas melanoma . Vaccinimelanoma . Selectimelanoma . JX-594,(GM-CSF) , is exte(GM-CSF) ,75.et al. composed of one systemic dose of vaccinia followed by three intra-peritoneal doses of anti-CTLA-4 therapy [The pre-clinical regiment established by Rojas  therapy . This re therapy . The autet al. have used HER2/neu expressing breast cancer cell lines to demonstrate that wild type VSV promoted survival of mice with palpable tumors [Vesicular stomatitis virus (VSV) is an enveloped, negative strand RNA virus, and similarly to NDV, exploits defective IFN signaling pathways to selectively infect and induce apoptosis in tumor cells . It alsoe tumors . Howevere tumors .regs suppressive activity [Previous studies from our laboratory have highlighted the therapeutic potential of onco-immunotherapy using a systemically delivered VSV as a platform for expressing a complementary DNA (cDNA) library generated from normal/tumor tissue. We have shown that VSV-cDNA libraries are capable of vaccinating against TAAs expressed on tumors ,80. Furtactivity . These sCharacterization of oncolytic viruses engineered to encode antibodies against immune checkpoint has not been well explored until recently using measles virus. Measles virus is negative strand RNA virus and can cause highly contagious exanthemous measles disease . Howeveret al., B16 cell lines were generated to express CD20 to allow for measles virus entry and to study the therapeutic effects of measles virus to control melanoma growth in an immunocompetent mouse model [regs infiltration within the tumor [In a study published by Engeland se model . The authe tumor .Research in our laboratory is also focused on exploring strategies to boost reovirus oncolytic therapy. Reovirus is a double stranded RNA virus with direct oncolytic activity against a variety of tumor types. The anti-tumor activity is directly associated with innate immune activation by virus replication in tumors and generation of adaptive anti-tumor immune response ,87. In pet al. has shown that combining intra-tumoral reovirus (Reolysin) with systemic anti-PD-1 antibody delivery was far superior in establishing tumor control than either of the monotherapies [in vitro depletion studies demonstrated that the combination therapy was able to ablate the suppressive effects of Tregs, indicating that perhaps anti-PD-1 antibody affected Tregs biology [In a subcutaneous melanoma tumor model, Rajani herapies . Treatme biology . MoreoveOverall, pre-clinical data support that combination of oncolysis and systemic immune checkpoint inhibition leads to significant synergy between the two therapies to effectively establish tumor control and therapy is dependent on immune effectors. However, several issues regarding the use of immune checkpoint inhibitors in the context of onco-immunotherapy and their predicted therapeutic efficacy arise from these studies. First, the timing of administration of checkpoint inhibitors needs to be carefully considered. Administering checkpoint inhibitors early during oncolytic therapy may act to enhance the anti-tumor, adaptive T cell response to clear the tumor; however it may also de-suppress both innate and adaptive anti-viral response, which in turn, would restrict viral spread and oncolysis. In contrast, administration of checkpoint inhibitors later in the combination regiment would augment both anti-tumor immune response and anti-viral response to effectively control virus and tumor growth.et al. [et al. [The pre-clinical data by Gao et al.  and Roja [et al.  provide vs. systemic delivery of checkpoint inhibitors, evaluation of location specific expression of checkpoint targets is needed. Targeting checkpoint molecules which are expressed in lymph nodes, such as CTLA-4, may require systemic delivery of checkpoint antibodies. Intra-tumoral delivery may result in optimal therapy with checkpoint antibodies targeting PD-L1, which is expressed on many tumors [Next, one must consider the route of delivery when combining oncolytic therapy with anti-checkpoint inhibitors. To study the effects of local y tumors ,27 and iy tumors .et al. has shown that tumors in type I receptor knockout mice IFNAR1 -/-, were resistant to anti-tumor effects of NDV and anti-CTLA-4 [The third aspect entails delineating the role of type I IFN in the context of oncolytic therapy and checkpoint blockade. There are three types of IFN families. Type I IFNs are secreted in response to viral infections within cells and trigger a cascade of gene expression and signal transduction events to interfere with the virus replication cycle and elicit an anti-viral state . In addii-CTLA-4 , indicatTherefore, a number of factors including the tumor type, the oncolytic virus, the checkpoint inhibitor, the relative timing of administration of the agents, and the induction of immune responses, are likely to contribute to the overall therapeutic effects of immune checkpoint inhibitor therapy with oncolytic virus. Each oncolytic virus induces different TLR activators  and elicversus anti-tumoral immune mechanisms will provide insight into the basic biology of the immune system.In pre-clinical studies, it is important to systematically evaluate factors which may play complementary or opposing roles to define the feasibility and viability of each virus with immune checkpoint agents. In addition, studies designed to delineate anti-viral ICP34.5 and ICP47 genes deleted in order to enhance tumor selective replication and immune-stimulating capabilities of the virus [https://clinicaltrials.gov). This approval would further provide impetus for the clinical development and regulatory approval of other oncolytic viruses, which are in various phases of clinical development. There are numerous immune checkpoint inhibitors that are entering clinical development which target molecules other than CTLA-4 or PD-L1/PD-1. It would be interesting to see if similar synergy is established with antibodies targeting checkpoint molecules such as LAG-3, TIM-3, and B & T lymphocyte attenuator with oncolytic viruses. Given the recent pre-clinical studies, there is precedence that the onco-immunotherapy can be potentiated with checkpoint blockade to achieve superior patient outcomes. Given these developments, onco-immunotherapy is entering exciting prospects as we expect to see initiation of several clinical trials combining oncolytic viruses with checkpoint blockade. We are entering an exciting phase in the development of onco-immunotherapy, as the FDA recently approved the sales of talimogene laherparepvec (T-VEC), a herpes virus encoding GM-CSF for treating melanoma ,100. Thehe virus . The safhe virus ,102 and


Accumulating evidence suggests that gut microbiota affects brain development and its function. It is well known that compared with conventional mice (SPF), germ-free (GF) mice display higher exploratory behavior, which normalizes after bacterial colonization. However, little is known about the underlying mechanisms and first critical steps initiating microbiota-gut-brain communication, which lead to establishment of normal behavior.To investigate the role of immune system in the establishment of normal behavior after bacterial colonization.E. coli JM83, and compared them to SPF mice, using the light-dark preference and tail suspension tests. Levels of brain-derived neurotrophic factor (BDNF) and c-Fos expression were measured by immunofluorescence in the hippocampus and amygdala. Colonic and brain gene expression were assessed using a NanoString technology. The immunodeficient MyD88-/- Ticam1- and SCID mice were used to study the role of the innate and adaptive immune systems. To demonstrate the role of the dendritic cells (DCs), we measured behavior before and after mono-colonization with E. coli JM83 in GF mice treated with cosalane and fingolimod, that inhibit DCs activation and migration, respectively. Brain levels of CD11b, CD11c and CD103 as DCs markers was assessed by immunofluorescence.We assessed behavior in GF mice before and after colonization with SPF microbiota, Altered Schaedler Flora (ASF) or the single bacterial strain E. coli JM83 showed similar normalization of behavior as those colonized with SPF microbiota. Mono-colonization with E. coli reduced both BDNF and c-fos levels in the hippocampus and amygdala. While colonization of GF SCID mice induced same change in behavior as in wild-type mice, GF MyD88-/-Ticam1-/- mice did not alter their behavior. Mono-colonization affected multiple genes in the colon and the brain, associated with innate immunity and neural plasticity. Treatment with both cosalane and fingolimod prevented behavioral changes after colonization, which was paralleled by absence of CD11b+CD103+CD11c+ cells in the brain, otherwise found in high numbers in control mono-colonized mice and absent in germ-free mice.Compared to SPF mice, GF mice showed higher exploratory and less depressive-like behavior. The ex-germ-free mice colonized with ASF microbiota, or mono-colonized with The innate immune system, through activation and migration of intestinal dendritic cells into the brain, initiates the neuro-immune signaling within the gut-brain axis and leads to normalization of behavior after bacterial colonization. Our findings may impact several psychiatric conditions, in which altered innate immune signaling has been implicated.CIHR, OtherBalsam Family FoundationNone Declared


High-performance THz photodetection is unprecedentedly accessed by integrating a topological Dirac (Weyl) semimetal in a carefully designed antenna at deep-subwavelength scales. For example, III–V or group IV semiconductors, widely exploited in several optical devices, exhibit an energy bandgap out of the THz spectral range, and this specific strongly hampers the devising of active THz optoelectronic components.In recent years, there has been a surge of interest in the THz spectral regime (from 300 Hz to 30 THz) since efficient electronic and photonic materials are traditionally not available in this frequency range. Conventional electronic materials are too slowly responsive for THz optoelectronics, and mainstream optoelectronic and photonic devices  operating in the visible and infrared ranges cannot be trivially recast to the THz band owing to intrinsic physical constraints3. However, despite the THz responsivity, metamaterials  exhibit undesired deep-subwavelength spatial modulations4, which can be detrimental for miniaturized on-chip optoelectronic devices. An alternate fashion of technology-amenable THz materials was inspired by graphene5, closely followed up by three-dimensional (3D) topological insulators10, where linearly dispersing fermions can be excited by low-energy photons. Recently, a new frontier of topological materials has emerged. Especially, topological Dirac (Weyl) semimetals have attracted a good deal of attention since they exhibit topologically protected crossing points, termed Dirac (Weyl) nodes, between four-fold degenerate (two-fold non-degenerate) linearly dispersing energy bands in their 3D electronic structure11. Topological semimetals offer unprecedented perspectives to tune the light-matter interaction at extremely low photon energy (even down to the THz regime)13. In this context, a subclass of van der Waals solids consisting of transition-metal dichalcogenides TMX2 , plays an outstanding role since they may assume allotropic phases hosting Lorentz-violating (type-II) topological Dirac or Weyl fermions in bulk crystal14 and potentially in the form of thin-films on substrates15. In these materials, the Dirac (Weyl) nodes are no longer pinned to the high-symmetry points of the crystal lattice, and they emerge at the boundary between electron and hole pockets arising from band intersections with the Fermi surface.To overcome these limitations, research efforts are currently focused on novel THz materials with unusual and advantageous properties. THz metamaterials, designer-made composite materials showing a resonant response at the desired THz frequency, provide a suitable platform to manipulate THz radiation16, Wang et al. set a milestone in THz technology by proposing the integration of the type-II Dirac semimetal PtSe2 in a high-performance THz photodetector, realizing a suitable nanometric antenna layout , and high anisotropic response. On the other hand, as a semimetal, PtSe2 can suffer from limited photo-electric conversion within a deep-subwavelength area and bias-induced dark current leakage. To overcome these latter drawbacks, the Authors propose an asymmetric metallization confining the optically active nanogap slit in the sub-skin-depth regime (λ/104) within the antenna device architecture. Breaking the in-plane symmetry of the electrode layout effectively results in an enhanced funneling of low-energy photons and photo-induced conduction at zero bias. The considered PtSe2-based photodetector exhibit a colossal photo-response, viz., light absorption in the THz range (from 0.1 to 0.3 THz) with responsivity exceeding 0.2 A/W. In addition, noise-equivalent power (NEP) less than 38 pW/Hz, and temporal response of the order of 1 μs are achieved by coupling the PTSe2 with graphene, so as to alleviate the energy barrier at the contact. These figures of merit hold promise for PtSe2 as outstanding THz material and pave the way for integrating other topological materials in miniaturized on-chip optoelectronic devices. Taking the PtSe2 antenna as a prototypical device, new perspectives for devising ultra-efficient low-energy optoelectronic components can thus be one step closer to the realization stage and to the deployment of efficient and ready-to-use THz products that are advantageous for biomedical, remote sensing, and security applications.In ref.


Multisystem Inflammatory Syndrome (MIS) is a rare but increasingly recognized complication of SARS-CoV-2 infection, usually presenting 2 to 6 weeks after the onset of COVID-19 infection symptoms and affecting mainly children. However, there have been reported several cases of a similar multisystem inflammatory syndrome in adults (MIS-A). We describe the case of a previously healthy 28-year-old male who presented with a clinical profile with multiorgan involvement within four weeks after confirmed SARS-CoV-2 infection, suggestive for multisystem inflammatory syndrome (MIS-A). The clinical presentation included persistent high grade of fever, gastrointestinal and mucocutaneous lesions, lymphadenopathy, elevated cardiac and inflammatory biomarkers, cytopenia and shock. This case report illustrates the wide range of presentations, diagnosis, and treatment modalities of multisystem inflammatory syndrome. The pathophysiology and the mechanisms by which SARS-CoV-2 triggers an abnormal immune response leading to MIS remain poorly understood. Better characterization of MIS-A and early recognition of MIS is important because it is associated with high mortality if left untreated. Multisystem inflammatory syndrome (MIS) is a rare but severe, potentially life-threatening condition, presenting from 2 to 6 weeks after the onset of COVID-19 infection symptoms, and affecting mainly children ,4. The pThe patient was a 28-year-old previously healthy man presented to the emergency department in the Hospital of Lithuanian University of Health Sciences with high-grade fever (39–40 °C), persistent for five days, acute abdominal pain, vomiting and diarrhea, which, after 3 days, were followed by: maculopapular rash on patient’s trunk and extremities, neck swelling, lymphadenopathy and bilateral conjunctival injections. Six weeks prior to these clinical manifestations, the patient was infected with a mild SARS-CoV-2 infection and completely recovered within 1 week. After a few hours in the emergency department, he was admitted to an intensive care unit for treatment of hemodynamic instability. New-onset heart failure and vasoplegia that progressed into shock with multiorgan dysfunction were diagnosed. The patient received supportive treatment with dynamic hemodynamic-driven preload resuscitation, complex vasopressor and inotrope support. He developed a severe hypoxic respiratory failure due to pulmonary edema which required mechanical ventilation . LaboratTreatment at Day 2: intravenous immunoglobulin  IV; broad spectrum antibiotics ; inotropic and vasoactive treatment ; fluid resuscitation; respiratory support; continuous veno-venous hemofiltration (with sorbent adsorption); antiplatelet and anticoagulant therapies (prophylaxis). Treatment at Day 4: GCS was added, methylprednisolone  IV. Treatment at Day 7: anti-IL-6 monoclonal antibody was added, tocilizumab  IV; broad-spectrum antibiotics ; anticoagulants for PE (low molecular weight heparin—nadroparine 5700 anti Xa IU x2 SQ). IV—intravenous infusion; SQ—subcutaneous injection; IVIG—intravenous immunoglobulin; GCS—glucocorticosteroid; anti-IL-6 monoclonal antibody, PE—pulmonary embolism.Multisystem inflammatory syndrome (MIS) is a rare complication of SARS-CoV-2 infection, usually presenting from 2 to 6 weeks after the onset of the COVID-19 infection symptoms and affecting mainly children. It is now known that adult patients with current or previous SARS-CoV-2 infection can develop a hyperinflammatory syndrome ,13. The MIS of COVID-19 shares many similarities with other inflammatory diseases, such as Kawasaki disease and toxic shock syndrome, associated with a cytokine storm suggestive of a macrophage activation syndrome, therefore it can create great difficulty to differentiate between them ,15. The The pathophysiology of MIS-A is complex and involves endothelial damage, thromboinflammation, immune dysregulation (dysregulating the innate and adaptive host immune responses) as well as the renin-angiotensin-aldosterone system, and/or viral persistence in extra-pulmonary tissues . The datPatients with SARS-CoV-2-associated MIS respond to immune modulation with IVIG and GCS and show increased inflammatory markers, thus the disease is likely immunologically mediated. According to the guidelines from the American College of Rheumatology . The higTalking about COVID-19 vaccination, current knowledge shows that COVID-19 vaccination is the best protection from MIS . AlthougThese findings suggest that MIS-A occurs in the post-acute COVID-19 period with a heterogeneous clinical presentation, likely owing to a dysregulated immune response. Prompt immune-modulatory treatment is needed in order to stabilize the disease and to avoid severe and life-threatening complications. Uncontrolled MIS has a high mortality, and it is of great importance to properly and early diagnose and start the treatment. The importance of vaccination among all eligible persons is based on the growing evidence that COVID-19 vaccination is associated with a high level of protection against MIS in children and adults.