The genetic architecture of complex traits is multifactorial. Genome-wide association studies (GWASs) have identified risk loci for complex traits and diseases that are disproportionately located at the non-coding regions of the genome. On the other hand, we have just begun to understand the regulatory roles of the non-coding genome, making it challenging to precisely interpret the functions of non-coding variants associated with complex diseases. Additionally, the epigenome plays an active role in mediating cellular responses to fluctuations of sensory or environmental stimuli. However, it remains unclear how exactly non-coding elements associate with epigenetic modifications to regulate gene expression changes and mediate phenotypic outcomes. Therefore, finer interrogations of the human epigenomic landscape in associating with non-coding variants are warranted. Recently, chromatin-profiling techniques have vastly improved our understanding of the numerous functions mediated by the epigenome and DNA structure. Here, we review various chromatin-profiling techniques, such as assays of chromatin accessibility, nucleosome distribution, histone modifications, and chromatin topology, and discuss their applications in unraveling the brain epigenome and etiology of complex traits at tissue homogenate and single-cell resolution. These techniques have elucidated compositional and structural organizing principles of the chromatin environment. Taken together, we believe that high-resolution epigenomic and DNA structure profiling will be one of the best ways to elucidate how non-coding genetic variations impact complex diseases, ultimately allowing us to pinpoint cell-type targets with therapeutic potential. Complex traits or diseases are considered to be influenced by interactions between environmental stimuli and regulation of multiple genes. Indeed, correlating allelic frequencies with complex trait variations through case-control genome-wide association studies made it abundantly clear that etiological dissection of complex diseases is non-trivial, and complex diseases are pleiotropic and polygenic. ,2,3. TheIn addition, the epi/genome can be influenced by the environment factors ,8,9. In The epigenomic elements can be broadly defined by regions of open chromatin including cis-regulatory elements, such as insulators, promoters, enhancers, and trans-regulatory binding sites for transcription factors (TFs), as well as histone modification marks that orchestrate a regulatory ensemble, under a dynamic chromatin topology, capable of modulating the transcriptome without altering the nucleotide sequences per se. In turn, the chromatin states and structures are largely influenced by heritable, but also reversible, chemical modifications to DNA and histones, collectively referred to as epigenetic modifications ,7. The eThe chromatin is structurally and functionally active . The two2+ or NAD+ cofactors to remove the acetyl groups, restabilizing the chromatin structure [More stable nucleosome post-translational modifications are facilitated by ATP-dependent chromatin remodeling complexes, such as SWI/SNF (Switch/Sucrose non-fermentable) and nucleosome remodeling and deacetylase complex (NuRD). More commonly, histone-remodeling enzymes, such as histone acetyl- or methyl- transferases, can lead to covalent modifications at the N-terminal tails or core of the histone proteins . The acttructure . TherebyIn general, histone modification patterns at regulatory sites, such as at promoters, can effect local chromatin permissiveness to TFs in regulating proximal gene activity, while large-scale histone modifications and nucleosome redistribution either directly or indirectly leading to the remodeling of chromatin accessibility landscapes can impact long-range gene regulation, and can be assessed by ATAC-seq (assay for transposase-accessible chromatin coupled to sequencing) or ChIA-PET . Moreover, these interactions can be reversible  or stable to define cell lineages  .The non-coding elements commonly effect distal gene expression through 3-dimensional (3D) chromatin interactions or loops, involving shifts in the chromatin topology. Chromatin loops spatially juxtapose functional loci and gene promoters to facilitate long-distance gene expression or insulate genomic regions with diverse chromatin states. These higher-order chromatin interactions can be mapped by chromatin conformation techniques, such as the 3C or Hi-C C. Of note, the CCCTC-binding factor (CTCF) is a transcription factor that colocalize with ring-shaped cohesin complexes to organize the formation of 3D chromatin loops C, as welRegions of open chromatin include coding and non-coding aspects of the genome. Interestingly, they harbor the majority of the genome-wide significant risk variants associated with neuropsychiatric disorders ,2,3, andDNase-seq leverages the DNase I enzyme that digests only the open chromatin regions, and not the nucleosome-packed inactive heterochromatin, generating DNase I hypersensitive sites (DHSs). These sites encompass cis-regulatory elements, locus control regions, and transcription factor binding sites, allowing identification of functional non-coding elements. Optimal DNase I digestion is carried out to enrich for the nucleosome-free regions from the isolated nuclei. To reduce random shearing, DNase I digested DNA is embedded in low-melt gel agarose plugs, followed by synthesis of blunt ends. The extracted chromatin is ligated to biotinylated linkers for subsequent enrichment of small DNA fragments using streptavidin columns, followed by PCR amplification and hybridization to microarrays (DNase-Chip)  or high-DNase-based high-throughput analyses of open chromatin have been widely employed to investigate regulatory functions of the non-coding regions and non-coding disease risk loci ,62,63. ELikewise, collectively employing multiple databases such as ENCODE, REC, and fetal DHSs, resulted in the association of thousands of noncoding SNPs to functional DHS sites, either directly or in LD (76%), for hundreds of complex diseases, and reproducibly, 93% of DHS SNPs overlapped TF binding sites. The candidate DHSs harboring disease risk variants were among those that mediated changes in chromatin accessibility and associated with distal gene promoters. The associations of gene promoter with DHSs were based on the significant correlations (Pearson correlation coefficient > 0.7) in their DNase I hypersensitivity signals within 500 kbps radius. This further suggested that functional DHSs that were found to be associated with complex disease risk variants could regulate distal gene promoters . Taken tSince the disruption of TF binding sites is considered to be an important mechanism by which non-coding variants mediate disease pathogenesis ,63, manyAlthough footprinting approaches rely on the ability of TF bound sites to be more resistant to cleavage by DNase digestion, accumulating evidence suggests that TFs with shorter DNA residence time leave minimal footprints , illustrHuman-specific DHSs were defined as regions with human-specific increase in DNase-seq signal compared to non-human primates. These DHSs were shown to be cell-type specific (present largely in one cell-type) and primarily enriched at distal enhancers . NotablyA stratified LD score regression can be employed to estimate contributions of functional epigenetic elements to heritability of complex traits. Using this approach, active DHSs were shown to explain higher proportions of complex trait heritability compared to coding regions . MoreoveTo examine cell-type specific differences in epigenomic signatures, a large number of biological replicates are required as produced by ENCODE; however, this may not be feasible for the primary tissues. Furthermore, deconvolution approaches require specific epigenetic markers for distinct cell-types, which remain approximative at best. More sensitive approaches that can allow unbiased cell-type specific investigations are inclusive of single-cell investigations.Single-cell DNase sequencing (scDNase-seq) has been shown to generate cell-type specific DHSs. Briefly, this method involves flow cytometry based single-cell sorting, DNase I digestion, and addition of circular carrier DNA to minimize loss of digested short fragments, followed by preferential amplification of small DNA fragments and sequencing . This meFAIRE-seq, like DNase-seq, maps open regions of the chromatin. It relies on crosslinking protein bound chromatin with formaldehyde followed by nuclei isolation and lysis, sonication, and reversal of cross-links to obtain 200–1000 bp fragments. Finally, phenol-chloroform extraction can separate the organic phase containing unused covalently-linked protein complexes, from the aqueous phase with protein-free DNA. The isolated DNA can subsequently be paired with quantitative amplification (qPCR), hybridized to microarrays, or libraries can be prepared for high-throughput sequencing . A combination of DNase-seq and FAIRE-seq in human cell lines encompassed 9% of human genome across cell-types and captured significantly more TF binding sites than either technique by itself. Despite the mostly overlapping nucleosome-free regions between the two techniques, there is a degree of uniqueness to each approach. FAIRE-seq captured more distal regulatory sites enriched in H3K4me1 histone marks, while DNase-seq captured open regions more proximal to TSSs enriched in H3K4me3 and H3K9ac histone marks. Together, these complementary approaches resulted in a higher-resolution mapping of cis-regulatory elements. Interestingly, open chromatin regions shared across cell lines were generally proximal to TSSs and enriched for CTCF binding sites. On the other hand, open chromatin associated with specific cell types was relatively depleted of CTCF binding sites but enriched for major cell-type defining TFs thought to coordinate cell-type specific gene expression . TherefoThe differential properties of the FAIRE-seq and DNase-seq in mapping cis-regulatory elements are likely the result of technical differences. These include distinct regulatory proteins bound at the open chromatin regions that could impact formaldehyde cross-linking in FAIRE-seq. Likewise, relative depletion of nucleosomes proximally to genes may be more susceptible to DNase I digestion .Given the accumulating evidence suggesting that risk SNPs in complex diseases are often located farther from gene bodies , FAIRE-sStaphylococcus aureus, which digests linker DNA and accessible chromatin between nucleosomes, without degrading the nucleosomes. A typical MNase-seq protocol involves crosslinking chromatin with formaldehyde to prevent digestion of histone bound DNA, nuclei isolation, micrococcal digestion to remove free DNA. Subsequently, cross-linking is reversed, and proteinase K digestion is used to release histone proteins. DNA is extracted with phenol-chloroform or spin columns and used as input for microarrays [One of the most popular methods to determine nucleosome occupancy is MNase-seq. Other similar methods include nucleosome occupancy and methylome sequencing (NOME-seq) that map nucleosome position along with DNA methylation  or site-roarrays , or highroarrays ,20. Employing MNase-seq in human cell lines showed that nucleosome occupancy is dependent on distinct DNA methylation and histone modification patterns . For exaIn general, and on an average across cell-types, most eukaryotic chromatin has a nucleosome repeat length of 185–195 bp, corresponding to ~147 bp of nucleosome DNA and ~45 bp of linker DNA. However, nucleosome spacing can also be indicative of specific cell-types and/or disease-states. For example, MNase-seq in distinct cell-types identified a shorter average nucleosome spacing in dorsal root ganglia neurons (~165 bp) compared to cortical astrocytes or oligodendrocyte precursor cells (~183 bp) . AnotherMNase-TSSs sequence capture is a modified technique to map nucleosome distribution surrounding only TSSs at a genome-wide scale. This approach identified nucleosome relocation around TSSs at early stages of the disease. This, in turn, was associated with aberrantly high TF binding and disruption of gene expression programs that mediate disease progression . Moreovede novo mutation datasets with MNase-seq-derived nucleosome maps revealed that non-coding regions at/around translationally stable nucleosome positioning across cell-types associate with significantly higher de novo mutation rates, INDELs, repeat elements, and a lower DNA replication fidelity of those sites [Combining human se sites . This fuRecently, single-cell MNase-seq has been able to obtain nucleosome positioning and chromatin accessibility profiles from single cells . BrieflyOther single-cell methods include scNOMe-seq that can measure both nucleosome occupancy and DNA methylation at a genome-wide scale . Multi-oATAC-seq can capture multi-nucleosome regions of open chromatin using at least 10 times less nuclei and can obtain a higher signal-to-noise ratio compared to the previously described DNase, FAIRE, or MNase-seq. Introduced by Buenrsotro et.al, ATAC-seq requires a prokaryotic Tn5 transposase charged with point mutations to increase its enzymatic activity and adaptors to tag accessible chromatin. Tn5 transposase is applied to the isolated nuclei in bulk. Specific primer pairs can be used to amplify the cut and tagged segments of DNA, which is then followed by high-throughput sequencing. A successful ATAC-seq library shows a laddering pattern with 200 bp periodicity, corresponding to segments of DNA devoid of one (200 bp) or more nucleosomes . With slATAC-seq has become a popular technique for studying DNA structure, not only because of its ease of use, but also because of its robust findings. For example, the Common Mind Consortium (CMC)-led study in postmortem human brain identified about 9% SNP heritability in schizophrenia in the open regions of chromatin. In addition, a four-fold increase in the SNP heritability for this illness was found when including evolutionarily conserved open regions . InteresSince ATAC-seq can be performed on small amounts of material, researchers have successfully used fluorescence-activated nuclei sorting (FANS) to isolate broad cell types based on antibodies against specific cell markers. Generating neuronal (NeuN+) and non-neuronal (NeuN-) populations from postmortem brain regions of healthy individuals showed that individual cell-types capture more than 50% of the variance in open chromatin brain regions, in contrast to biological sex that accounted for less than 2% variance . AdditioOverlapping risk loci with open chromatin regions revealed that neurons from the striatum and hippocampus were enriched for schizophrenia risk variants, while non-neuronal hippocampal regions were enriched for risk variants associated with major depressive disorder (MDD) . LikewisBdnf gene was found in the glutamatergic neurons, while open chromatin of Lhx6 gene was detected in the GABAergic neurons. In addition, cell-type specific open chromatin overlapped with regulatory regions of cell-type specific marker genes. Further, TF footprinting using ATAC-seq, such as DNase-seq, can predict binding of TFs at open chromatin. The footprinted TFs were associated with target genes by the distance of TF binding sites to TSSs. Moreover, the target genes of cell-type specific TFs were among those with cell-type specific open chromatin [Combining ATAC-seq with a more refined FANS approach by sorting for glutamatergic neurons, GABAergic neurons, oligodendrocytes, and microglia/astrocytes resulted in cell-type specific differentially open coding- and noncoding-regions . For exahromatin . These rThe open chromatin regions in glutamatergic neurons showed strong enrichments for risk variants associated with psychiatric phenotypes including schizophrenia and brain-related traits like neuroticism and intelligence . MoreoveSingle-cell or nucleus ATAC-sequencing (sc/sn-ATAC-seq) can capture cells that cannot be isolated through gene markers , as well as identify landscapes of rare cell-types and/or cell-states. Using the principles of bulk ATAC-seq, scATAC-seq requires a fluidics-based chip, where single cells are captured into individual wells, followed by Tn5 transposition and amplification. Single-cells are then barcoded for cell-identification, and subsequently pooled for library generation and next-generation sequencing (NGS) . AlternaA plate-based combinatorial barcoding approach called sci-ATAC-seq was established to allow multiplexing of high numbers of cells/nuclei. First, one-to-few nuclei are tagged with barcoded Tn5 in a single well of a 96-well plate, and then it is followed by a fixed number of successive barcoding events with different barcode and pools of nuclei, enabling multiplexing of cells, making it scalable and cost-efficient . This apThe use of bulk-ATAC-seq captured minimal enrichments for Alzheimer’s or Parkinson’s disease associated risk variants, however, combining it with snATAC-seq revealed five-fold enrichment of SNPs overlaying regions of open chromatin at cell-type specific regulatory loci . FurtherTaken together, the general patterns of chromatin accessibility and disease enrichments consistently show distal regulation of cell-type specific genes. Risk variants for psychosis-associated diseases are mainly enriched in the open regions of neurons, while neurodegenerative disease variants occur more consistently in open chromatin regions of non-neuronal cell-types. These findings hold true across distinct chromatin accessibility measuring approaches ,89,90.Mounting evidence suggests that histone-remodeling factors mediate open/closed chromatin states, which in-turn alters the binding of TFs and other cofactors in mediating gene expression. Moreover, these histone remodelers are capable of keeping regulatory regions in a stable configuration over time. The modification can even be maintained after passage of the replication fork, thereby, sustaining a long-term “epigenetic memory” over cell generations and preserving cell- or lineage-specific gene expression programs .Chromatin immunoprecipitation or ChIP is a widely employed technique for assaying protein-DNA interactions by using specific antibodies ,34,35. TChIP-seq has been used extensively to map important functional, non-coding regions of the genome, by either defining histone modifications to a chromatin state or mapping various transcription factors to genomic regions. In a seminal 2007 ChIP-seq study, genome-wide binding sites of a repressor element-1 silencing transcription factor (REST) were identified in the human T cell lines . In thisChIP-seq in neuroblastoma cell lines was used to probe genome-wide binding sites of TCF4, a transcription factor known to regulate the excitability of cortical pyramidal cells while its dysregulation has been associated with numerous cognitive deficits . This stMef2c overexpression in cortical neurons of adult mice improved cognitive performance after psychotogenic drug treatment [Super-enhancers are defined as broad stretches of multiple enhancers spanning open chromatin regions and strongly associated with histone acetylation signals. In addition, the super-enhancers are often found to be associated with a transcriptional coactivator, Med1. Typically, super-enhancers allow binding of cell-type specific TFs and regulation of cell-type specific gene expression . Of notereatment .CACNA1C, ADCYAP1, DPP10. Interestingly, increase in human-specific H3K4 methylation at the 5′ promoter of psychiatric-risk gene, DPP10, correlated with its downregulation via transcription of an antisense RNA [H3K4me3 ChIP-seq in human prefrontal cortical neurons, compared to chimps and macaques, revealed hundreds of human-specific methylation gains that correlated with dysregulation of genes implicated in psychiatric disorders, including ense RNA . Therefoense RNA . Interesense RNA . These fH3K4me3 ChIP-seq in neuronal and non-neuronal cells from prenatal, young, and elderly human prefrontal cortex revealed cell-type specific dynamic remodeling from mid-gestational to early postnatal life  but only minimal changes from adolescence to adulthood. Developmentally regulated H3K4me3-signals were within 2 kbps of age-related and synaptic genes. In addition, developmentally upregulated H3K4me3 peaks in cortical neurons were enriched for activity-dependent AP-1 motifs . LikewisRapid epigenetic changes specific to early-life at activity-dependent gene regulators  support Although ChIP-seq from tissue homogenates has highlighted potential disease-related targets, the lack of cell-type features can mask subtle histone-modifications, which might be driven by cellular composition rather than phenotype. As such, we have witnessed the development of single-cell ChIP-seq approaches using drop-seq based microfluidics and barcode multiplexing. Of note, cell-types clustered accurately based on H3K4me3 or H3K27me3 histone marks associated with permissive or repressive transcription . Indeed,ChIP-seq has some limitations, such as nonspecific DNA binding or uneven fragmentation that can contaminate immunoprecipitants leading to spurious or false-positive reads. Another limitation of X-ChIP is the requirement of sonication that can cause high background noise, necessitating higher cellular input for optimal signal-to-noise ratio . RecentlEscherichia coli Dam tethered to protein-of-interest, which can catalyze N6-methylation of adenines in GATC sequences present in their vicinity. The methylated regions are digested with DpnI, followed by microarray-hybridization or high-throughput sequencing [This technique uses quencing . DamID-squencing .ANK3 [Targeted DamID in embryonic mouse cortex characterizing CHD8 binding sites, a chromatin remodeler with de novo mutation associated with sporadic autism spectrum disorder, indicated that binding of CHD8 at distal enhancers regulates neurodevelopment-associated genes, e.g., ANK3 . LabelinANK3 .Single-cell DamID in human cell lines provided a “molecular contact memory” approach that was used to map fates of regulatory loci spatially interacting with nuclear lamina over time. This was also found to correlate with H3K9me2 histone marks for transcriptional repression . FurtherThe drawbacks of ChIP-seq and DamID have motivated development of alternative methodologies, including CUT&RUN. Briefly, unfixed nuclei are immobilized on magnetic beads and incubated with MNase-tethered staphylococcal protein A (pA-MN) that binds to antibodies targeting protein-of-interest. This is followed by Ca2+ ion treatment for induction of double-stranded DNA cleavage and centrifugation to separate protein-bound DNA for sequencing, generating long-range protein-interactions maps at single-bp resolution .Mecp2 conditional knockout mouse cortex. Thereby, site-specific methylation and MECP2 binding were found to be important mechanisms regulating postnatal long-term decommissioning of neuronal enhancers [The early postnatal maturation of the brain follows a precise epigenomic remodeling, and environment-prompted alterations in these stages are associated with increased susceptibility to diseases . CUT&RUNnhancers . On the Additionally, CUT&RUN for ARID1A, a subunit of SWI/SNF BAF chromatin remodeling complex, showed increased binding at FOS-bound postnatally induced enhancers by 3 weeks postnatally. Further, the binding of ARID1A at postnatally activated enhancers continued into the adulthood, which likely maintained them in an active configuration through nucleosome repositioning . The cul2+ ions for Tn5 activation and integration of adapters to protein binding sites, generating chromatin fragments ready for amplification and sequencing [An interesting alternative to CUT&RUN is cleavage under targets and tagmentation (CUT&TAG), which is principally the same but employs Tn5 transposase for tagmentation of DNA sequences near the binding sites of a regulatory protein, making it ultra-low-input and more sensitive than CUT&RUN . Brieflyquencing .Schizophrenia-associated genes identified from snRNA-seq in postmortem brains were found to be highly regulated by a few TFs , also overlapping GWAS risk loci . CUT&TAGIn biology, structure follows function, for example, chromosome territories (CT) compartmentalize gene rich and poor regions, and their shuffling has been reported in pathological states . FurtherSpatial interactions between any two genomic loci can be mapped using 3C, a technique introduced by Dekker et al. in 2002. Briefly, intact nuclei are fixed and cross-linked by formaldehyde resulting in formation of covalent bonds between physically interacting chromosomal segments bridged by proteins. Cross-linked chromatin is digested with restriction enzymes to retain only physically linked fragments, followed by ligation, reversal of cross-links to form chimera of interacting fragments and PCR amplification with locus-specific primers. A control without the ligation step validates physical interactions .The 3C-qPCR in postmortem cortical neurons showed 3D-interacting H3K4me3-peaks up to 1 Mb apart associated with human-specific increases in H3K4 methylation at loci implicated in neurodevelopmental disorders and encompassing psychiatric-risk genes . TherefoMHC (major histocompatibility) complexes, central to immune-related functions, have been strongly implicated in psychiatric disorders by GWAS , albeit Of note, ChIP-3C assay involving an antibody-based immunoprecipitation of cross-linked, physically interacting loops illustrates their functional roles. MECP2 ChIP-3C showed that MECP2 binding was necessary for chromatin loop-mediated gene silencing of imprinted genes . The abeXist were involved in 3D interactions with each other [Given that a regulatory locus can interact with multiple other loci, for example, an enhancer regulating expression of several genes, one-to-many mapping is particularly informative. The 4C-Chip, also known as 3C-on-Chip, takes the advantage of high-throughput microarray technology paired with the 3C technique . In addich other . The 4C-ch other ,123,124.Chromosome Conformation Capture Carbon Copy, also called 5C, maps interactions among many regulatory loci at the same time. Post-3C cross-linking and ligation, 5C employs a multiplexed ligation-mediated amplification using primers pairs that anneal across 3C-ligated junctions and can be paired with microarray or sequencing. For example, 10,000 5C primers can generate up to 25 million distinct chromatin interactions ,125.5C-seq has shown that long-distant spatial configuration disproportionally mediates gene expression in mammalian cells . A studyUsing 5C-seq to generate a CTCF connectome showed that most gene-enhancer connections anchored by CTCF in pluripotent cells are lost during embryonic differentiation and neural lineage-commitment . As suchHi-C provides an unbiased genome-wide mapping of all the genomic loci paired with high throughput sequencing. Briefly, cells are cross-linked with formaldehyde that results in covalent links between 3D-interacting chromatin fragments. Chromatin is digested with restriction enzymes and 5′-overhangs are filled. This is followed by addition of biotinylated residues and ligation, after which biotin-fragments are enriched with streptavidin beads and Hi-C libraries are constructed and sequenced. This technique was used to validate compartmentalization of human genome into A/B sections within the nucleus, where A is open, active, and accessible compared to B . Topology maps of human corticogenesis from distinct postmortem frontoparietal regions including cortical plate (comprising postmitotic neurons) and germinal zone  of mid-gestational fetuses were generated using Hi-C . IntegraPsychENCODE is a harmonized collection of transcriptomic, open chromatin, and Hi-C interactome data, across cortical brain regions for 1866 individuals. Altogether, 90,000 enhancer-promoter long-range interactions and cell-type specific 3D interactions within 2735 CTCF-bound TADs have been cataloged. From these data, a distinct pattern was reported for fetal and adult Hi-C connectome. Interestingly, it was found that eQTLs distal to gene promoters supported by Hi-C enhancer-promoter interactions had significantly higher association with gene expression than those eQTLs located within the gene promoter or exons and not supported by Hi-C interactions . Thus, HHi-C chromatin maps from 21 adult human tissues identified another major architectural feature called frequently interacting regions (FIREs), with significantly higher cis-connectivity and cell-type specificity. FIREs were detected to be mostly located in compartment A within TADs, and were found to be partly dependent on CTCF-cohesin complex for their formation . AdditioA low input easy-Hi-C protocol, which improves the resolution of proximity-ligation events through a biotin-free strategy, in situ proximity-ligation, and an extra exonuclease step to remove un-ligated contaminants, was successfully applied to both adult and fetal postmortem human brain tissues and cell lines . EmployiGiven a striking difference in percentage of gene loops between NPCs, neuronal, or glial cells , single-Single-cell chromatin topology maps using diploid chromatin conformation capture, Dip-C, demonstrated clustering of distinct cell-types based on cell-type specific enhancer-promoter 3D contacts . By elimH19/IGF2 gene locus [A major challenge in 3D reconstruction of diploid genome is accurately identifying chromosome haplotypes involved in spatial interactions. Since non-coding SNPs disrupting 3D chromatin interactions are identified in complex diseases ,119, spane locus , and 15qne locus . Furtherne locus . Taken tAlthough Hi-C can provide genome-wide mapping of chromatin contacts, it cannot provide precise functional roles mediated by chromosomal loops. Similar to 3C-ChIP, chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) allows genome-wide mapping of chromatin interactions mediated by regulatory proteins. Briefly, formaldehyde cross-linked DNA is sonicated and enriched for protein-of-interest with specific antibodies followed by proximity-based ligation with DNA linkers and extraction of 20bp paired-end tags for sequencing . The semAn ENCODE ChIA-PET study showed that cohesin-bound loops were present at a sub-TAD scale and cell-type specific cohesin-loops were enriched for disease risk loci, unlike invariant TAD boundaries across cell-types . FurtherChIA-PET requires millions of cells and greater read-depth for assaying 3D interactome, precipitating the development of other techniques. HiChIP (Hi-C with ChIP) involves cross-linking and digestion of DNA fragments in the intact nuclei for Hi-C library construction followed by ChIP. This technique generated cohesin-mediated interactome in human cell lines with 100-fold less nuclei input and 10-fold higher read-depth relative to ChIA-PET . H3K27acIn addition, long-range chromatin interactions can also be mapped by the proximity ligation-assisted ChIP-Seq (PLAC-seq), principally similar to HiChIP, and is more sensitive than ChIP  The majoGiven that non-coding genomic regions have been reported to be the hotspot of single-nucleotide or structural variants underlying complex traits, the integration of multi-omics approaches to profiling genomic architecture has identified functional roles of the non-coding causal risk variants in mediating complex diseases, particularly brain diseases. Gene-environment interactions mediated by activity-dependent changes at non-coding elements are found to be essential for normal brain development, whereas abnormal epigenetic changes at these regions during early-life may increase susceptibility to complex brain disorders. Moreover, human-specific genomic sequences that are under adaptive evolution include those non-coding elements that regulate genes important for cognitive functions but have also been found to harbor risk SNPs associated with psychiatric traits.Notably, identifying disease risk genes based on their linear proximity or linkage disequilibrium has been insufficient. Accumulating evidence has shown that most risk variants enrich in distal regulatory sites and regulate gene expression through 3D chromatin loops. Moreover, 3D interactions have been found to outperform other paradigms in linking risk genes to disease risk loci. Additionally, germline and/or de novo risk variants are found to often disrupt transcription factor recognition sequences at distal gene enhancers or associate with differential histone modifications patterns in modifying cell-type specific transcriptome. Importantly, examining cell-type specific epigenetic changes using single-cell investigations is imperative to untangling biological complexity of polygenic traits in a heterogenous brain tissue and to allow unbiased discovery of rare cell-types or novel regulatory elements.Overall, these findings illustrate the importance of employing chromatin profiling techniques in determining structures and functions of the chromatin environment. Moreover, these findings supported significant remodeling of chromatin states in driving altered gene expression networks underlying complex traits. Therefore, investigating open regulatory landscapes in cell- or cell-type specific manner using chromatin-profiling techniques is central to the quest of pinpointing epi/genetic targets associated with etiopathology of complex traits and diseases.


Recent double-blind, randomized, controlled trials reported that human umbilical cord-derived mesenchymal stem cell (MSC) infusions in COVID-19 patients with acute respiratory distress syndrome (ARDS) could diminish cytokine storm and lung damage. While these outcomes are significant, additional phase II/III trials are required to validate the efficacy of MSCs to improve the survival of COVID-19 patients with ARDS. Future studies also need to assess the efficacy of MSCs to prevent long COVID. SARS-CoV-2 infection is a worldwide pandemic currently with >219 million infected cases and >4.5 million deaths. Over 40 million people have already been infected in the United States, and >659,000 people have died of COVID-19 [Worldometers.info]. SARS-CoV-2 infection initially causes mild-to-moderate symptoms, which include fever, cough, loss of smell and taste, muscle aches, fatigue, and shortness of breath, but severe cases with acute respiratory distress syndrome (ARDS), cytokine storm, or pneumonia result in considerable mortality4. Older individuals and individuals with certain health conditions have a higher risk of developing severe COVID-19 and mortality5. Overactive immune response causing cytokine storm and immunothrombosis are believed to underlie severe COVID-197.Coronavirus disease 2019 (COVID-19), a grave acute respiratory illness, is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)9. Such infection results in a cytokine storm with elevations in multiple proinflammatory cytokines, leading to edema, air exchange dysfunction, ARDS, secondary infection, and death in many cases8. Because ACE2 expression is also present in other organs, including the heart, liver, kidney, and digestive system, infected patients develop other complications such as myocardial injury, arrhythmia, acute kidney injury, shock, and death from multiple organ dysfunction10. COVID-19 patients developing ARDS currently receive high-flow oxygen therapy, intensive care, and, frequently, mechanical ventilation14. Treating COVID-19 patients afflicted with ARDS, cytokine storm, or pneumonia is challenging despite several recent drug combinations showing variable efficacy17. On a positive note, vaccines against SARS-CoV-2 are now available, but it will take a significant amount of time to vaccinate the vast majority of the world population. While the vast majority of effort is currently aimed at reducing or eliminating infection through vaccination, there is also a need for reducing the mortality in severely ill patients with COVID-19. Therefore, safe and effective treatment for COVID-19 patients with severe complications such as ARDS, cytokine storm, or pneumonia is critical for saving lives.A widespread expression of the angiotensin-converting enzyme 2 (ACE2) receptor in the lung alveolar cells and capillary endothelial cells facilitates severe respiratory illness following SARS-CoV-2 infection19 have shown that infusions of human umbilical cord-derived mesenchymal stem cells (UC-MSCs) into COVID-19 patients resulted in better functional outcomes22. Leng and associates showed that seven patients with COVID-19 pneumonia displayed improved functional outcomes and recovered after an intravenous administration of clinical-grade human UC-MSCs8. Among the patients who received UC-MSC infusions, one had a critically severe type, four had severe types, and the other two had common types of pneumonia8. This study provided the first evidence of the promise of MSC therapy for saving the lives of COVID-19 patients developing severe complications and has resulted in the commencement of many clinical trials23. A few subsequent studies also found that the infusion of UC-MSCs was safe in COVID-19 patients22. The study by Iglesias and colleagues enrolled five patients with severe ARDS who have not shown improvements in their clinical conditions with the prevailing medical care for 48 h and displayed arterial oxygen partial pressure (PaO2)/fractional inspired oxygen (FiO2) values <100 mmHg19. The study also reported that MSCs mediated anti-inflammatory effects in the lungs, evident from an improved respiratory function with higher PaO2/FiO2 values19. Treatment of MSCs resulted in the survival of three patients who were extubated 9 days post-infusion. However, the lack of double-blind, randomized, controlled trials using MSCs has raised questions about the efficacy of MSCs among skeptics.Previously, studies from China and MexicoStem Cell Translational Medicine14. This study represents the first double-blind, phase I/IIa, randomized, controlled trial of MSC treatment for COVID-19 patients. Table 14. The study also did not find any serious adverse events in the 12 patients receiving UC-MSC infusions. The patients in the control and MSC treatment groups were matched for age and ARDS severity, which balanced the control and MSC treatment groups. The study also revealed that the cytokine storm could be restrained through MSC infusions in COVID-19 patients with ARDS. MSC infusions did not decrease the viral load but significantly dampened the concentration of multiple proinflammatory cytokines. This study’s critical takeaway message is that the survival of COVID-19 patients with ARDS could be improved substantially with MSC treatment. In all, 91% survival was seen in the MSC infusion group compared to 42% survival in the control group. This is the most impressive finding from this controlled trial. Since severe COVID-19 is widely believed to be due to the overactive immune response with cytokine storm causing other complications such as immunothrombosis, mini-strokes, and multiple organ failure, the ability of MSCs to modulate the immune response and improve the survival of patients with ARDS is a significant advance. Overall, this trial’s findings validate the results of previous trials19. The results of another randomized, double-blind, placebo-controlled phase II trial using UC-MSCs in COVID-19 patients with lung damage have been published recently24. UC-MSC infusions  in 49 patients resulted in a reduced lung lesion volume compared to 25 patients receiving the placebo24. These findings suggest that UC-MSC infusions could be employed as an adjunct therapy to the standard care of COVID-19 patients with lung damage.While the outcome of many more extensive double-blind clinical trials for severe COVID-19 patients is yet to be published, Lanzoni and colleagues’ study has been recently published by the journal 27. The rationale for employing MSC infusions for COVID-19 complications is the safety and efficacy of MSCs for immune system-mediated inflammatory diseases, such as graft-versus-host disease28, type 2 diabetes29, and spinal cord injury30. Immunomodulatory effects of MSCs are believed to underlie improved function after MSC infusions in multiple disease conditions, as MSCs secrete multiple paracrine factors, which positively interact with immune cells, facilitating immunomodulation32. The better outcome in COVID-19 patients after MSC infusions also appeared to comprise the robust anti-inflammatory activity of MSCs suppressing the cytokine storm. MSCs typically accumulate in the lungs after intravenous infusion, which likely facilitates the secretion of multiple paracrine factors33, leading to significant protection and rejuvenation of alveolar epithelial cells, and improved lung function in COVID-19 patients. Figure MSCs have been employed widely in cell therapy, which comprises numerous preclinical studies and a significant number of clinical trials14. However, another trial has validated some of the beneficial effects of MSC infusions in 49 COVID-19 patients24. Since UC-MSCs were used in both studies, as in the previous study8, it will be necessary to ascertain whether similar efficacy could be obtained from MSCs from other sources such as those derived from the bone marrow or adipose tissue. The average age of patients employed in these trials was ~59–60 years24. Therefore, it remains to be determined whether MSC infusions would be efficacious in much older COVID-19 patients . Furthermore, the dose employed was 40–100 million per infusion, with infusions separated by 72 h apart. Dose–response studies are needed in the future to improve the efficacy further. Since the primary endpoint was safety, efficacy measures in these studies were restricted to patient survival and cytokine levels in the blood14 or lung lesion volume and a 6-min walk test24. Future studies need to assess whether MSC infusions in COVID-19 patients with ARDS or other complications would prevent the symptoms of long COVID. Patients with long COVID  experience persistent brain-related problems, including brain fog typified by cognitive impairments, post-exertional malaise, and chronic fatigue, mimicking chronic multisymptom conditions, such as fibromyalgia35, chronic fatigue syndrome36, or Gulf War Illness37. Additional phase II/III trials evaluating the outcomes of MSC infusions for reducing mortality and preventing long COVID symptoms are needed. Also, while the advent of efficacious vaccines has reduced SARS-CoV-2 infections in many nations, SARS-CoV-2-infected survivors experiencing long COVID symptoms might benefit from MSC therapy because of the ability of MSCs to ease neuroinflammation and promote regeneration in organs, such as the lungs, brain, and heart.While the new clinical trial results provide further support for considering MSC infusions for COVID-19 complications, a few critical issues need to be addressed in future MSC therapy trials for COVID-19 patients with ARDS. The numbers of patients recruited in one of the trials were low


Early detection of SARS-CoV-2 infection is critical to reduce asymptomatic and presymptomatic transmission, curb the spread of variants, and maximize treatment efficacy. Low-analytical-sensitivity nasal-swab testing is commonly used for surveillance and symptomatic testing, but the ability of these tests to detect the earliest stages of infection has not been established. In this study, conducted between September 2020 and June 2021 in the greater Los Angeles County, California, area, initially SARS-CoV-2-negative household contacts of individuals diagnosed with COVID-19 prospectively self-collected paired anterior-nares nasal-swab and saliva samples twice daily for viral-load quantification by high-sensitivity reverse-transcription quantitative PCR (RT-qPCR) and digital-RT-PCR assays. We captured viral-load profiles from the incidence of infection for seven individuals and compared diagnostic sensitivities between respiratory sites. Among unvaccinated persons, testing saliva with a high-analytical-sensitivity assay detected infection up to 4.5 days before viral loads in nasal swabs reached concentrations detectable by low-analytical-sensitivity nasal-swab tests. For most participants, nasal swabs reached higher peak viral loads than saliva but were undetectable or at lower loads during the first few days of infection. High-analytical-sensitivity saliva testing was most reliable for earliest detection. Our study illustrates the value of acquiring early (within hours after a negative high-sensitivity test) viral-load profiles to guide the appropriate analytical sensitivity and respiratory site for detecting earliest infections. Such data are challenging to acquire but critical to designing optimal testing strategies with emerging variants in the current pandemic and to respond to future viral pandemics. Early detection of SARS-CoV-2 infection is needed to reduce asymptomatic and presymptomatic transmission, including the introduction and spread of new viral variants. More than half of transmission events occur from presymptomatic or asymptomatic persons . Early d3–2 to 103 copies/mL of sample, whereas tests with low analytical sensitivity have LOD values equivalent to ∼105 to 107 copies/mL  early in infection; therefore, only tests with high analytical sensitivity would reliably detect virus in saliva.Tests with high analytical sensitivity can detect low levels of molecular components of the virus  in a sample. Analytical sensitivity is described by the limit of detection (LOD) of a test . LOD of SARS-CoV-2 diagnostic tests are described in various units; the most directly comparable among tests are units that report the number of viruses  or viral RNA copies per milliliter of sample. Viral RNA copies/mL are roughly equivalent to genome copy equivalents/mL (GCE/mL) or nucleic-acid detectable units/mL (NDU/mL). These LOD values are tabulated by the U.S. Food and Drug Administration (FDA) and vary by ≥5 orders of magnitude between tests . Tests wpies/mL 912. Imporpies/mL 9 demonstrpies/mL 9 and thenpies/mL 9 and othepies/mL 9, 16 showSampling site or specimen type may also be critical to early detection. Other respiratory viruses have been shown to have detection rates that vary by sampling site , which h20–24–N1 and N2 gene positivity using a high-sensitivity assay. When a transmission event was observed (a previously SARS-CoV-2-negative participant tested positive in at least one sample type), we quantified viral loads in all samples prospectively collected from that participant for at least 2 weeks from their first positive result. Quantification was performed via quantitative reverse-transcription PCR (RT-qPCR), with a subset of measurements validated by reverse-transcription droplet digital PCR (RT-ddPCR), capturing the early and full course of acute SARS-CoV-2 infection with high sensitivity.To understand the required test sensitivity and the optimal sample type for earliest SARS-CoV-2 detection, we designed a case-ascertained study of household transmission with high-frequency sampling of both saliva and anterior-nares nasal swabs. Building on our earlier work , we enroRefer to the supplemental material for detailed methods.Acquisition of participant data was performed as described previously . SymptomParticipants self-collected nasal-swab and saliva samples in the Spectrum SDNA-1000 saliva collection kit , which contains 1.5 mL of liquid buffer, at home twice per day (after waking up and before going to bed), per the manufacturer’s guidelines. A parent or legal guardian assisted all minors with collection and were instructed to wear a face covering during supervision.Samples were stored at 4°C and equilibrated to room temperature before being processed with extraction protocols.Participant saliva and anterior-nares swab samples were extracted using the KingFisher Flex 96 instrument (Thermo Fisher Scientific) with the MagMax Viral Pathogen I nucleic acid isolation kit  guided by Thermo Fisher technical notes for SARS-CoV-2 modification and saliva.RT-qPCR was performed as previously described  using thqC) of duplicate RT-qPCRs using the equations obtained from calibration curves of contrived samples—healthy human saliva or nasal fluid spiked with heat-inactivated SARS-CoV-2 particles. See the supplemental material methods for additional details.For samples defined as positive by assay guidelines from the CDC , viral lQuantification was also performed by reverse-transcription droplet digital PCR (RT-ddPCR) on both the calibration curve samples , Fig. S2N1 gene qC of below 26 were sent to Chan Zuckerberg Biohub for SARS-CoV-2 viral genome sequencing, a modification of Deng et al. (github.com/cov-lineages/pangolin). Chan Zuckerberg Biohub submitted the resulting genomes to GISAID.Saliva and nasal-swab samples with an g et al.  as descrg et al. . Sequencg et al.  using PhData availability. Data are available on CaltechDATA at https://data.caltech.edu/records/1942.qC) to viral load   and an Rral load  and 63 pral load  across aNext, to quantify the viral load at the earliest stage of infection, we analyzed the viral loads in the saliva and nasal swabs of participants who were negative in both sample types upon enrollment and became positive during their participation in the study . We exteRNaseqP C values remained consistent throughout the collection period for saliva and swabs for most participants , while the nasal swab remained negative, and days before the participant reported any symptoms. As measured, viral load in nasal-swab samples reached the level of LOD of low-analytical-sensitivity tests 1.0 days after the first saliva positive samples (pink triangle). This same pattern of earlier detection in high-sensitivity saliva was observed in five of the other six participants; high-sensitivity saliva was 2.5 days earlier (3 to 104) in saliva for 4 days, while nasal swabs remained negative by high-sensitivity measurements. Nasal viral loads spiked above 1010 copies/mL while the participant’s only symptoms were mild congestion/runny nose.In six out of seven participants, high-analytical-sensitivity saliva testing would have been superior for early detection of SARS-CoV-2 infection compared with the predicted performance of nasal-swab tests with low analytical sensitivity. This prediction was made by evaluating when nasal-swab viral loads entered the LOD range of nasal-swab tests with low analytical sensitivity. In the seventh participant, the first positive high-analytical-sensitivity saliva test was detected at the same time point that the first nasal-swab test reached a viral load likely to be detected by a low-analytical-sensitivity nasal-swab test . In the  earlier , 3.0 day earlier , 6.0 day earlier , 4.5 day earlier , and 2.5 earlier . The max earlier . This paEven with high-analytical-sensitivity nasal-swab testing, only one participant tested positive in nasal swab before saliva . In thisTwo participants  and E ha8 viral copies/mL (N1 gene target) while SARS-CoV-2 RNA remained undetectable in nasal swab, even by the high-analytical-sensitivity assay used here.Remarkably  is not authorized (at the time of this writing) for the sample collection of nasal swabs. The solution in the Spectrum SDNA-1000 kits is a guanidinium-based inactivating and stabilizing buffer that preserves viral RNA but eliminated the opportunity to also perform viral culture. Third, we have paired data for saliva and anterior-nares nasal swabs but do not compare nasopharyngeal (NP) swabs, sputum, or other lower-respiratory specimens. We do not know whether other sampling sites, such as NP swabs or oropharyngeal swabs, would have provided earlier or later detection than saliva. Fourth, we are inferring the ability of tests with low analytical sensitivity to detect infections based on the quantified viral load in the participant samples and the LODs reported by the FDA for the diagnostic platforms. Fifth, some degradation may have occurred in some samples . Sixth, all samples were self-collected, which may result in lower-quality specimens.Conclusions. By rapidly enrolling household members at high risk for contracting COVID-19 and having them self-sample twice daily in paired respiratory sites, we observed patterns in SARS-CoV-2 viral load in the earliest days of infection. All seven participants tested negative in saliva and nasal swabs upon enrollment, demonstrating that we captured the earliest detectable SARS-CoV-2 viral load (within 12 h) in both sample types. Our data set helps inform diagnostic testing strategies by showing differences in viral loads in paired nasal swabs and saliva samples at high temporal resolution during the early days and presymptomatic phases of infection.We made five conclusions from our study.First, choosing the correct respiratory sampling site is critical for earliest detection of SARS-CoV-2 infection. In our study, alignment of longitudinal data to the first day of positivity clearly shows the superiority of high-sensitivity saliva testing for detection in the first 5.5 days of infection . Given oSecond, our data explain the conflicting results in the literature comparing test performance in paired respiratory sites, with some studies showing that nasal swabs outperform saliva , 23, 40 42–7 to 108 copies/mL) viral load in saliva samples while the paired nasal swab was either negative (3 copies/mL) viral load  Additional longitudinal studies are needed that include high-frequency collection from multiple respiratory sites using quantitative assays with high analytical sensitivity. (ii) Policy makers need to use such quantitative data to revise and optimize screening testing guidelines to ensure early detection of SARS-CoV-2 infections and reduction of transmission. (iii) Innovation is needed to produce rapid point-of-care tests with high analytical sensitivity for a range of sample types  at a price point to enable global distribution. (iv) Quantitative studies of the kinetics of early-stage viral loads in each respiratory site  must be updated with the emergence of new SARS-CoV-2 variants.We hope our data, important work by others in this area , 51, 52,


RNA binding proteins (RBPs) have a broad biological and physiological function and are critical in regulating pre-mRNA posttranscriptional processing, intracellular migration, and mRNA stability. QKI, also known as Quaking, is a member of the signal transduction and activation of RNA (STAR) family, which also belongs to the heterogeneous nuclear ribonucleoprotein K- (hnRNP K-) homology domain protein family. There are three major alternatively spliced isoforms, QKI-5, QKI-6, and QKI-7, differing in carboxy-terminal domains. They share a common RNA binding property, but each isoform can regulate pre-mRNA splicing, transportation or stability differently in a unique cell type-specific manner. Previously, QKI has been known for its important role in contributing to neurological disorders. A series of recent work has further demonstrated that QKI has important roles in much broader biological systems, such as cardiovascular development, monocyte to macrophage differentiation, bone metabolism, and cancer progression. In this mini-review, we will focus on discussing the emerging roles of QKI in regulating cardiac and vascular development and function and its potential link to cardiovascular pathophysiology. Transcriptional and posttranscriptional modifications are critical in regulating gene expression . Posttracis-regulatory nucleotide sequences and trans-acting factors  family, which belongs to the hnRNP K-homology domain protein family . QKI is v mouse line is a well-known spontaneous mutant mouse line, in which a 1 Mb promoter/enhancer region is deleted at the upstream of the Qki transcription start site and results in myelination defect in the central nervous system  are capable of differentiating into monolayer cardiomyocyte sheets but have dramatically weaker and asynchronous spontaneous-beating activity. Transcriptomic analysis at a single-cell resolution confirms that hESC-QKIdel largely maintains pluripotency, self-renewal activity, and early cardioprogenitor differentiation ability but has a significant defect in becoming functional cardiomyocytes. Genes involved in contractile function, such as ACTN2 (α-actinin), MYL2 (myosin light chain 2), MYH7 (β-myosin heavy chain), ANKRD1 (ankyrin repeat domain 1), TNNI3 (troponin I3), ACTA1 , and TAGLN (transgelin), are downregulated significantly in cardiomyocytes derived from hESCs-QKIdel, suggesting that the altered contractile function in QKI-deficient cardiomyocyte sheet is mainly caused by the altered regulation of sarcomere genes. Interestingly, a replicate multivariate analysis of transcript splicing (rMATS) revealed altered alternative splicing in Z-line proteins, such as ACTN2, NEBL, ABLIM1, PDLIM5, and the proteins connecting the Z-line to the M-line in the sarcomere, such as TITIN. The Z-line provides an essential anchor for sarcomerogenesis and contractile function (ACTN2 splicing creates a premature STOP codon that leads to the activation of non-sense-mediated decay (NMD) and consequently to the striking downregulation of ACTN2 expression -induced cell death in cardiomyocytes by antagonizing the elevation of the proapoptotic transcription factor FoxO1 in response to cardiac injury . FoxO1 aQKI expression can be strongly induced in vascular smooth muscle cells (VSMCs) in response to vascular injury and is shown to be involved in VSMC dedifferentiation via its regulation of alternative splicing of Myocd, a well-known master transcription factor in smooth muscle differentiation . PerturbA recent study demonstrated that QKI-6 regulates HDAC7 splicing and promotes VSMC differentiation from induced pluripotent stem cells (iPSCs). Remarkably, QKI-6-overexpressing smooth muscle cells show a greater contractile ability, and when cocultured with QKI-5-overexpressing endothelial cells, QKI-6-overexpressing smooth muscle cells exhibit a greater angiogenic potential , suggestQKI is highly expressed in both macro- and microvascular endothelial cells . Severalin vitro, which likely occurs via its activity in binding to the 3′UTR of VE-cadherin and β-catenin mRNA and the regulation of VE-cadherin and β-catenin mRNA translation (Interestingly, QKI-7 can be significant upregulated in mouse iPSC-derived endothelial cell when exposed to hyperglycemia mimicking diabetic pathophysiological condition . Importanslation . CollectThis mini review summarizes a series of recent exciting data on the physiological role of QKI in regulating numerous important features of cardiovascular development and function. As shown in XC and JY drafted the manuscript. DC and DX revised the manuscript. ZZ, YL, and WS finalized the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Intestinal necrosis and perforation are potentially life-threatening medical conditions that can lead to bacterial sepsis and systemic inflammatory response syndrome. Various etiologies can compromise the intestinal wall causing leakage of luminal contents including enteric bacteria and precipitate aggressive immunological responses including the complement system and neutrophils. RLS-0071 is a peptide inhibitor of the classical and lectin pathways and known modulator of various neutrophil mediated effectors including myeloperoxidase activity and NETosis.In this study we evaluated the extent to which immunomodulation via inhibition of the complement system and neutrophil effectors would alter survival in the setting of intestinal necrosis. Adolescent male Long-Evans rats were subject to cecal ligation and puncture (CLP) with one cohort receiving 40 mg/kg of RLS-0071 thirty minutes after surgery while the control group received no treatment. Survival of the rats was then assessed up to 5 days after surgery. Animals treated with RLS-0071 demonstrated nearly 1.5-fold increase in survival compared to the untreated group. In order to further elucidate the increase in survival we explored inflammatory responses as assessed by markers of NETosis i.e., free DNA in plasma, and the pro-inflammatory cytokine, IL-6. A reduction in blood levels of free DNA and the inflammatory cytokine IL-6 were observed for animals treated with RLS-0071.RLS-0071 increases survival of rats after cecal ligationKaplan-Meier survival curve assessment. The red line indicates the outcome after 75% CLP in animals not receiving treatment (n=13), whereas red curves represent the outcome after animals received a single dose of 40 mg/kg RLS-0071 (n=11).RLS-0071 reduces free DNA levels in the bloodPlasma was isolated before surgery (pre-bleed) (n=7) and from animals subject to CLP with (n=4) and without (n=3) RLS-0071 administration 24 hours post-surgery. Plasma samples were incubated with PicoGreen. Fluorescence was read at an excitation wavelength of 485 nm and an emission wavelength of 520nm in a microplate reader. Data are means and standard error of the meansRLS-0071 reduces IL-6 levels in the bloodPlasma was isolated before surgery (pre-bleed) (n=8) and from animals subject to CLP with (n=3) and without (n=5) RLS-0071 administration 24 hours post-surgery. Plasma samples were analyzed in an IL-6 ELISA according to the manufacturer’s instructions. Data are means and standard error of the means.The results of these experiments demonstrate that RLS-0071 can increase survival after intestinal perforation by multi-pronged modulation of complement activation, neutrophil immune mechanisms and cytokine mediated inflammatory responses.Kenji Cunnion, MD, MPH, ReAlta Life Sciences Inc  Parvathi Kumar, MBBS, ReAlta Life Sciences Inc (Employee) Brittany Lassiter, BS, ReAlta Life Sciences Inc (Employee) Katherine LaValle, DVM, ReAlta Life Sciences Inc (Employee) Neel Krishna, PhD, ReAlta Life Sciences Inc


MOFs applications in indoor-air VOCs pollutants cleaning, however, are less concerned partially because of limited diffusion of both gaseous pollutant molecules and photo-induced active species in very porous MOFs structures. In this mini-review, we focus on the major breakthroughs of MOFs as photocatalysts for the effective removal of indoor-air VOCs such as aldehydes, aromatics and short-chain alcohols. According to their nature of photoactive centers, herein MOFs photocatalysts are divided into two categories to comment, that is, MOFs with variable valence metal nodes as direct photoactive centers and MOFs with non-variable valence metal nodes but after combining other photoactive variable valence metal centers as excellent concentrated and concerted electron-transfer materials. The mechanisms and current challenges of the photocatalytic degradation of indoor-air VOC pollutants by these MOFs will be discussed as deeply as possible.With their outstanding diversities in both structures and performances, newly emerging metal-organic frameworks (MOFs) materials are considered to be the most promising artificial catalysts to meet multiple challenges in the fields of energy and environment. Especially in absorption and conversion of solar energy, a variety of MOFs can be readily designed to cover and harvest the sun irradiation of ultraviolet (UV), visible and near-infrared region through tuning both organic linkers and metal nodes to create optimal photocatalytic efficiency. Nowadays, a variety of MOFs were successfully synthesized as powerful photocatalysts for important redox reactions such as water-splitting, CO Garcia oxylate) . From thoxylate) . Howeveroxylate) , especiaoxylate) .4+/3+, Fe3+/2+, Cu2+/1+ as nodes, they act as photo-induced redox sites by themselves directly to initiate indoor VOCs degradation. For the latter, because MOFs are made of non-variable valence metal such as Zr4+, Zn2+ ion as nodes, they must combine other photoactive centers in which MOFs themselves act as excellent concentrated and concerted ET role to enhance photo-degradation. Some paragon examples of these two kinds of MOFs photocatalysts applied in decomposition of representative indoor air VOCs will be discussed in detail. The motivation of this mini-review is based on MOFs photocatalytic materials recently emerging of the outstanding diversities in both porous structures and performances that may just meet two challenges, namely, the restricted lighting-area and difficult diffusion of trace VOCs in the elimination of indoor air pollutants. To demonstrate and comment on photocatalytic removal of indoor air pollutants in terms of metal node redox properties of MOFs is the main innovation of this review.In this mini-review, we will focus on two kinds of MOFs photocatalysts for typical indoor air VOCs treatments according to whether the metal nodes in MOFs are or not photoactive centers. For the former, since MOFs materials bear variable valence metal ion such as Ti•OH radicals  in traditional photocatalytic degradation of aqueous organic pollutants is invalid. Only the direct hvb+ oxidative degradation of VOCs is left behind . At this time, gaseous O2 will play pivotal roles: one is to rapidly capture, remove the conduction-band electron (ecb−) so as to efficiently generate more hvb+ to oxidize VOCs; another is directly adding to the VOCs• intermediates (VOCs• + O2 → VOCs-•OO), or the secondary products with ecb− including O2−•, •OOH, H2O2 by ET or the singlet 1O2 via energy transfer, which will diffuse in the pores and react with VOCs pollutants. The hvb+ cannot leave the surface to react with VOCs via indirect solvent-relay route, while O2 and its secondary active species can diffuse and migrate in MOFs channels. Thus, the design of MOFs structure will mainly focus on the O2 diffusion channels and favorable reaction sites (see 2/g) can assist to pre-concentrate and store a large amount of VOCs. Once upon sunlight irradiation, the enriched VOCs can conveniently diffuse from the internal pores to the surface to be photo-degraded  Due to the gas-solid interface reaction traits, the highly effective oxidative degradation nearly for any organics by highly active ites see  (Li et aites see . 2) Fromaded see  (Saouma aded see . 3) Combaterials . For theency see . The cri6 octahedral coordination and Ti4+/Ti3+ redox similar to the star heterogeneous photocatalyst TiO2, Ti-based MOFs are investigated in depth for the purpose of the semiconductor photocatalysts for VOCs removal. By a microwave heating procedure, NH2-MIL-125 (Ti) was hybridized with graphene oxide (GO) to form a heterojunction photocatalyst (2-MIL-125(Ti) sample exhibits much enhanced photo-degradation activity of gaseous acetaldehyde under visible-light irradiation (λ > 420 nm) in comparison with mechanically mixed two components and unmodified NH2-MIL-125(Ti). The authors attributed the superior photocatalytic performance of the hybridized photocatalyst to the enhanced visible-light absorbance, more electron carrier density, more efficient charge carrier transfer and less electron-hole recombination. This example indicates that the formation of heterojunction can considerably improve the MOFs photocatalyst performance in VOCs remediation.Due to the TiOcatalyst . As-prepZhao et al. developed a N/Zn co-decoration strategy to strengthen the photocatalytic activity of MIL-125(Ti) towards indoor-air acetaldehyde oxidative degradation . N and Z2-MIL-125(Ti) nanohybrid photocatalyst can realize ultra-rapid degradation of acetaldehyde even under high humidity (4 ionic-liquid was exploited to dissolve the PANI to oligomers and graft the in-situ formed PANI oligomers to NH2-MIL-125(Ti). The smart-grafted IL-PANI/NH2-MIL-125(Ti) exhibits strong interfacial π-π conjugation and van der Waals interactions between NH2-MIL-125(Ti) and PANI oligomer. Besides, the IL-treated PANI/interfacial displays more defects such as ligand vacancies and Ti3+ sites thereby created much enhanced Lewis acidity, which is critically important for effective adsorption of acetaldehyde with Lewis basicity (O lone pair). Interestingly, water molecules can enhance the performance of this photocatalyst. From the electron paramagnetic resonance (EPR) trapping experiments, H2O participates in ROS generation by reacting with O2−• to yield more active hydroxyl radicals to oxidize acetaldehyde. The IL-treated PANI-Ti-MOFs exhibited much increased affinity to acetaldehyde and acetic acid in comparison with CO2, which improves the retention of the intermediate acetic acid in the photocatalyst reactive site and the desorption of the final innocuous CO2 product.The same group discovered that by an ionic-liquid assisted synthetic procedure, defect-rich polyaniline (PANI)/NHhumidity . [EMIM]Bin-situ solvothermal synthesis of NH2-UiO-66 (Zr) in the presence of pre-formed TiO2 nanoparticles, a TiO2/NH2-UiO-66 (Zr) composite photocatalyst was successfully prepared. . The authors proposed that due to the affinity of styrene with Zr-MOF’s linker 2-aminoterephthalate by strong π-π interaction, the VOC (styrene) could be concentrated in the NH2-UiO-66 interior pores. The encapsulated TiO2 photoactive sites can contact more conveniently and react with the neighboring styrene inside the porous structure in comparison with styrene in the outer surface. Besides the easier diffusion and reaction of VOCs substrate, the photo-generated electrons and holes could be transferred and separated between NH2-UiO-66 (Zr) and encapsulated TiO2. Namely, the elaborate conjugation of semiconductors with stable Zr-MOFs significantly improves its photocatalytic activity for indoor VOCs degradation through concerted strong interfacial effects.Apart from MIL-125(Ti)-based MOFs materials, their Zr-MOF counterparts exhibit much enhanced stability towards humidity and long-term light-irradiation. Thus, it is also highly desirable to apply Zr-MOFs for photocatalytic VOCs degradation especially in highly humid environment. To just take advantage of the hydrolytic stability, if proper semiconductor or metal nanoparticles as photoactive centers were hybridized with Zr-MOFs, their photocatalytic performances can be considerably improved. By an repared. . Ultra-s2-UiO-66-NH2 composite photocatalyst for gaseous toluene and acetaldehyde removal. 4 in the presence of pre-formed UiO-66-NH2, more effective photocatalytic removal of toluene can be achieved comparing with TiO2 and unmodified UiO-66-NH2. -porphyrin, a novel Fe@PCN-224 photocatalyst was constructed  ions into the center of porphyrin units of PCN-224 MOF photocatalyst bonded Zr-oxo cluster with structed . Its phoZn-based zeolite imidazole framework ZIF-8 is another kind of highly stable MOFs against hydrolytic and photo-irradiation conditions . To expl2 comparing with its original ZIF-8 parent. Both the augmentation of light absorption, transmittance and formaldehyde adsorption result in much enhanced photocatalytic removal efficiency of indoor HCHO. Interestingly, water in this system can accelerate the generation of strongly oxidative H2O2 species thereby enhance the degradation of formaldehyde in moist air, by which further boosts its activity and stability for this porous photocatalyst under humid environment.Although ZIF-8 photocatalyst can provide excellent performance under UV irradiation, it rarely has visible-light activity. To create its visible-light activity, a post-modification strategy was applied to fine-tune the chemical structure of the ZIF-8 photocatalyst.. After h2-MIL-101(Fe) MOFs also act as efficient photocatalyst for VOCs degradation under irradiation conditions. Cu-based HKUST-1 MOF material merged with anatase TiO2 semiconductor to form a core/shell structure with Cu3(BTC)2  as the core and ultra-thin anatase film as a shell  photocatalyst was reported to effectively remove the toluene in hermetic space  process. Moreover, the Fe3-μ3-oxo cluster can directly absorb the visible-light and initiate an electron-hole separation event. Photo-induced electron and hole migrate separately to the catalyst surface to react with the adsorbed O2 and H2O molecules, yielding strongly oxidative ROS species such as O2−• and •OH to degrade toluene into CO2 and H2O by consecutive radical addition, hydroxylation, ring-opening and chain-cleavage processes.As an earth-abundant and non-toxic element, iron-based materials are considered as promising catalysts for remediation of indoor air. NHic space . The micTi-Based MOFs Photocatalysts for Degradation of Indoor Air VOCs and Other Metal-Based MOFs Photocatalysts for Degradation of Indoor Air VOCs section is now summarized in 2 to improve its light harvesting (2 groups. 2) Location of the Pt and metalloporphyrin cocatalyst in MOF composites can significantly boost electron−hole separation  Photllutants . All of llutants .2. Such poor mineralization results in the accumulation of organic intermediates on the surface of photocatalysts, which usually leads to catalyst deactivation and blocks the catalytic cycle. Although there have been some MOFs-based photocatalysts with excellent stability for moisture under long-term illumination, the weak coordinative bonds still restrain the discoveries of more powerful MOFs-based photocatalysts for VOCs removal. Lastly, up till now, there still lacks MOFs photocatalysts to realize indoor VOCs removal under NIR irradiation due to the low-energy of NIR photon not enough cleaving the high-energy VOCs chemical bonds. This considerably decreases the solar-light-utilization efficiency as NIR possesses about half of the overall energy of sunlight. The doping, co-doping, dye-sensitization can be useful methods to extend absorption spectrum and enhance charge-carrier separation. The utilization of the organic ligands with electron donor-acceptor structure is another potential tactic to enhance the visible-light absorption or even to induce NIR absorptivity. The merging of two-photon absorbing organic moiety may initiate its NIR photocatalytic ability for indoor VOCs degradation (Despite some successful applications of MOFs photocatalysts for the removal of indoor-air VOCs pollutants , there sradation . Althoug


To the Editor: We read with interest the article by Amit et al. (https://www.pfizer.com). Reverse transcription PCR of a nasopharyngeal swab sample after fatigue persisted beyond 1 week of vaccination showed a positive result for SARS-CoV-2. The patient later showed development of severe coronavirus disease (COVID-19) pneumonia, requiring admission to an intensive care unit.We report a 60-year-old previously healthy man who had isolated, progressively worsening fatigue starting 1 day after receiving a second dose of mRNA-based vaccine (Pfizer/BioNTech, HCWs are at a higher risk for exposure and more likely to get tested if symptoms develop after vaccination, as in the study by Amit et al. (COVID-19 vaccines are efficacious in reducing transmission of symptomatic COVID-19 (


To evaluate the predictive ability of a deep learning-based algorithm to determine long-term best-corrected distance visual acuity (BCVA) outcomes in neovascular age-related macular degeneration (nARMD) patients using baseline swept-source optical coherence tomography (SS-OCT) and OCT-angiography (OCT-A) data.In this phase IV, retrospective, proof of concept, single center study, SS-OCT data from 17 previously treated nARMD eyes was used to assess retinal layer thicknesses, as well as quantify intraretinal fluid (IRF), subretinal fluid (SRF), and serous pigment epithelium detachments (PEDs) using a novel deep learning-based, macular fluid segmentation algorithm. Baseline OCT and OCT-A morphological features and fluid measurements were correlated using the Pearson correlation coefficient (PCC) to changes in BCVA from baseline to week 52.Total retinal fluid  volume at baseline had the strongest correlation to improvement in BCVA at month 12 . Fluid was subsequently sub-categorized into IRF, SRF and PED, with PED volume having the next highest correlation  to BCVA improvement. Average total retinal thickness in isolation demonstrated poor correlation . When two features, mean choroidal neovascular membranes (CNVM) size and total fluid volume, were combined and correlated with visual outcomes, the highest correlation increased to PCC = 0.695 (p = 0.002).In isolation, total fluid volume most closely correlates with change in BCVA values between baseline and week 52. In combination with complimentary information from OCT-A, an improvement in the linear correlation score was observed. Average total retinal thickness provided a lower correlation, and thus provides a lower predictive outcome than alternative metrics assessed. Clinically, a machine-learning approach to analyzing fluid metrics in combination with lesion size may provide an advantage in personalizing therapy and predicting BCVA outcomes at week 52. Age-related macular degeneration (ARMD) remains the leading cause of blindness worldwide, with the prevalence expected to rise to 288 million by 2040 . The maiOptical coherence tomography angiography (OCT-A) has revealed further retinal anatomical and vascular structures to track nARMD progression . Our 202Machine learning has bridged the gap in OCT imaging interpretation of biomarkers and nARMD disease monitoring. Rohm et al. in 2018 found that deep learning networks showed good visual predictions at 3-months following anti-VEGF treatment for nARMD . SchleglWhile AI models have postulated promise in OCT fluid segmentation and have demonstrated predictive value in anti-VEGF treatment outcomes, there are currently no trials demonstrating machine learning with swept-source OCT images in concordance with OCT-A analysis for nARMD patients. Therefore, our study was conducted as a follow-up, phase IV, proof-of-concept trial to evaluate a novel deep learning algorithm to determine BCVA outcomes in nARMD patients using both OCT and OCT-A imaging.Patients who previously completed the CANADA study were enrolled in this phase IV, retrospective, open-label proof-of-concept study. Analysis, as part of this study, began in July 2020 and ended in December 2020. The selection of participants for the CANADA study has been previously reported . Twenty-The key inclusion criteria were: (1) ≥ 25 years of age; (2) diagnosis of nARMD; (3) treatment naïve and (4) previously enrolled in the CANADA study. Exclusion criteria included patients with uncontrolled systemic hypertension or thromboembolic events  within 6 months from baseline, ocular conditions affecting visual acuity besides nARMD , ocular or periocular infection or active intraocular inflammation, hypersensitivity to aflibercept or any ingredient in the formulation, previous ocular surgery (including cataract extraction or YAG capsulotomy) within 3 months from baseline, planned ocular surgery throughout the study or previous treatments of laser photocoagulation or intravitreal anti-VEGF or steroid treatments. Pregnant women, nursing women, or patients unwilling to provide informed consent were also excluded.Participants were not treated as part of the ONTARIO study; however, participants were enrolled from the CANADA study where they were treated with initial 3 monthly injections (loading phase) of intravitreal aflibercept 2 mg at baseline, month 1 and month 2, then every other month for a total of 12 months (52 weeks).As part of the CANADA study, all patients underwent SS-OCT-A testing using the Topcon Triton Swept Source OCT  to identify CST and lesion size at baseline . The SS-Baseline 7 mm x 7 mm 3D macula volume scans containing 512 A-scans and 256 B-scans were segmented using a prototype version of the Orion software . This performs layer segmentation as a pre-processing step to fluid segmentation that uses a deep learning-based algorithm. Performance of the fluid segmentation has previously been validated relative to two expert graders (SS and JO) .A deep learning-based algorithm was implemented to segment fluid regions within each OCT-volume. As reported previously, the method takes each OCT B-scan as input, and a segmentation mask is generated automatically using Orion software  , 17. The2) at baseline.All data are presented as either mean or mean ± SD. The BCVA was converted to the logarithm of the minimum angle of resolution (logMAR) for statistical purposes. Counting fingers vision was given a value of 1.85, and hand motions vision was given a value of 2.30 . StatistTwenty-three patients (25 eyes) completed the CANADA study and were enrolled in this subsequent study. One patient was excluded based on image quality and subsequent layer segmentation issues. Therefore, the final cohort consisted of 22 patients (24 eyes). The average age of the patients was 76 years (range 48–89 years). Fifteen of the 22 treated patients were female and 7 of the 22 treated patients were male. Fifteen of the 24 eyes were pseudophakic and 2 of 24 eyes were phakic at baseline and were pseudophakic at week 52. For the entire cohort, the mean BCVA at baseline was 20/125 and improved to 20/80 by week 52 (p<0.001).Twenty-two SS-OCT volumes of the macula, comprising 5,632 images from the 22 nARMD subjects in the final cohort, were used to quantify IRF, SRF and fluid in serous PEDs. Assessment used 10-fold cross-validation, where each fold ensured no subject eye data was in both the training and testing data. In each fold, the training set used data augmentation such that one volume, comprising 256 B-scans, was replicated six times using random scaling, rotations and shifting. Given these were large input volumes and a heterogeneous input data set, the reported validation compared excellently to manual segmentations. Results showed strong correlation in all fluid volumes between the algorithm and the manually labeled data .2). Of the final cohort of 22 patients (24 eyes) used in the fluid segmentation, 17 patients (18 eyes) had OCT-A data due to data or segmentation issues in 5 patients [OCT-A analysis was performed as part of the CANADA study to determine the mean CNVM lesion size (μmpatients . There w2) from 3 mm OCT-A scans was very low . When two features were combined and correlated with visual outcomes, the best correlation increased to PCC = 0.695 (p = 0.002) using mean CNVM size and total fluid volume  . Fluid wd volume .Bland-Altman plots were used to assess the differences in agreement. In looking at the limits of agreement (LOA) in the Bland-Altman plots, we can evaluate the bias between the mean differences. The use of AI to automate the analysis of ocular images and allow quantification of retinal biomarkers has increased in popularity globally. Previous studies in nARMD have used spectral domain OCT (SD-OCT) to quantify fluid and used this data to predict BCVA outcomes . Our stuAssociating structural parameters derived from OCT data to functional outcomes has been a mainstay of clinical OCT since the ground-breaking PrONTO study . DevelopCorrelation in isolation provides no information about the differences in agreement and thus additional plots need to be employed . In thisThe use of swept-source scans has the potential to increase the precision and recall of our automated algorithm as swept-source devices use a narrower wavelength of light and captures 100,000 A-scans per second, rather than ~68,000 A-scans that are captured by SD-OCT devices . The incDue to its historical use in clinical trials, CST is currently used extensively for assessing treatment response and determining the next treatment date , 29. TheQualitative features of OCT-A scans, including medusa form, sea-fan form, pruned vascular tree pattern, tangled network pattern and vascular loop, can be assessed without the need for adjuvant software , 31. A lSeveral OCT and OCT-A biomarkers have been linked to visual outcomes in nARMD, including, but not limited to, branching patterns, total number of branch points, junction density, hyperreflective foci –38. In tThis study demonstrates the clinical implementation of a novel, deep learning-based algorithm and the importance of including OCT-A quantitative parameters to existing fluid analysis algorithms to increase the predictive power. Delineating lesion size on OCT-A scans either requires time-intensive manual segmentation or additional automated software, however the inclusion of OCT-A-related parameters may be key to accurately predicting long-term visual outcomes.


Genome-wide association studies (GWAS) have identified and reproduced thousands of diseases associated loci, but many of them are not directly interpretable due to the strong linkage disequilibrium among variants. Transcriptome-wide association studies (TWAS) incorporated expression quantitative trait loci (eQTL) cohorts as a reference panel to detect associations with the phenotype at the gene level and have been gaining popularity in recent years. For nicotine addiction, several important susceptible genetic variants were identified by GWAS, but TWAS that detected genes associated with nicotine addiction and unveiled the underlying molecular mechanism were still lacking. In this study, we used eQTL data from the Genotype-Tissue Expression (GTEx) consortium as a reference panel to conduct tissue-specific TWAS on cigarettes per day (CPD) over thirteen brain tissues in two large cohorts: UK Biobank  = 142,202) and the GWAS & Sequencing Consortium of Alcohol and Nicotine use , then meta-analyzing the results across tissues while considering the heterogeneity across tissues. We identified three major clusters of genes with different meta-patterns across tissues consistent in both cohorts, including homogenous genes associated with CPD in all brain tissues; partially homogeneous genes associated with CPD in cortex, cerebellum, and hippocampus tissues; and, lastly, the tissue-specific genes associated with CPD in only a few specific brain tissues. Downstream enrichment analyses on each gene cluster identified unique biological pathways associated with CPD and provided important biological insights into the regulatory mechanism of nicotine dependence in the brain. The past decade has witnessed an explosion in genome-wide association studies (GWAS) research, which identified thousands of robust, reproducible genetic risk variants associated with complex diseases and traits ,2. TheseCYP2A6 . For GSCAN, only meta-analyzed GWAS summary data were available.GWAS & Sequencing Consortium of Alcohol and Nicotine use (GSCAN): a meta-analysis of up to 35 GWAS cohorts of European ancestry including around 1.2 million individuals (depending on traits) . SmokingIn this study, we performed TWAS analysis on two large cohorts that include both genotype and nicotine addiction phenotype data:Cigarettes per day (CPD) is one of the well-known traits related to nicotine addiction and is widely used in many published studies ,31. We uhttps://hakyimlab.org/post/2020/01/07/predictdb-transcriptome-prediction-model-repository/, accessed on 16 November 2021) for use in our study.The reference panel of the eQTL cohort used to perform TWAS analysis was obtained from the Genotype-Tissue Expression (GTEx) project (version 8) . It inclIn this study, we first conducted tissue-specific TWAS (TS-TWAS) of CPD for each of the 13 brain tissues by combining GWAS data with the eQTL reference panel and then performed meta-analysis to combine the TS-TWAS results and categorize the CPD associated genes by their meta-patterns across the tissues . Below, www.cog-genomics.org/plink/1.9/, accessed on 16 November 2021) [p-value below 0.001, and missing genotype rate at 5%, and excluded individuals with more than 2% missing genotypes. The UKBB cohort had relatively low prevalence of self-reported health conditions  and light-to-moderate consumptions of alcohol and cannabis  [p-value for the g-th gene in s-th tissue as In the first step, we conducted TS-TWAS for each of the 13 brain tissues using S-PrediXcan . For UKBer 2021)  under ancannabis ; thus, wer 2021) . For GSCer 2021) . We inteer 2021) , an exteer 2021)  that usep-values from multiple studies while taking the study to study heterogeneity into account. In this paper, we treated different brain tissues as studies and applied AW-Fisher’s method to meta-analyze the TS-TWAS results from all S = 13 brain tissues. The null hypothesis in meta-analysis is commonly considered asp-value of the g-th gene in the s-th tissue from TS-TWAS, p-value of the observed weighted statistic p-value among all possible weights. For inference, there is no closed-form distribution for AW-Fisher’s statistics under the null, so permutation tests and importance sampling are used to obtain the p-values Adaptively weighted (AW)-Fisher’s method  is a meter paper .p-value < 0.05) helped us understand the unique functions for each category of genes associated with nicotine addiction but with different cross-tissue patterns. We also applied the S-MultiXcan method [After meta-analysis, we focused on genes passing an FDR threshold of 0.05 in both cohorts  and performed downstream analysis. We categorized the genes by their meta-patterns across brain tissues using hierarchical clustering with Ward linkage on −log10ogy (GO) , Kyoto Eogy (GO) , and Reaogy (GO) . Top enrn method  for multWe first performed TS-TWAS and then meta-analyzed the TS-TWAS results over 13 brain tissues by AW-Fisher’s method. The meta-analysis of TS-TWAS identified 48 genes significantly associated with CPD at FDR < 0.05 common in both UKBB and GSCAN cohorts . CompariCHRNA5, NCKIPSD, and SIRT6. The second cluster consisted of 8 genes including PSMA4 and RPRD2, which are highly expressed and associated with CPD in forebrain regions such as frontal cortex BA9, anterior cingulate BA24, and hindbrain regions including the cerebellum and cerebellar hemisphere. The third cluster included 10 heterogeneous genes only associated with CPD in very few specific brain tissues.Gene categorization by meta-patterns identified three clusters of genes common to both cohorts : (i) homCHRNA5) was mainly enriched in pathways related to presynaptic and postsynaptic nicotinic acetylcholine receptors  was enriched in pathways related to proteasomal activity , intercellular bivalent cations Mg2+ , and chromosome segregation  that can be highly impacted by cigarette smoking to inhibit proteasomal activity, cause mental disorders disease, and induce segregation anomalies separately reported in previous studies [CHRNA3) was enriched in pathways GO:MF acetylcholine-activated cation-selective channel activity and GO:CC acetylcholine-gated channel complex. Genes with different meta-patterns across tissues were functionally specific that are worth further investigation in future studies.We then performed pathway analysis to each cluster of identified genes. Cluster 1 of homogeneous genes . Cluste studies ,40,41. CCHRNA5, PSMA4, and CHRNA3, were identified and their cross-brain-tissue-association patterns were revealed. To the best of our knowledge, our study was the first comprehensive meta-analysis of TWAS on nicotine addiction across 13 major brain tissues, investigated and validated in two large-scale epidemiological cohorts (UKBB and GSCAN).In this study, we used an eQTL reference panel from GTEx to conduct meta-analysis of TS-TWAS on nicotine addiction over 13 brain tissues in two large cohorts, UKBB and GSCAN. The meta-analysis was shown to be more powerful than the multi-tissue TWAS method implemented in S-MultiXcan , by deteCHRNA5. Previous GWAS have identified multiple reproducible variants in CHRNA5 [CHRNA5 was highly expressed in all brain tissues, and its association with CPD was consistent throughout the brain. The second cluster was marked by the proteasome gene PSMA4. Its association with CPD was prominent in the frontal lobe , cerebellum/cerebellar hemisphere, and hippocampus tissues. The enriched biological pathways were driven by PSMA4, playing a central role in decreasing neuronal proteasome activity [CHRNA3, was observed in the third cluster with tissue-specific association with CPD only in putamen basal ganglia and nucleus accubens basal ganglia. The elevated dopamine activity in the basal ganglia region of cigarette smokers has been identified for the enriched pathways driven by CHRNA3 in the previous studies [The first cluster of genes was enriched in pathways related to presynaptic and postsynaptic nicotinic acetylcholine receptors, as marked by the gene n CHRNA5 ,31, whicn CHRNA5 . We furtactivity . Another studies ,45. ThesTWAS are getting popular over recent years as a promising complement to GWAS by incorporating the functional annotation information and analyzing association with the trait at the gene level. Despite the foreseen success, most TWAS methods to date are tissue specific and ignore the similarity in transcription regulation across tissues, usually having a limited effective sample size and, thus, being underpowered . Our stuLastly, our results are based on analyzing two big cohorts, UKBB and GSCAN, so are more generalizable to the general population. Though the UKBB cohort consists of mainly healthier participants, health volunteer selection bias will not largely affect the credibility of results, as has been noted in several previous studies ,54,55. F