Comprehensive Sequence Analysis Research Articles

Phylogeny, alkenone profiles and ecology of Isochrysidales subclades in saline lakes: Implications for paleosalinity and paleotemperature reconstructions

Long-chain alkenones (LCAs) produced by phylogenetically classified Groups 1 and 3 Isochrysidales are increasingly used for paleotemperature and/or paleosalinity reconstructions in oligohaline lakes and marine environments. However, there are considerable difficulties in the paleoenvironmental interpretation of LCAs from Group 2 Isochrysidales thriving in saline lakes. The biggest challenge lies in our poor understanding of the complexity and ecological niches of individual Group 2 subclades in saline lakes. Here, we perform comprehensive analysis of haptophyte-specific 18S rRNA sequences and distributions of LCAs, and long-chain alkenoates (LCEs) in surface sediments and suspended particulate matter (SPM) from 37 saline lakes in northern China. These lakes span a large salinity gradient from 0.5 to 308‰. Combined with published genomic data of Group 2 Isochrysidales, our phylogenetic analysis reveals three Group 2 subclades occupying distinct ecological niches: one ice-related bloomer Group 2i and two warm-season bloomers Groups 2w1 and 2w2. Group 2i, the earliest seasonal bloomer, frequently co-occurs with Group 2w1 in sediments from saline lakes with relatively low to intermediate salinity waters, whereas Group 2w2 blooms in hypersaline waters. Based on existing data, C39:4 methyl alkenone is a chemotaxonomic biomarker for Group 2i. %C37:4 (relative abundance of C37:4 to the total C37 alkenones) values in the three Group subclades follow the order: Group 2i > Group 2w2 > Group 2w1. The %C37:4 in sediment cores of saline lakes does not directly record past salinity changes, but instead reflects variable contributions in production by these three subclades. This could indirectly and partially reflect overall salinity changes in some lakes dominated by Groups 2i and 2w1, but can be more complicated in lakes dominated by other assemblages. For our sites, we also demonstrate that direct use of C37 alkenone unsaturation indices (U37K, U37K', and U37K'') for paleotemperature reconstructions in saline lakes is generally not feasible, except for cases where alkenone-producing Isochrysidales are dominated by one single species/subclade and seasonal production effects can be circumvented. We propose two possible alternative proxies for paleotemperature reconstructions in saline lakes: 1) unsaturation ratios of C41 and C42 alkenones, as these compounds are predominantly produced by a limited number of Group 2 species, such as Isochrysis nuda (Liao et al., 2020); 2) C38Et/C36OEt ratio (ratio of C38 ethyl alkenones and C36 ethyl alkenoates), which appears to have similar temperature sensitivity for Groups 2w1 and 2w2, in lakes with no Group 2i inputs. Our study provides new insights into the phylogenetic classifications of Group 2 Isochrysidales and their ecological/environmental niches, which are fundamental for more quantitative and rigorous applications of LCAs and LCEs in saline lakes as paleosalinity and paleotemperature proxies.

Mesenchymal Stromal Cells (MSCs) from Myelodysplastic Syndromes (MDS) Are Not Clonally Mutated I n Vivo

IntroductionThere is increasing evidence for an active role of the bone marrow (BM) microenvironment in the pathogenesis of Myelodysplastic Syndromes (MDS). Genetically engineered murine models have shown that isolated mutations in the BM niche can disrupt the non-mutated hematopoietic compartment and induce MDS-like phenotypes. However, it is still unclear whether primary MDS in humans may possibly be associated with acquired mutations non-hematopoietic BM stroma cells. Although chromosomal aberrations and mutations have been described in in ex vivo expanded MSC cultures from MDS and AML patients, little validation has been performed to address whether such molecular lesions were not clonal outgrowths resulting from the strenuous and massively expansive cell culture procedures.Materials and MethodsWe performed whole exome sequencing on paired ex vivo expanded MSCs and native BM samples of n=98 MDS and associated myeloid neoplasia cases treated at the Department of Hematology and Oncology of the Medical Faculty Mannheim, Heidelberg University, Germany (median age 73 years, range 44-86). As controls, we included a cohort of n=28 samples from healthy subjects (median age 75 years, range 36-84). MSCs were expanded adherently on plastic dishes by seeding 5x10e6 mononuclear cells in StemMACS MSC Expansion Medium XF (Miltenyi Biotec) for a median of 34 days, (95% confidence interval 22-50d). Whole exome sequencing was carried out using Nextera DNA Flex Tagmentation kit (Illumina) with IDT xGene Research probe v1 at a median coverage at 88x with BM MNC as germline control accounting for possible LOH in the BM sample. Validation experiments were performed by deep re-sequencing of single CFU-F colonies (n=4 patients), sequencing of serial cultures (n=7 patients) and re-sequencing of primary sorted native bone marrow MSCs from n=9 patients.ResultsIn the exome sequencing analyses of ex vivo expanded MSCs we discovered multiple recurrent mutations in MSCs of MDS patients including but not limited to genes such as ZFX (n=8/98) and RANK (n=5/98). MSCs from MDS patients displayed an overall higher mutational burden and increased replicative stress as determined by gH2AX and RPA staining, which correlated with the mutational burden and shorter telomeres as compared to healthy controls. The analysis of mutational signatures revealed that MDS MSCs were distinct compared to healthy MSCs. Furthermore, we found that MDS MSCs displayed increased senescence assessed by flow bGAL staining and associated inflammatory gene expression determined by IL6 qPCR/ELISA for n=32 cases.To investigate whether acquired mutations in MSCs were driven by the ex vivo expansion we performed individualized amplicon based deep re-sequencing of serial culture passages and different BM aspirations for n=7 patients as well as single colony re-sequencing in n=4 patient cases. Furthermore, we re-sequenced primary sorted CD45-,CD235a-,CD31+/-,CD271+/- BM cells of n=9 cases. All of these validation experiments indicated that the discovered mutations were associated with expansion in culture and but not present in clonally relevant cell populations in the primary BM in vivo.DiscussionTogether with previously published data of the BM niche of myeloid neoplasms, our results add to the notion that MSCs in MDS are molecularly and functionally altered. Nevertheless, our current comprehensive sequencing analyses leave little doubt that if acquired mutations in the stroma of MDS patients play a role in MDS disease initiation at all, then at such a low clonal and possibly locally confined level, that they are not detectable with currently feasible sample acquisition and methodology. In our current study, we discovered no evidence for acquired mutations in BM derived MSCs in MDS. DisclosuresSchmitt: Affimed GmbH: Research Funding. Flach: Gilead: Current Employment. Hofmann: BMS: Honoraria; Amgen: Honoraria; Novartis: Honoraria. Nowak: Pharmaxis: Current holder of individual stocks in a privately-held company, Research Funding; Celgene: Honoraria; AbbVie: Other: Investigator on funded clinical trial; Tolero Pharma, Pharmaxis, Apogenix: Research Funding; Affimed: Research Funding; Takeda: Honoraria.

Emergence and expansion of highly infectious spike protein D614G mutant SARS-CoV-2 in central India

COVID-19 has emerged as global pandemic with largest damage to the public health, economy and human psyche.The genome sequence data obtained during the ongoing pandemic are valuable to understand the virus evolutionary patterns and spread across the globe. Increased availability of genome information of circulating SARS-CoV-2 strains in India will enable the scientific community to understand the emergence of new variants and their impact on human health. The first case of COVID-19 was detected in Chambal region of Madhya Pradesh state in mid of March 2020 followed by multiple introduction events and expansion of cases within next three months. More than 5000 COVID-19 suspected samples referred to Defence Research and Development Establishment, Gwalior, Madhya Pradesh were analyzed during the nation -wide lockdown and unlock period. A total of 136 cases were found positive over a span of three months that included virus introduction to the region and its further spread. Whole genome sequences employing Oxford nanopore technology were generated for 26 SARS-CoV-2 circulating in 10 different districts in Madhya Pradesh state of India. This period witnessed index cases with multiple travel histories responsible for introduction of COVID-19 followed by remarkable expansion of virus. The genome wide substitutions including in important viral proteins were identified. The detailed phylogenetic analysis revealed the circulating SARS-CoV-2 clustered in multiple clades including A2a, A4 and B. The cluster-wise segregation was observed, suggesting multiple introduction links and subsequent evolution of virus in the region. This is the first comprehensive whole genome sequence analysis from central India, which revealed the emergence and evolution of SARS-CoV-2 during thenation-wide lockdown and unlock.

A holistic fault impact analysis of the high-performance sequences of operation for HVAC systems: Modelica-based case study in a medium-office building

ASHRAE Guideline 36: High-performance sequences of operation (SOO) for Heating, Ventilation, and Air-conditioning (HVAC) Systems has been demonstrated to save 17%-30% energy under ideal simulation environments. However, HVAC systems are susceptible to various types of faults in a real building operation. There are no existing studies that pertain to a comprehensive fault impact analysis of the high-performance control sequences suggested by ASHRAE Guideline 36 for HVAC systems. How these sequences handle and adapt to the various types of faults is still largely unknown. In this context, a comprehensive fault impact analysis and robustness assessment of the high-performance control sequences is conducted. A Modelica-based medium office virtual testbed is developed following the air-side and the plant-side SOO. A total of 359 fault scenarios in three different seasonal operating conditions (cooling, shoulder, and heating seasons) are injected into the baseline model. The evaluated key performance indexes (KPIs) include the operational cost, source energy, site energy, control loop quality, thermal comfort, ventilation, and power system metrics. The faults of the most negative impact are identified for different seasonal operating conditions over all the KPIs. The results also show that high-performance control sequences are well adapted for the vast majority (∼90%) of all the fault scenarios over all the KPIs in this study.

High-Throughput Sequencing Reveals the Regulatory Networks of Transcriptome and Small RNAs During the Defense Against Marssonina brunnea in Poplar

MicroRNAs are implicated in the adjustment of gene expression in plant response to biotic stresses. However, the regulatory networks of transcriptome and miRNAs are still poorly understood. In the present study, we ascertained the induction of genes for small RNA biosynthesis in poplar defense to a hemibiotrophic fungus Marssonina brunnea and afterward investigated the molecular regulatory networks by performing comprehensive sequencing analysis of mRNAs and small RNAs in M. brunnea-inoculated leaves. Differentially expressed genes in M. brunnea-infected poplar are mainly involved in secondary metabolisms, phytohormone pathways, the recognition of pathogens, and MAPK pathway in the plant, with real-time quantitative PCR (qPCR) validating the mRNA-seq results. Furthermore, differentially expressed miRNAs, such as MIR167_1-6, MIR167_1-12, MIR171_2-3, MIR395-13, MIR396-3, MIR396-16, MIR398-8, and MIR477-6, were identified. Through psRobot and TargetFinder programs, MIR167-1-6, MIR395-13, MIR396-3, MIR396-16, and MIR398-8 were annotated to modulate the expression of genes implicated in transportation, signaling, and biological responses of phytohormones and activation of antioxidants for plant immunity. Besides, validated differentially expressed genes involved in lignin generation, which were phenylalanine ammonia-lyase, ferulate-5-hydroxylase, cinnamyl alcohol dehydrogenase, and peroxidase 11, were selected as targets for the identification of novel miRNAs. Correspondingly, novel miRNAs, such as Novel MIR8567, Novel MIR3228, Novel MIR5913, and Novel MIR6493, were identified using the Mireap online program, which functions in the transcriptional regulation of lignin biosynthesis for poplar anti-fungal response. The present study underlines the roles of miRNAs in the regulation of transcriptome in the anti-fungal response of poplar and provides a new idea for molecular breeding of woody plants.

Activation of the gut microbiota-kynurenine-liver axis contributes to the development of nonalcoholic hepatic steatosis in nondiabetic adults.

The contribution of gut-liver signaling to the development of non-alcoholic hepatic steatosis (NHS) in non-diabetic adults remains unclear. We therefore performed comprehensive 16S ribosomal RNA sequencing and fecal metabolomics analyses in 32 controls and 59 non-diabetic adults with NHS and performed fecal microbiota transplantation into germ-free mice using controls and NHS patients as donors. Compared to controls, the abundance of the genera Collinsella and Acinetobacter were higher, while that of Lachnospira was lower, in NHS subjects. Fecal metabolomics analysis showed decreased L-tryptophan levels and increased abundance of the tryptophan metabolite kynurenine in individuals with NHS. Correlation analysis showed that kynurenine levels positively associated with the abundance of Collinsella and Acinetobacter. ROC analysis demonstrated that the combination of tryptophan and kynurenine could discriminate NHS patients from controls with good statistical power [P < 0.05; AUC = 0.833 (95% CI, 0.747 to 0.918)]. Supporting a key role of dysbiotic gut microbiota in NHS development, incipient hepatic steatosis and increased kynurenine levels were observed in GF mice colonized with samples from NHS patients. These results indicate that enhanced kynurenine production resulting from altered gut microbiota composition contributes to NHS in nondiabetic adults and suggest the relevance of tryptophan metabolites as diagnostic biomarkers.

A comprehensive RRM domains sequence analysis from nucleolin proteins in plants

A comprehensive RRM domains sequence analysis from nucleolin proteins in plants

An additional NF-κB site allows HIV-1 subtype C to evade restriction by nuclear PYHIN proteins

SUMMARYSubtype C is the most prevalent clade of human immunodeficiency virus type 1 (HIV-1) worldwide. The reasons for this are poorly understood. Here, we demonstrate that a characteristic additional third nuclear factor κB (NF-κB) binding site in the long terminal repeat (LTR) promoter allows subtype C HIV-1 strains to evade restriction by nuclear PYHIN proteins, which sequester the transcription factor Sp1. Further, other LTR alterations are responsible for rare PYHIN resistance of subtype B viruses. Resistance-conferring mutations generally reduce the dependency of HIV-1 on Sp1 for virus production and render LTR transcription highly responsive to stimulation by NF-κB/p65. A third NF-κB binding site increases infectious virus yield in primary CD4+ T cells in an γ-interferon-inducible protein 16 (IFI16)-dependent manner. Comprehensive sequence analyses suggest that the frequency of circulating PYHIN-resistant HIV-1 strains is increasing. Our finding that an additional NF-κB binding site in the LTR confers resistance to nuclear PYHIN proteins helps to explain the dominance of clade C HIV-1 strains.

Comprehensive sequence and structure analysis of algal lipid catabolic enzyme Triacylglycerol lipase: an in silico study to vitalize the development of optimum engineered strains with high lipid productivity

Microalgae as an alternative renewable resource for biofuel production have captured much significance. Nonetheless, its economic viability is a field of major concern for researchers. Unraveling the lipid catabolic pathway and gaining insights into the sequence-structural features of its primary functioning enzyme, Triacylglycerol lipase, will impart valuable information to target microalgae for augmented lipid content. In the present study, a genome-wide comparative study on putative Triacylglycerol lipase (TAGL) enzyme from algal species belonging to varied phylogenetic lineages was performed. The comprehensive sequence analysis revealed that TAGL comprises of three distinct conserved domains, such as, Patatin, Class III Lipase, and Abhydro_lipase, and also confirmed the ubiquitous presence of GXSXG motif in the sequences analyzed. In the absence of a crystal structure of algal TAGL till date, we developed the first 3D model of patatin domain of TAGL from an oleaginous microalga, Phaedactylum tricornutum, employing homology modeling, docking and molecular dynamic simulations methods. The domain-substrate complex having the low-ranking docking score revealed the binding of palmitic acid to the TAGL patatin domain surface with strong hydrogen bond interactions. The simulation results implied that the substrate-complexed patatin domain and the free enzyme adopted a more stable conformation after 40 ns. This is the first ever attempt to provide in-silico insights into the structural and dynamical insights on catalytic mechanism of the TAGL patatin domain. Subsequently, these findings aided our understanding on their structural stability, folding mechanism and protein-substrate interactions, which could be further utilized to design site-specific mutagenic experiments for engineering microalgal strains. Communicated by Ramaswamy H. Sarma

Assembly and annotation of whole-genome sequence of Fusarium equiseti

Fusarium equiseti is a plant pathogen with a wide range of hosts and diverse effects, including probiotic effects. However, the molecular mechanisms underlying these effects remain unclear, hindering its effective utilization. The final assembly included 16 scaffolds of contiguous sequence without gaps. The total sequence length was 40,776,005 bp, and the GC content of 48.01%. In total, we annotated the putative function of 13,134 genes, accounting for 94.97% of the candidate genes. We identified two and 23 candidate genes that are likely involved in the production of mycotoxins zearalenone and trichothecene, respectively. A comparative genomic analysis supported the high quality of the F. equiseti assembly. Our comprehensive analysis of whole-genome sequence will serve as a valuable resource for future studies of expression, regulation, function and evolution of the genes of F. equiseti as well as studies into disease prevention and control.

Acquisition of an Additional NF-κB Site Allows HIV-1 Subtype C to Evade Restriction by Nuclear PYHIN Proteins

Subtype C is the most prevalent clade of HIV-1 worldwide. The reasons for this are poorly understood. Here, we demonstrate that a characteristic additional third NF-κB binding site in the LTR promoter allows subtype C HIV-1 strains to evade restriction by nuclear PYHIN proteins, which sequester the transcription factor Sp1. We also show that other LTR alterations are responsible for rare PYHIN resistance of subtype B viruses. Resistance-conferring mutations generally reduce the dependency of HIV-1 on Sp1 for virus production and render LTR transcription highly responsive to stimulation by NF-κB/p65. A third NF-κB binding site increased infectious virus yield in primary CD4+ T cells in an IFI16 dependent manner. Comprehensive sequence analyses suggest that the frequency of circulating PYHIN-resistant HIV-1 strains is increasing. Our finding that an additional NF-κB binding site in the LTR confers resistance to nuclear PYHIN proteins helps to explain the dominance of clade C HIV-1 strains.

Conserved functional motifs of the nuclear receptor superfamily as potential pharmacological targets

Nuclear receptors (NRs) are one of the most diverse and well-reported family of proteins. They are involved in numerous cellular processes as they play pivotal roles in cell signaling and the cell cycle. The participation of NRs in various applications in medicine and biology has greatly attracted the interest of the pharmaceutical industry for the discovery of novel and/or improved drugs for the treatment of several diseases, including cancer, diabetes or infertility. In the present study, in an effort to elucidate the molecular function of this superfamily and to identify novel pharmacological targets, a comprehensive sequence and structural analysis was performed using all available information from a repertoire of depositories. Functional conserved motifs were identified and analyzed with regards to their potential roles and implications in a number of biological processes. The essential differences among them were also addressed and discussed. In addition, these motifs were characterized in the main groups of the NRs, such as that of the steroid hormone receptors.

Genomic Determinants Encode the Reactivity and Regioselectivity of Flavin-Dependent Halogenases in Bacterial Genomes and Metagenomes.

ABSTRACTHalogenases create diverse natural products by utilizing halide ions and are of great interest in the synthesis of potential pharmaceuticals and agrochemicals. An increasing number of halogenases discovered in microorganisms are annotated as flavin-dependent halogenases (FDHs), but their chemical reactivities are markedly different and the genomic contents associated with such functional distinction have not been revealed yet. Even though the reactivity and regioselectivity of FDHs are essential in the halogenation activity, these FDHs are annotated inaccurately in the protein sequence repositories without characterizing their functional activities. We carried out a comprehensive sequence analysis and biochemical characterization of FDHs. Using a probabilistic model that we built in this study, FDHs were discovered from 2,787 bacterial genomes and 17 sediment metagenomes. We analyzed the essential genomic determinants that are responsible for substrate binding and subsequent reactions: four flavin adenine dinucleotide-binding, one halide-binding, and four tryptophan-binding sites. Compared with previous studies, our study utilizes large-scale genomic information to propose a comprehensive set of sequence motifs that are related to the active sites and regioselectivity. We reveal that the genomic patterns and phylogenetic locations of the FDHs determine the enzymatic reactivities, which was experimentally validated in terms of the substrate scope and regioselectivity. A large portion of publicly available FDHs needs to be reevaluated to designate their correct functions. Our genomic models establish comprehensive links among genotypic information, reactivity, and regioselectivity of FDHs, thereby laying an important foundation for future discovery and classification of novel FDHs.IMPORTANCE Halogenases are playing an important role as tailoring enzymes in biosynthetic pathways. Flavin-dependent tryptophan halogenases (Trp-FDHs) are among the enzymes that have broad substrate scope and high selectivity. From bacterial genomes and metagenomes, we found highly diverse halogenase sequences by using a well-trained profile hidden Markov model built from the experimentally validated halogenases. The characterization of genotype, steady-state activity, substrate scope, and regioselectivity has established comprehensive links between the information encoded in the genomic sequence and reactivity of FDHs reported here. By constructing models for accurate and detailed sequence markers, our work should guide future discovery and classification of novel FDHs.

Comparative analysis of human coronaviruses focusing on nucleotide variability and synonymous codon usage patterns

The prevailing COVID-19 pandemic has drawn the attention of the scientific community to study the evolutionary origin of Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2). This study is a comprehensive quantitative analysis of the protein-coding sequences of seven human coronaviruses (HCoVs) to decipher the nucleotide sequence variability and codon usage patterns. It is essential to understand the survival ability of the viruses, their adaptation to hosts, and their evolution.The current analysis revealed a high abundance of the relative dinucleotide (odds ratio), GC and CT pairs in the first and last two codon positions, respectively, as well as a low abundance of the CG pair in the last two positions of the codon, which might be related to the evolution of the viruses. A remarkable level of variability of GC content in the third position of the codon among the seven coronaviruses was observed. Codons with high RSCU values are primarily from the aliphatic and hydroxyl amino acid groups, and codons with low RSCU values belong to the aliphatic, cyclic, positively charged, and sulfur-containing amino acid groups. In order to elucidate the evolutionary processes of the seven coronaviruses, a phylogenetic tree (dendrogram) was constructed based on the RSCU scores of the codons. The severe and mild categories CoVs were positioned in different clades. A comparative phylogenetic study with other coronaviruses depicted that SARS-CoV-2 is close to the CoV isolated from pangolins (Manis javanica, Pangolin-CoV) and cats (Felis catus, SARS(r)-CoV). Further analysis of the effective number of codon (ENC) usage bias showed a relatively higher bias for SARS-CoV and MERS-CoV compared to SARS-CoV-2. The ENC plot against GC3 suggested that the mutational bias might have a role in determining the codon usage variation among candidate viruses.A codon adaptability study on a few human host parasites (from different kingdoms), including CoVs, showed a diverse adaptability pattern. SARS-CoV-2 and SARS-CoV exhibit relatively lower but similar codon adaptability compared to MERS-CoV.

Distribution of ε-Poly-l-Lysine Synthetases in Coryneform Bacteria Isolated from Cheese and Human Skin.

ε-Poly-l-lysine is a potent antimicrobial produced through fermentation of Streptomyces and used in many Asian countries as a food preservative. It is synthesized and excreted by a special nonribosomal peptide synthetase (NRPS)-like enzyme called Pls. In this study, we discovered a gene from cheese bacterium Corynebacterium variabile that showed high similarity to the Pls from Streptomyces in terms of domain architecture and gene context. By cloning it into Streptomyces coelicolor with a Streptomyces albulus Pls promoter, we confirmed that its product is indeed ε-poly-l-lysine. A comprehensive sequence analysis suggested that Pls genes are widely spread among coryneform actinobacteria isolated from cheese and human skin; 14 out of 15 Brevibacterium isolates and 10 out of 12 Corynebacterium isolates contain it in their genomes. This finding raises the possibility that ε-poly-l-lysine as a bioactive secondary metabolite might be produced and play a role in the cheese and skin ecosystems.IMPORTANCE Every year, microbial contamination causes billions of tons of food wasted and millions of cases of illness. ε-Poly-l-lysine has potent, wide-spectrum inhibitory activity and is heat stable and biodegradable. It has been approved for food preservation by an increasing number of countries. ε-Poly-l-lysine is produced from soil bacteria of the genus Streptomyces, also producers of various antibiotic drugs and toxins and not considered to be a naturally occurring food component. The frequent finding of pls in cheese and skin bacteria suggests that ε-poly-l-lysine may naturally exist in cheese and on our skin, and ε-poly-l-lysine producers are not limited to filamentous actinobacteria.

Comprehensive Analysis of CDR3 Sequences in Gluten-Specific T-Cell Receptors Reveals a Dominant R-Motif and Several New Minor Motifs.

Gluten-specific CD4+ T cells are drivers of celiac disease (CeD). Previous studies of gluten-specific T-cell receptor (TCR) repertoires have found public TCRs shared across multiple individuals, biased usage of particular V-genes and conserved CDR3 motifs. The CDR3 motifs within the gluten-specific TCR repertoire, however, have not been systematically investigated. In the current study, we analyzed the largest TCR database of gluten-specific CD4+ T cells studied so far consisting of TCRs of 3122 clonotypes from 63 CeD patients. We established a TCR database from CD4+ T cells isolated with a mix of HLA-DQ2.5:gluten tetramers representing four immunodominant gluten epitopes. In an unbiased fashion we searched by hierarchical clustering for common CDR3 motifs among 2764 clonotypes. We identified multiple CDR3α, CDR3β, and paired CDR3α:CDR3β motif candidates. Among these, a previously known conserved CDR3β R-motif used by TRAV26-1/TRBV7-2 TCRs specific for the DQ2.5-glia-α2 epitope was the most prominent motif. Furthermore, we identified the epitope specificity of altogether 16 new CDR3α:CDR3β motifs by comparing with TCR sequences of 231 T-cell clones with known specificity and TCR sequences of cells sorted with single HLA-DQ2.5:gluten tetramers. We identified 325 public TCRα and TCRβ sequences of which 145, 102 and 78 belonged to TCRα, TCRβ and paired TCRαβ sequences, respectively. While the number of public sequences was depended on the number of clonotypes in each patient, we found that the proportion of public clonotypes from the gluten-specific TCR repertoire of given CeD patients appeared to be stable (median 37%). Taken together, we here demonstrate that the TCR repertoire of CD4+ T cells specific to immunodominant gluten epitopes in CeD is diverse, yet there is clearly biased V-gene usage, presence of public TCRs and existence of conserved motifs of which R-motif is the most prominent.

Genetic Diversity and Comprehensive Phylodynamic Analysis of Whole Corona Viruses Genomes

Covid-19 is a rapidly emerging viral respiratory disease. The infection of corona virus disease (COVID-19) is&nbsp;initiated by the strains of severe acute respiratory syndrome. Corona virus 2 (SARS-CoV-2), is a highly&nbsp;transmittable viral infection that was first emerged in the of Wuhan, a city of China and ultimately extent in&nbsp;whole world. The World health organization (WHO) affirmed this infection as a global public health disaster.&nbsp;Genomic analysis showing that this novel SARSCoV-2 is hereditarily linked with severe acute respiratory&nbsp;syndrome-like (SARS-like) bat viruses indicating that bats might be the primary source of this epidemic infection.&nbsp;In this review we have summarized a comprehensive phylodynamic analysis of whole corona virus genome&nbsp;sequences in order to analyze the pathogenicity and emergence of novel COVID-19 infection and previous&nbsp;human coronaviruses. Our main objective is to describe the genetic relationships of the 2019-nCoV with other&nbsp;genera of coronavirus and to find out supposed recombination contained by sarbecovirus subgenus.&nbsp;Keywords: COVID-19, Phylogeny, SARS-CoV, MERS-CoV, Coding Sequence (CDS).

A novel homozygous SLC13A5 whole-gene deletion generated by Alu/Alu-mediated rearrangement in an Iraqi family with epileptic encephalopathy.

Biallelic loss-of-function (LoF) of SLC13A5 (solute carrier family 13, member 5) induced deficiency in sodium/citrate transporter (NaCT) causes autosomal recessive developmental epileptic encephalopathy 25 with hypoplastic amelogenesis imperfecta (DEE25; MIM #615905). Many pathogenic SLC13A5 single nucleotide variants (SNVs) and small indels have been described; however, no cases with copy number variants (CNVs) have been sufficiently investigated. We describe a consanguineous Iraqi family harboring an 88.5kb homozygous deletion including SLC13A5 in Chr17p13.1. The three affected male siblings exhibit neonatal-onset epilepsy with fever-sensitivity, recurrent status epilepticus, global developmental delay/intellectual disability (GDD/ID), and other variable neurological findings as shared phenotypical features of DEE25. Two of the three affected subjects exhibit hypoplastic amelogenesis imperfecta (AI), while the proband shows no evidence of dental abnormalities or AI at 2 years of age with apparently unaffected primary dentition. Characterization of the genomic architecture at this locus revealed evidence for genomic instability generated by an Alu/Alu-mediated rearrangement; confirmed by break-point junction Sanger sequencing. This multiplex family from a distinct population elucidates the phenotypic consequence of complete LoF of SLC13A5 and illustrates the importance of read-depth-based CNV detection in comprehensive exome sequencing analysis to solve cases that otherwise remain molecularly unsolved.

Profile of gut flora in hypertensive patients with insufficient sleep duration.

Recently, the contribution of both insufficient sleep duration and gut microbiome dysbiosis to hypertension (HTN) have been revealed, yet the profile of gut flora in hypertensive patients with insufficient sleep duration remains unknown. To examine this condition, the specific shifts in the fecal microbiome of 53 participants with or without HTN were investigated. The patients were divided into those who slept short (≤6 h) or optimal (6-9 h) duration per day. Comprehensive metagenomic sequencing analysis of fecal specimens was performed in healthy controls with sufficient sleep (s-CTR, n = 10), healthy controls with insufficient sleep (ins-CTR, n = 6), hypertensive patients with sufficient sleep (s-HTN, n = 25), and HTNs complicated by short sleep duration (ins-HTN, n = 12). We found that the α-diversity and β-diversity were quite similar between s-HTN and ins-HTN. Similarities were also observed in the enterotype distribution between s-HTN and ins-HTN subjects. In addition, the enrichment of gut bacteria was evident, such as Fusobacterium mortiferum and Roseburia inulinivorans in ins-HTN subjects. Several functional modules that were distinct between s-HTN and ins-HTN subjects were identified, which were unique to hypertensive patients with insufficient sleep duration. Overall, the data demonstrated that the gut microbial features were largely maintained in hypertensive participants with insufficient sleep duration.

Gut microbiota determines the social behavior of mice and induces metabolic and inflammatory changes in their adipose tissue

The link between the gut microbiota and social behavior has been demonstrated, however the translational impact of a certain microbiota composition on stable behavioral patterns is yet to be elucidated. Here we employed an established social behavior mouse model of dominance (Dom) or submissiveness (Sub). A comprehensive 16S rRNA gene sequence analysis of Dom and Sub mice revealed a significantly different gut microbiota composition that clearly distinguishes between the two behavioral modes. Sub mice gut microbiota is significantly less diverse than that of Dom mice, and their taxa composition uniquely comprised the genera Mycoplasma and Anaeroplasma of the Tenericutes phylum, in addition to the Rikenellaceae and Clostridiaceae families. Conversely, the gut microbiota of Dom mice includes the genus Prevotella of the Bacteriodetes phylum, significantly less abundant in Sub mice. In addition, Sub mice show lower body weight from the age of 2 weeks and throughout their life span, accompanied with lower epididymis white adipose tissue (eWAT) mass and smaller adipocytes together with substantially elevated expression of inflammation and metabolic-related eWAT adipokines. Finally, fecal microbiota transplantation into germ-free mice show that Sub-transplanted mice acquired Sub microbiota and adopted their behavioral and physiological features, including depressive-like and anti-social behaviors alongside reduced eWAT mass, smaller adipocytes, and a Sub-like eWAT adipokine profile. Our findings demonstrate the critical role of the gut microbiome in determining dominance vs. submissiveness and suggest an association between gut microbiota, the eWAT metabolic and inflammatory profile, and the social behavior mode.