Comprehensive Sequence Analysis Research Articles

Revisiting Aspergillus terreus MTCC6324 recombinant alcohol oxidase (rAOx): enhanced in-silico insight into structure, function, and substrate sequestration mechanism

Alcohol oxidase (AOx) enzymes have gained significant attention for their potential in industrial applications due to their unique ability to catalyze irreversible oxidation of diverse alcohol substrates without external co-factors. This study revisits and enhances in-silico work on Aspergillus terreus MTCC6324 recombinant AOx (rAOx) enzyme, combining artificial intelligence (AlphaFold), molecular docking (AutoDock Vina) techniques and Molecular dynamics (MD) simulations (Desmond). Comprehensive sequence analysis revealed a high degree of conservation among 23 AOx amino acid sequences from various Aspergillus species highlights conserved regions, affirming its GXGXXG Rossmann fold motif. AlphaFold-predicted 3D structure of rAOx demonstrated improved stereo-chemical stability compared to I-TASSER predicted structure with 87.6% amino acid residues in most favourable region of Ramachandran plot compared to 79.5%, respectively. Molecular docking revealed the binding affinities of co-factors FAD and diverse alcohol substrates, with cinnamyl alcohol exhibiting robust interaction with rAOx holoenzyme. MD simulations further elucidate the stability and dynamics of rAOx-FAD-cinnamyl alcohol complex over 100 nanoseconds. The simulations showcase FAD’s stable binding within the protein core and highlights transient substrate interactions, dissociating within the active site after 75 ns suggesting a substrate sequestration mechanism. The study unveils substrate sequestration mechanism wherein cinnamyl alcohol exhibits temporary binding, leading to quick detachment from active site, mimicking reported exponential kinetics. This study not only validates previous findings but also offers a comprehensive understanding of intricate dynamics governing rAOx enzymatic activity. The improved sequence-to-structure prediction and detailed molecular insights into substrate sequestration provide a valuable foundation for future experimental investigations and rational design of bio-catalytic processes.

Genomic landscape of adult testicular germ cell tumours in the 100,000 Genomes Project

Testicular germ cell tumours (TGCT), which comprise seminoma and non-seminoma subtypes, are the most common cancers in young men. In this study, we present a comprehensive whole genome sequencing analysis of adult TGCTs. Leveraging samples from participants recruited via the UK National Health Service and data from the Genomics England 100,000 Genomes Project, our results provide an extended description of genomic elements underlying TGCT pathogenesis. This catalogue offers a comprehensive, high-resolution map of copy number alterations, structural variation, and key global genome features, including mutational signatures and analysis of extrachromosomal DNA amplification. This study establishes correlations between genomic alterations and histological diversification, revealing divergent evolutionary trajectories among TGCT subtypes. By reconstructing the chronological order of driver events, we identify a subgroup of adult TGCTs undergoing relatively late whole genome duplication. Additionally, we present evidence that human leukocyte antigen loss is a more prevalent mechanism of immune disruption in seminomas. Collectively, our findings provide valuable insights into the developmental and immune modulatory processes implicated in TGCT pathogenesis and progression.

Transcriptome-wide analysis for glucocorticoid receptor-mediated mRNA decay reveals various classes of target transcripts

The glucocorticoid receptor (GR) can bind to DNA or RNA, eliciting transcriptional activation/repression or rapid mRNA degradation, respectively. Although GR-mediated transcriptional regulation has been well-characterized, the molecular details of rapid mRNA degradation induced by glucocorticoids (GCs) are not yet fully understood. Here, we demonstrate that GC-induced GR-mediated mRNA decay (GMD) takes place in the nucleus and the cytoplasm, acting on pre-mRNAs and mRNAs. We also performed cross-linking and immunoprecipitation coupled with high-throughput sequencing (CLIP-seq) analysis for GMD factors (GR, YBX1, and HRSP12) and mRNA sequencing (mRNA-seq) analysis to identify endogenous GMD substrates. Our comprehensive CLIP-seq and mRNA sequencing analyses reveal that a range of cellular transcripts containing a common binding site for GR, YBX1, and HRSP12 are preferential targets for GMD, suggesting possible new functions of GMD in various biological events.

Chemical composition determination and transcriptomic analyses provide insight into the differences between wild and grafted Semen Ziziphi Spinosae

Semen Ziziphi Spinosae (SZS) is a traditional Chinese herbal medicine widely used to treat insomnia and anxiety in clinical practice. Currently, the demand for SZS is increasing every year, but the production of wild SZS is unstable due to environmental factors. Grafting sour jujube scions onto sour jujube or jujube tree stocks can achieve a high production rate within a short period of time. However, the effects of grafting on the quality of SZS have not been reported. This study investigated the differences between wild-type and grafted SZS from three aspects: phenotype, chemical composition, and molecular mechanism. The findings revealed that the grafted specimens were generally larger in morphology and lighter in color than the wild-type samples. The dimensions of both the grafted specimens were generally larger than those of the wild specimens. The HPLC-ELSD results revealed that the three main chemical components in the grafted SZS, namely, spinosin, jujuboside A, and jujuboside B, had higher contents than their wild-type counterparts. Comprehensive transcriptome sequencing analysis and KEGG annotation revealed that DEG enrichment between grafted and wild-type SZS occurred mainly during stress resistance and rootstock scion healing. There were 23 DEGs that may encode enzymes involved in the biosynthetic pathway of flavonoids and 21 genes encoding terpenoid saponins. Further investigation revealed that the expression of the genes C4H, CHS, CHI, and F3’5’H in the flavonoid biosynthesis pat.hway and HMGR, MVK, MVD, and FPPS in the saponin biosynthesis pathway accounted for the difference in quality between grafted and wild SZS. Furthermore, WGCNA identified 15 core genes related to medicinal ingredients between grafted and wild SZS. These results provide support for further research on the differences in the quality of medicinal ingredients between grafted and wild SZS.

Virome Characterization of Native Wild-Rice Plants Discovers a Novel Pathogenic Rice Polerovirus With World-Wide Circulation.

Pandemics originating from zoonotic viruses have posed significant threats to human health and agriculture. Recent discoveries have revealed that wild-rice plants also harbour viral pathogens capable of severely impacting rice production, a cornerstone food crop. In this study, we conducted virome analysis on ~1000 wild-rice individual colonies and discovered a novel single-strand positive-sense RNA virus prevalent in these plants. Through comprehensive genomic characterization and comparative sequence analysis, this virus was classified as a new species in the genus Polerovirus, designated Rice less tiller virus (RLTV). Our investigations elucidated that RLTV could be transmitted from wild rice to cultivated rice via a specific insect vector, the aphid Rhopalosiphum padi, causing less tiller disease symptoms in rice plants. We generated an infectious cDNA clone for RLTV and demonstrated systemic infection of rice cultivars and induction of severe disease symptoms following mechanical inoculation or stable genetic transformation. We further illustrated transmission of RLTV from stable transgenic lines to healthy rice plants by the aphid vector, leading to the development of disease symptoms. Notably, our database searches showed that RLTV and another polerovirus isolated from a wild plant species are widely circulating not only in wild rice but also cultivated rice around the world. Our findings provide strong evidence for a wild plant origin for rice viruses and underscore the imminent threat posed by aphid-transmitted rice Polerovirus to rice cultivar.

Cost-saving approach with screening of selected variants in genetic diagnosis in Turkish pediatric familial Mediterranean fever patients: a single center longitudinal study.

The aim of this study was to investigate whether a short exon screening consisting of selected variants could confirm the diagnosis in patients with a preliminary diagnosis of familial Mediterranean fever (FMF), thus providing a cost-saving alternative to a comprehensive MEditerranean FeVer (MEFV) gene sequence analysis test. This observational study on pediatric patients focused on clinically suspected FMF cases without prior genetic analysis. Participants met the Turkish pediatric FMF criteria. They underwent short exon screening for M694V, M680I, V726A, and E148Q variants. Those who were heterozygous or negative on short exon screening received further MEFV gene sequence analysis. The study involved 1557 patients. Pathogenic variants in both alleles of the MEFV gene were found in 611 patients (39.2%), and a high-penetrance variant in heterozygosity or an E148Q variant on the other allele was found in 643 patients (41.3%). A further 189 patients (12.1%) had one or two E148Q variants. Short-exon screening was negative in 114 patients (7.6%). Of the 876 patients who underwent MEFV gene sequence analysis, additional variants were found in 72 of the 762 initially heterozygous patients. Of the 114 initially negative patients, 34 had homozygous or compound heterozygous variants, and 74 had heterozygous variants. Ultimately, only 6 patients yielded negative results in the MEFV gene sequence analysis. The short exon screening for common MEFV mutations offers a practical and cost-saving alternative to comprehensive MEFV gene sequence analysis in populations with a high prevalence of FMF.

46P Congenital myopathies and muscular dystrophies in the UK: a comprehensive next generation sequencing analysis of frequencies over an 8-year period (2016-2023)

46P Congenital myopathies and muscular dystrophies in the UK: a comprehensive next generation sequencing analysis of frequencies over an 8-year period (2016-2023)

Fibroblasts Regulate the Transformation Potential of Human Papillomavirus-positive Keratinocytes.

Persistent human papillomavirus (HPV) infection is necessary but insufficient for viral oncogenesis. Additional contributing co-factors, such as immune evasion and viral integration have been implicated in HPV-induced cancer progression. It is widely accepted that HPV+ keratinocytes require co-culture with fibroblasts to maintain viral episome expression, yet the exact mechanisms for this have yet to be elucidated. Here we present comprehensive RNA sequencing and proteomic analysis demonstrating that fibroblasts not only support the viral life cycle, but reduce HPV+ keratinocyte transformation. Our co-culture models offer novel insights into HPV-related transformation mechanisms.

Correction: Association study of GBA1 variants with MSA based on comprehensive sequence analysis -Pitfalls in short-read sequence analysis depending on the human reference genome-

Correction: Association study of GBA1 variants with MSA based on comprehensive sequence analysis -Pitfalls in short-read sequence analysis depending on the human reference genome-

Diversification and conservation of DNA binding specificities of SPL family of transcription factors.

SQUAMOSA Promoter-Binding Protein-Like (SPL) transcription factors play vital roles in plant development and stress responses. In this study, we report a comprehensive DNA Affinity Purification sequencing (DAP-seq) analysis for 14 of the 16 SPL transcription factors in Arabidopsis thaliana, providing valuable insights into their DNA-binding specificities. We performed Gene Ontology (GO) analysis of the target genes to reveal their convergent and diverse biological functions among SPL family proteins. Comparative analysis between the paralogs AtSPL9 and AtSPL15 revealed differences in their binding motifs, suggesting divergent regulatory functions. Additionally, we expanded our investigation to homologs of AtSPL9/15 in Zea mays (ZmSBP8/30) and Triticum aestivum (TaSPL7/13), identifying conserved and unique DNA-binding patterns across species. These findings provide key resources for understanding the molecular mechanisms of SPL transcription factors in regulating plant development and evolution across different species.

MATR3 promotes liver cancer progression by suppressing DHX58–mediated type I interferon response

MATR3 is a nuclear matrix protein implicated in various cancers; however, its specific role in tumor progression remains unclear. The study utilized the TCGA database to reveal that MATR3 expression is upregulated in liver cancer and is correlated with poor prognosis. Functionally, MATR3 promoted liver cancer cell proliferation and metastasis. Comprehensive RNA sequencing analysis showed that MATR3 significantly affected the type I IFN signaling pathway and DHX58 is a downstream target of MATR3. Further experiments showed that MATR3 bound to DHX58 mRNA through its RRM structural domain and recruited YTHDF2, an m6A reader, leading to degradation of DHX58 mRNA and suppression of the type I IFN signaling pathway. The knockout of MATR3 in liver cancer cells triggered a natural immune response that stimulated CD8+ T cells to eliminate liver cancer cells. This study demonstrated that MATR3 downregulates type I IFN signaling in liver cancer cells through m6A modification and inhibits immune cell infiltration within tumors. These findings expand our understanding of the role of MATR3 in liver cancer.

The complete genomic sequencing of a Klebsiella pneumoniae isolate derived from the blood of a patient suffering from severe neurological problems in Zhengzhou, China.

This study presents the comprehensive analysis of the genomic sequence of Klebsiella pneumoniae strain FAHZZU7042hy, having a 5,690,191 bp chromosome size. This strain was obtained from a blood sample of a patient suffering from severe neurological problems in Zhengzhou, China, 2023.

Qki5 safeguards spinal motor neuron function by defining the motor neuron-specific transcriptome via pre-mRNA processing

Many RNA-binding proteins (RBPs) are linked to the dysregulation of RNA metabolism in motor neuron diseases (MNDs). However, the molecular mechanisms underlying MN vulnerability have yet to be elucidated. Here, we found that such an RBP, Quaking5 (Qki5), contributes to formation of the MN-specific transcriptome profile, termed "MN-ness," through the posttranscriptional network and maintenance of the mature MNs. Immunohistochemical analysis and single-cell RNA sequencing (scRNA-seq) revealed that Qki5 is predominantly expressed in MNs, but not in other neuronal populations of the spinal cord. Furthermore, comprehensive RNA sequencing (RNA-seq) analyses revealed that Qki5-dependent RNA regulation plays a pivotal role in generating the MN-specific transcriptome through pre-messenger ribonucleic acid (mRNA) splicing for the synapse-related molecules and c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) signaling pathways. Indeed, MN-specific ablation of the Qki5 caused neurodegeneration in postnatal mice and loss of Qki5 function resulted in the aberrant activation of stress-responsive JNK/SAPK pathway both in vitro and in vivo. These data suggested that Qki5 plays a crucial biological role in RNA regulation and safeguarding of MNs and might be associated with pathogenesis of MNDs.

Gynostemma pentaphyllum saponins shield mice from peanut allergy by modulation of gut microbiota: A novel approach for peanut allergy management

BackgroundFood allergies, particularly peanut (PN) allergies, are a growing concern, with fatal anaphylaxis incidents often reported. While palforzia is the sole FDA-approved drug for managing PN allergies, it is not universally effective. PurposeThis study aimed to investigate the potential of Gynostemma pentaphyllum saponins (GpS) as a novel therapeutic agent for PN allergy through modulation of gut microbiota, addressing the limitations of current treatments. MethodsTo elucidate the role of GpS on peanut allergy, we first built a PN-sensitized C57BL/6J model mice. Through comprehensive sequencing analysis, we identified Parabacteroides distasonis as a key bacterium triggering PN sensitization. Employing the same mouse model, GpS was evaluated for its effects on anaphylactic symptoms, serum immunoglobulin levels, and allergy-related biomarkers. 16S rRNA sequencing and transcriptomic analysis were applied to investigate the impact of GpS on the host's gut epithelium and microbiome. ResultsGpS treatment effectively reduced anaphylactic symptoms in PN-sensitized mice, as shown by decreased IgG1, total IgE, and PN-specific IgE levels. It also modulated the immune response by suppressing proinflammatory cytokines (IL-1β, IFN-γ, IL-21) and chemokines (CCL5, CCL12, CCL17, CCL22), while enhancing anti-inflammatory cytokines (IL-4, IL-10, IL-12, IL-13). Fecal microbial transplant from GpS-treated Model mice to PN-sensitized mice displayed anti-peanut allergy effects. Additionally, the administration of GpS-enhanced bacteria (Clostridium aldenese or Lactobacillus murinus), alleviated anaphylactic symptoms and reduced serum allergy markers in PN-sensitized mice. ConclusionTo conclude, we revealed the intestinal environment, signaling molecules, mucosal cytokines, and commensal microbial profiles in the peanut-sensitized mouse model. We further presented evidence for the protective effect of GpS against PN allergen sensitization by downregulating a series of food-allergy-associated biomarkers and cytokines via the modulation of gut bacteria. More importantly, supported by both in vitro and in vivo experiments, we demonstrated that the protective effect of GpS against PN-allergy is through the enhancement of two commensal bacteria, Clostridium aldenese, and Lactobacillus murinus.

Development of novel humanized VHH synthetic libraries based on physicochemical analyses

Due to the high affinity and specificity of antibodies toward antigens, various antibody-based applications have been developed. Recently, variable antigen-binding domains of heavy-chain antibodies (VHH) have become an attractive alternative to conventional fragment antibodies due to their unique molecular characteristics. As an antibody-generating strategy, synthetic VHH libraries (including humanized VHH libraries) have been developed using distinct strategies to constrain the diversity of amino acid sequences. In this study, we designed and constructed several novel synthetic humanized VHH libraries based on biophysical analyses conducted using the complementarity determining region-grafting method and comprehensive sequence analyses of VHHs deposited in the protein data bank. We obtained VHHs from the libraries, and hit clones exhibited considerable thermal stability. We also found that VHHs from distinct libraries tended to have different epitopes. Based on our results, we propose a strategy for generating humanized VHHs with distinct epitopes toward various antigens by utilizing our library combinations.

Aspiration of acidified milk induces milk allergy by activating alveolar macrophages in mice

BackgroundEpidemiological studies have identified associations between gastroesophageal reflux (GER) and cow's milk allergy (CMA) in infants. However, the role of GER in the development of CMA remains poorly understood. Our primary objectives were to develop a mouse model that suggests GER as a potential pathogenic mechanism for CMA and to elucidate the immunological mechanisms that connect lung innate immunity with CMA. MethodsMice were exposed to cow's milk (CM) treated with hydrochloric acid through repeated aspiration into their airways. Subsequently, they were challenged by intraperitoneal injection of CM extract. The immunological mechanisms were investigated using comprehensive single-cell RNA sequencing (scRNA-seq) analysis of the lungs, combined with the use of genetically modified mice. ResultsMice exposed to CM mixed with hydrochloric acid via airway sensitization developed CMA, as evidenced by the production of antigen-specific IgE and IgG antibodies, and the induction of anaphylaxis upon systemic antigen administration. In contrast, aspiration of CM alone did not induce CMA. scRNA-seq analysis revealed potential roles of alveolar macrophages in response to hydrochloric acid. Mice lacking the TLR4 pathway were protected from developing CMA. ConclusionsWe have developed a novel mouse model for CMA that utilizes the natural antigen and follows the physiological airway sensitization pathway, thus potentially resembling clinical scenarios. This model, named the acidified milk aspiration-induced allergy model, has the potential to shed light on the role of early innate immunity by analyzing a more physiological model.

Antigen-driven Convergent Evolution of Polysaccharide-specific "DH-less" B Cells in Glycoconjugate Immunized Mice.

Glycoconjugate vaccines elicit robust anti-polysaccharide Ab response by recruiting T-cell help. Multiple doses of glycoconjugate vaccine are required to induce long-lasting immunity. The characteristics of anti-polysaccharide Ab response have been reported previously. However, the effect of glycoconjugate booster immunization on anti-polysaccharide and anti-carrier protein Ab repertoire remains poorly understood. In this study, we used clinically relevant pneumococcal capsular polysaccharide type 14 (PCP14) conjugated with cross-reactive material 197 (CRM197) as a model glycoconjugate Ag (PCP14-CRM197). We performed a comprehensive sequence analysis of mouse mAbs generated against PCP14 and CRM197 following immunization with one or three doses of PCP14-CRM197. Analysis of the paired Ig H and L chain transcripts revealed that anti-PCP14 Ab repertoire is extremely restricted. The reoccurrence of five replacement mutations at identical positions in anti-polysaccharide mAbs generated from different mice provided evidence for Ag-driven selection in PCP14-specific B cells. Convergent evolution was observed wherein distinct V(D)J rearrangements resulted in identical or nearly identical CDR3 in anti-PCP14 mAbs. Abs that lacked DH encoded amino acids dominated the anti-PCP14 Ab response. In contrast, anti-CRM197 Ab response was quite diverse, with fewer mutations compared with the anti-PCP14 mAbs, suggesting that conjugation of the polysaccharide to a carrier protein interferes with the development of carrier protein-specific Ab responses. Our findings provide molecular insights into the maturation of Ab responses driven by booster doses of glycoconjugate. This has fundamental implications for the design of glycoconjugate vaccines, especially where the development of Ab response against the carrier protein is also crucial.

Investigating the emergence of a zoonotic virus: phylogenetic analysis of European bat lyssavirus 1 in the UK.

European bat lyssavirus 1 (EBLV-1, Lyssavirus hamburg) is predominantly detected in serotine bats (Eptesicus serotinus) and is responsible for the majority of bat rabies cases in mainland Europe. A passive bat rabies surveillance scheme detected the virus in a serotine bat in the UK for the first time in October 2018. As of May 2024, 34 cases have been reported, 20 of which involved contact with an animal and 5 reported human contact. We investigated the emergence of EBLV-1 by undertaking comprehensive sequence analysis and Bayesian phylogenetics, based on complete virus genomes of 33 UK sequences and 108 sequences covering six countries in mainland Europe (1968-2023), including 21 French EBLV-1-positive RNA samples sequenced for this study. Sequence analysis revealed extreme similarity among UK EBLV-1 sequences (99.9%-100%), implying a single source of introduction rather than multiple independent introductions. Bayesian analysis revealed that the UK EBLV-1 sequences shared their most recent common ancestor with an EBLV-1 sequence from a serotine bat detected in Brittany, France, in 2001, with an estimated date of divergence of 1997. Within the UK sequences, the earliest divergence was estimated to occur in 2007. This study provides valuable insights into the molecular epidemiology of an emerging zoonotic pathogen and improved understanding of the risks posed to public and animal health.

Reproducible gut microbial signatures in bipolar and schizophrenia spectrum disorders: A metagenome-wide study

BackgroundNumerous studies report gut microbiome variations in bipolar disorder (BD) and schizophrenia spectrum disorders (SSD) compared to healthy individuals, though, there is limited consensus on which specific bacteria are associated with these disorders. MethodsIn this study, we performed a comprehensive metagenomic shotgun sequencing analysis in 103 Dutch patients with BD/SSD and 128 healthy controls matched for age, sex, body mass index and income, while accounting for diet quality, transit time and technical confounders. To assess the replicability of the findings, we used two validation cohorts (total n = 203), including participants from a distinct population with a different metagenomic isolation protocol. ResultsThe gut microbiome of the patients had a significantly different β-diversity, but not α-diversity nor neuroactive potential compared to healthy controls. Initially, twenty-six bacterial taxa were identified as differentially abundant in patients. Among these, the previously reported genera Lachnoclostridium and Eggerthella were replicated in the validation cohorts. Employing the CoDaCoRe learning algorithm, we identified two bacterial balances specific to BD/SSD, which demonstrated an area under the receiver operating characteristic curve (AUC) of 0.77 in the test dataset. These balances were replicated in the validation cohorts and showed a positive association with the severity of psychiatric symptoms and antipsychotic use. Last, we showed a positive association between the relative abundance of Klebsiella and Klebsiella pneumoniae with antipsychotic use and between the Anaeromassilibacillus and lithium use. ConclusionsOur findings suggest that microbial balances could be a reproducible method for identifying BD/SSD-specific microbial signatures, with potential diagnostic and prognostic applications. Notably, Lachnoclostridium and Eggerthella emerge as frequently occurring bacteria in BD/SSD. Last, our study reaffirms the previously established link between Klebsiella and antipsychotic medication use and identifies a novel association between Anaeromassilibacillus and lithium use.

Association study of GBA1 variants with MSA based on comprehensive sequence analysis -Pitfalls in short-read sequence analysis depending on the human reference genome.

Multiple system atrophy (MSA) is a neurodegenerative disorder characterized by various combinations of autonomic failure, parkinsonism, and cerebellar ataxia. To elucidate variants associated with MSA, we have been conducting short-read-based whole-genome sequence analysis. In the process of the association studies, we initially focused on GBA1, a previously proposed susceptibility gene for MSA, to evaluate whether GBA1 variants can be efficiently identified despite its extraordinarily high homology with its pseudogene, GBA1LP. To accomplish this, we conducted a short-read whole-genome sequence analysis with alignment to GRCh38 as well as Sanger sequence analysis and compared the results. We identified five variants with inconsistencies between the two pipelines, of which three variants (p.L483P, p.A495P-p.V499V, p.L483_M489delinsW) were the results of misalignment due to minor alleles in GBA1P1 registered in GRCh38. The miscalling events in these variants were resolved by alignment to GRCh37 as the reference genome, where the major alleles are registered. In addition, a structural variant was not properly identified either by short-read or by Sanger sequence analyses. Having accomplished correct variant calling, we identified three variants pathogenic for Gaucher disease (p.S310G, p.L483P, and p.L483_M489delinsW). Of these variants, the allele frequency of p.L483P (0.003) in the MSA cases was higher than that (0.0011) in controls. The meta-analysis incorporating a previous report demonstrated a significant association of p.L483P with MSA with an odds ratio of 2.92 (95% CI; 1.08 - 7.90, p = 0.0353).