Bioinformatics and biomedical informatics with ChatGPT: year one review.

The year 2023 marked a significant surge in the exploration of applying large language model (LLM) chatbots, notably ChatGPT, across various disciplines. We surveyed the applications of ChatGPT in various sectors of bioinformatics and biomedical informatics throughout the year, covering omics, genetics, biomedical text mining, drug discovery, biomedical image understanding, bioinformatics programming, and bioinformatics education. Our survey delineates the current strengths and limitations of this chatbot in bioinformatics and offers insights into potential avenues for future development.

Increased interleukin-9 and Th9 cells in patients with refractory Graves' disease and interleukin-9 polymorphisms are associated with autoimmune thyroid diseases.

Introduction: Autoimmune thyroid diseases (AITDs) are prevalent disorders, primarily encompassing Graves' disease (GD) and Hashimoto's thyroiditis (HT). Despite their common occurrence, the etiology of AITDs remains elusive. Th9 cells, a new subset of CD4+T cells with immunomodulatory properties, have been linked to the development of various autoimmune diseases. However, research on the role of Th9 cells in AITDs is limited. Methods: We investigated the expression of Th9 cells,their functional cytokine IL-9, and transcription factor IRF4 in peripheral blood mononuclear cells (PBMCs) and plasma of AITD patients and healthy controls. Additionally, we explored the genetic association between four loci polymorphisms (rs31564, rs2069879, rs1859430, and rs2069868) of the IL-9 gene and AITDs. Results: We reported, for the first time, that refractory GD patients exhibited elevated mRNA levels of IL-9 and IRF4 in PBMCs, increased IL-9 protein levels in plasma, and a higher proportion of Th9 cells in peripheral blood when compared to normal controls. Furthermore, human recombinant IL-9 protein was found to enhance IFN-g secretion in PBMCs from both GD patients and normal controls. At the genetic association level, after adjusting for age and sex, the rs2069879 polymorphism exhibited a significant association with AITDs under an additive model (P<0.001, OR= 0.05, 95% CI=0.03-0.08). Discussion: Our results reveal that Th9 cells may exert a pivotal role in the pathogenesis and progression of refractory GD and HT, and IL-9 holds promise as a novel therapeutic target for the management of AITDs.

cellMarkerPipe: Cell Marker Identification and Evaluation Pipeline in Single Cell Transcriptomes.

Assessing marker genes from all cell clusters can be time-consuming and lack systematic strategy. Streamlining this process through a unified computational platform that automates identification and benchmarking will greatly enhance efficiency and ensure a fair evaluation. We therefore developed a novel computational platform, cellMarkerPipe (https://github.com/yao-laboratory/cellMarkerPipe), for automated cell-type specific marker gene identification from scRNA-seq data, coupled with comprehensive evaluation schema. CellMarkerPipe adaptively wraps around a collection of commonly used and state-of-the-art tools, including Seurat, COSG, SC3, SCMarker, COMET, and scGeneFit. From rigorously testing across diverse samples, we ascertain SCMarker's overall reliable performance in single marker gene selection, with COSG showing commendable speed and comparable efficacy. Furthermore, we demonstrate the pivotal role of our approach in real-world medical datasets. This general and opensource pipeline stands as a significant advancement in streamlining cell marker gene identification and evaluation, fitting broad applications in the field of cellular biology and medical research.

A Roadmap for the Human Gut Cell Atlas.

The number of studies investigating the human gastrointestinal tract using various single-cell profiling methods has increased substantially in the past few years. Although this increase provides a unique opportunity for the generation of the first comprehensive Human Gut Cell Atlas (HGCA), there remains a range of major challenges ahead. Above all, the ultimate success will largely depend on a structured and coordinated approach that aligns global efforts undertaken by a large number of research groups. In this Roadmap, we discuss a comprehensive forward-thinking direction for the generation of the HGCA on behalf of the Gut Biological Network of the Human Cell Atlas. Based on the consensus opinion of experts from across the globe, we outline the main requirements for the first complete HGCA by summarizing existing data sets and highlighting anatomical regions and/or tissues with limited coverage. We provide recommendations for future studies and discuss key methodologies and the importance of integrating the healthy gut atlas with related diseases and gut organoids. Importantly, we critically overview the computational tools available and provide recommendations to overcome key challenges.

HLA-DRA Gene Polymorphisms Are Associated with Graves' Disease as an Autoimmune Thyroid Disease.

Background: Autoimmune thyroid disease (AITD), one of the most prevalent organ-specific autoimmune diseases, mainly includes Graves' disease (GD) and Hashimoto's thyroiditis (HT). This study was aimed at researching the association between AITD and single nucleotide polymorphisms (SNPs) of the HLA-DRA gene. Methods: Using Hi-SNP high-throughput sequencing technology, we detected the distribution of three SNPs (rs3177928, rs7197, and rs3129878) of HLA-DRA genotypes in 1033 AITD patients (634 GD and 399 HT ones) and 791 healthy volunteers in Chinese Han Population. Chi-square test, multivariate logistic regression, and haplotype analysis were performed by SPSS and Haploview software to analyze the relationship between HLA-DRA gene polymorphisms and AITD. Results: The results show that allele frequency and genotype distribution of rs3177928 and rs7197 were correlated with AITD and GD compared with the healthy control group, but not with HT. Rs3177928 and rs7197 were correlated with AITD and HT in the allele model, dominant model, and overdominant model before and after gender and age adjustment, but not with HT. In addition, we found that two loci (rs3177928 and rs7197) constituted a linkage disequilibrium (LD) region, and haplotype AA was associated with AITD and GD. However, we found no association between rs3129878 and AITD. Conclusion: Our study is the first to find that rs3177928 and rs7197 of HLA-DRA are significantly correlated with AITD and GD in the Chinese Han population. This will help further reveal the pathogenesis of AITD and provide new candidate genes for the prediction or treatment of AITD.

Profiling a Community-Specific Function Landscape for Bacterial Peptides Through Protein-Level Meta-Assembly and Machine Learning.

Small proteins, encoded by small open reading frames, are only beginning to emerge with the current advancement of omics technology and bioinformatics. There is increasing evidence that small proteins play roles in diverse critical biological functions, such as adjusting cellular metabolism, regulating other protein activities, controlling cell cycles, and affecting disease physiology. In prokaryotes such as bacteria, the small proteins are largely unexplored for their sequence space and functional groups. For most bacterial species from a natural community, the sample cannot be easily isolated or cultured, and the bacterial peptides must be better characterized in a metagenomic manner. The bacterial peptides identified from metagenomic samples can not only enrich the pool of small proteins but can also reveal the community-specific microbe ecology information from a small protein perspective. In this study, metaBP (Bacterial Peptides for metagenomic sample) has been developed as a comprehensive toolkit to explore the small protein universe from metagenomic samples. It takes raw sequencing reads as input, performs protein-level meta-assembly, and computes bacterial peptide homolog groups with sample-specific mutations. The metaBP also integrates general protein annotation tools as well as our small protein-specific machine learning module metaBP-ML to construct a full landscape for bacterial peptides. The metaBP-ML shows advantages for discovering functions of bacterial peptides in a microbial community and increases the yields of annotations by up to five folds. The metaBP toolkit demonstrates its novelty in adopting the protein-level assembly to discover small proteins, integrating protein-clustering tool in a new and flexible environment of RBiotools, and presenting the first-time small protein landscape by metaBP-ML. Taken together, metaBP (and metaBP-ML) can profile functional bacterial peptides from metagenomic samples with potential diverse mutations, in order to depict a unique landscape of small proteins from a microbial community.

Deletion of murine Rhoh leads to de-repression of Bcl-6 via decreased KAISO levels and accelerates a malignancy phenotype in a murine model of lymphoma.

RHOH/TFF, a member of the RAS GTPase super family, has important functions in lymphopoiesis and proximal T cell receptor signalling and has been implicated in a variety of leukaemias and lymphomas. RHOH was initially identified as a translocation partner with BCL-6 in non-Hodgkin lymphoma (NHL), and aberrant somatic hypermutation (SHM) in the 5' untranslated region of the RHOH gene has also been detected in Diffuse Large B-Cell Lymphoma (DLBCL). Recent data suggest a correlation between RhoH expression and disease progression in Acute Myeloid Leukaemia (AML). However, the effects of RHOH mutations and translocations on RhoH expression and malignant transformation remain unknown. We found that aged Rhoh-/- (KO) mice had shortened lifespans and developed B cell derived splenomegaly with an increased Bcl-6 expression profile in splenocytes. We utilized a murine model of Bcl-6 driven DLBCL to further explore the role of RhoH in malignant behaviour by crossing RhohKO mice with Iµ-HABcl-6 transgenic (Bcl-6Tg) mice. The loss of Rhoh in Bcl-6Tg mice led to a more rapid disease progression. Mechanistically, we demonstrated that deletion of Rhoh in these murine lymphoma cells was associated with decreased levels of the RhoH binding partner KAISO, a dual-specific Zinc finger transcription factor, de-repression of KAISO target Bcl-6, and downregulation of the BCL-6 target Blimp-1. Re-expression of RhoH in RhohKOBcl-6Tg lymphoma cell lines reversed these changes in expression profile and reduced proliferation of lymphoma cells in vitro. These findings suggest a previously unidentified regulatory role of RhoH in the proliferation of tumour cells via altered BCL-6 expression. (250).

Identification of lncRNA and mRNA Expression Profile in Relapsed Graves' Disease.

Background: Graves' disease (GD) is a common autoimmune disease, and its pathogenesis is unclear. Studies have found that the occurrence of GD is related to the immune disorder caused by the interaction of genetic susceptibility and environmental factors. The CD4+ T cell subset is closely related to the immune disorder of GD. LncRNAs are RNA molecules with a length of more than 200 nt and are involved in a variety of autoimmune diseases. However, the roles of lncRNAs in recurrent GD are still elusive. The purpose of this study is to identify lncRNA and mRNA expression profile in relapsed Graves' disease. Method: CD4+ T cells from 12 recurrent GD and 8 healthy controls were collected for high-throughput sequencing. The gene-weighted co-expression network analysis (WGCNA) was used to construct the co-expression module relevant to recurrent GD, and the key genes in the module were verified by RT-PCR. Results: There are 602 upregulated lncRNAs and 734 downregulated lncRNAs in CD4+ T cells in recurrent GD patients compared with the healthy controls. The module most relevant to GD recurrence was constructed using WGCNA, and the key genes in the module were verified by RT-PCR. We found that the expression of RPL8, OAS2, NFAT5, DROSHA, NONHSAT093153.2, NONHSAT118924.2, and NONHSAT209004.1 was significantly decreased in GD group (p < 0.001, p < 0.001, p < 0.01, p < 0.05, p < 0.001, p < 0.05, and p < 0.01, respectively). Conclusion: LncRNAs are closely related to the recurrence of GD. For the first time, we constructed the expression profile of lncRNAs and mRNAs in CD4+ T cells in recurrent GD patients.

Systemic Proteomic Analysis Reveals Distinct Exosomal Protein Profiles in Rheumatoid Arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) is a complex disease with unknown pathogenesis. In recent years, fewer have paid attention to the broad spectrum of systemic markers of RA. The aim of this study was to identify exosomal candidate proteins in the pathogenesis of RA. METHODS: Totally, 12 specimens of plasma from 6 RA patients and 6 age- and gender-matched controls from the Chinese population were obtained for nanoscale liquid chromatography coupled to tandem mass spectrometry (nano-LC-MS/MS) analysis to identify exosomal profiles. RESULTS: A total of 278 exosomal proteins were detected. Among them, 32 proteins were significantly upregulated (FC ≥ 2.0 and P < 0.05) and 5 proteins were downregulated (FC ≤ 0.5 and P < 0.05). Bioinformatics analysis revealed that transthyretin (TTR), angiotensinogen (AGT), lipopolysaccharide-binding protein (LBP), monocyte differentiation antigen CD14 (CD14), cartilage oligomeric matrix protein (COMP), serum amyloid P (SAP/APCS), and tenascin (TNC) can interact with each other. Subsequently, these cross-linked proteins may be mainly involved in the inflammatory-related pathways to mediate the onset of RA. Noteworthy, the LBP/CD14 complex can promote the expression of IL-8 and TNF-α, eventually leading to the development of RA. CONCLUSIONS: Our findings suggest distinct plasmatic exosomal protein profiles in RA patients. These proteins not only take important parts in the vicious circle in the pathogenic process of RA but also serve as novel biomarkers in RA diagnosis and prognosis.

Augmenting and directing long-range CRISPR-mediated activation in human cells.

Epigenetic editing is an emerging technology that uses artificial transcription factors (aTFs) to regulate expression of a target gene. Although human genes can be robustly upregulated by targeting aTFs to promoters, the activation induced by directing aTFs to distal transcriptional enhancers is substantially less robust and consistent. Here we show that long-range activation using CRISPR-based aTFs in human cells can be made more efficient and reliable by concurrently targeting an aTF to the target gene promoter. We used this strategy to direct target gene choice for enhancers capable of regulating more than one promoter and to achieve allele-selective activation of human genes by targeting aTFs to single-nucleotide polymorphisms embedded in distally located sequences. Our results broaden the potential applications of the epigenetic editing toolbox for research and therapeutics.

ZNF410 represses fetal globin by singular control of CHD4.

Known fetal hemoglobin (HbF) silencers have potential on-target liabilities for rational ß-hemoglobinopathy therapeutic inhibition. Here, through transcription factor (TF) CRISPR screening, we identify zinc-finger protein (ZNF) 410 as an HbF repressor. ZNF410 does not bind directly to the genes encoding γ-globins, but rather its chromatin occupancy is concentrated solely at CHD4, encoding the NuRD nucleosome remodeler, which is itself required for HbF repression. CHD4 has two ZNF410-bound regulatory elements with 27 combined ZNF410 binding motifs constituting unparalleled genomic clusters. These elements completely account for the effects of ZNF410 on fetal globin repression. Knockout of ZNF410 or its mouse homolog Zfp410 reduces CHD4 levels by 60%, enough to substantially de-repress HbF while eluding cellular or organismal toxicity. These studies suggest a potential target for HbF induction for ß-hemoglobin disorders with a wide therapeutic index. More broadly, ZNF410 represents a special class of gene regulator, a conserved TF with singular devotion to regulation of a chromatin subcomplex.

Transcription factor competition at the γ-globin promoters controls hemoglobin switching.

BCL11A, the major regulator of fetal hemoglobin (HbF, α2γ2) level, represses γ-globin expression through direct promoter binding in adult erythroid cells in a switch to adult hemoglobin (HbA, α2ß2). To uncover how BCL11A initiates repression, we used CRISPR-Cas9, dCas9, dCas9-KRAB and dCas9-VP64 screens to dissect the γ-globin promoters and identified an activator element near the BCL11A-binding site. Using CUT&RUN and base editing, we demonstrate that a proximal CCAAT box is occupied by the activator NF-Y. BCL11A competes with NF-Y binding through steric hindrance to initiate repression. Occupancy of NF-Y is rapidly established following BCL11A depletion, and precedes γ-globin derepression and locus control region (LCR)-globin loop formation. Our findings reveal that the switch from fetal to adult globin gene expression within the >50-kb ß-globin gene cluster is initiated by competition between a stage-selective repressor and a ubiquitous activating factor within a remarkably discrete region of the γ-globin promoters.

Motif-Raptor: a cell type-specific and transcription factor centric approach for post-GWAS prioritization of causal regulators.

MOTIVATION: Genome-wide association studies (GWASs) have identified thousands of common trait-associated genetic variants but interpretation of their function remains challenging. These genetic variants can overlap the binding sites of transcription factors (TFs) and therefore could alter gene expression. However, we currently lack a systematic understanding on how this mechanism contributes to phenotype. RESULTS: We present Motif-Raptor, a TF-centric computational tool that integrates sequence-based predictive models, chromatin accessibility, gene expression datasets and GWAS summary statistics to systematically investigate how TF function is affected by genetic variants. Given trait-associated non-coding variants, Motif-Raptor can recover relevant cell types and critical TFs to drive hypotheses regarding their mechanism of action. We tested Motif-Raptor on complex traits such as rheumatoid arthritis and red blood cell count and demonstrated its ability to prioritize relevant cell types, potential regulatory TFs and non-coding SNPs which have been previously characterized and validated. AVAILABILITY AND IMPLEMENTATION: Motif-Raptor is freely available as a Python package at: https://github.com/pinellolab/MotifRaptor. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Dissecting ELANE neutropenia pathogenicity by human HSC gene editing.

Severe congenital neutropenia (SCN) is a life-threatening disorder most often caused by dominant mutations of ELANE that interfere with neutrophil maturation. We conducted a pooled CRISPR screen in human hematopoietic stem and progenitor cells (HSPCs) that correlated ELANE mutations with neutrophil maturation potential. Highly efficient gene editing of early exons elicited nonsense-mediated decay (NMD), overcame neutrophil maturation arrest in HSPCs from ELANE-mutant SCN patients, and produced normal hematopoietic engraftment function. Conversely, terminal exon frameshift alleles that mimic SCN-associated mutations escaped NMD, recapitulated neutrophil maturation arrest, and established an animal model of ELANE-mutant SCN. Surprisingly, only -1 frame insertions or deletions (indels) impeded neutrophil maturation, whereas -2 frame late exon indels repressed translation and supported neutrophil maturation. Gene editing of primary HSPCs allowed faithful identification of variant pathogenicity to clarify molecular mechanisms of disease and encourage a universal therapeutic approach to ELANE-mutant neutropenia, returning normal neutrophil production and preserving HSPC function.

Epigenetic Alterations in Keratinocyte Carcinoma.

Basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) are both derived from epidermal keratinocytes but are phenotypically diverse. To improve the understanding of keratinocyte carcinogenesis, it is critical to understand epigenetic alterations, especially those that govern gene expression. We examined changes to the enhancer-associated histone acetylation mark H3K27ac by mapping matched tumor-normal pairs from 11 patients (five with BCC and six with SCC) undergoing Mohs surgery. Our analysis uncovered cancer-specific enhancers on the basis of differential H3K27ac peaks between matched tumor-normal pairs. We also uncovered biological pathways potentially altered in keratinocyte carcinoma, including enriched epidermal development and Wnt signaling pathways enriched in BCCs and enriched immune response and cell activation pathways in SCCs. We also observed enrichment of transcription factors that implicated SMAD and JDP2 in BCC pathogenesis and FOXP1 in SCC pathogenesis. On the basis of these findings, we prioritized three loci with putative regulation events (FGFR2 enhancer in BCC, intragenic regulation of FOXP1 in SCC, and WNT5A promoter in both subtypes) and validated our findings with published gene expression data. Our findings highlight unique and shared epigenetic alterations in histone modifications and potential regulators for BCCs and SCCs that likely impact the divergent oncogenic pathways, paving the way for targeted drug discoveries.

Therapeutic base editing of human hematopoietic stem cells.

Base editing by nucleotide deaminases linked to programmable DNA-binding proteins represents a promising approach to permanently remedy blood disorders, although its application in engrafting hematopoietic stem cells (HSCs) remains unexplored. In this study, we purified A3A (N57Q)-BE3 base editor for ribonucleoprotein (RNP) electroporation of human-peripheral-blood-mobilized CD34+ hematopoietic stem and progenitor cells (HSPCs). We observed frequent on-target cytosine base edits at the BCL11A erythroid enhancer at +58 with few indels. Fetal hemoglobin (HbF) induction in erythroid progeny after base editing or nuclease editing was similar. A single therapeutic base edit of the BCL11A enhancer prevented sickling and ameliorated globin chain imbalance in erythroid progeny from sickle cell disease and ß-thalassemia patient-derived HSPCs, respectively. Moreover, efficient multiplex editing could be achieved with combined disruption of the BCL11A erythroid enhancer and correction of the HBB -28A>G promoter mutation. Finally, base edits could be produced in multilineage-repopulating self-renewing human HSCs with high frequency as assayed in primary and secondary recipient animals resulting in potent HbF induction in vivo. Together, these results demonstrate the potential of RNP base editing of human HSPCs as a feasible alternative to nuclease editing for HSC-targeted therapeutic genome modification.

Proteomics Screening of Differentially Expressed Cytokines in Tears of Patients with Graves' Ophthalmopathy.

BACKGROUND: The current study aimed at exploring the cytokine profile in the tears of patients with Graves' ophthalmopathy (GO). METHODS: Tears were sampled from the eyes of 7 patients with active GO and 7 healthy volunteers using filter paper. Then the levels of up to 34 cytokines in the tears of each subject were detected using high-throughput protein microarray technology in line with the introduction. RESULTS: The results of cytokine protein microarray screening showed that 10 proteins, namely, CD40, CD40 Ligand, GITR, IL-12p70, IL-1 beta, IL-2, IL-21, IL-6, MIP-3 alpha and TRANCE, were overexpressed (with fold change >1.20) and 3 proteins, namely, GM-CSF, IL-1 sRI and IL-13 were downregulated (with fold change < 0.83) in GO patients. In addition, the protein levels of CD40 and CD40 ligand (CD40L) were significantly different between GO patients and healthy controls (P=0.028 and 0.011, respectively). Further Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of differently expressed proteins showed that these proteins were involved in biological functions including biological processes (positive regulation of cytokine production, JAK-STAT cascade and leukocyte proliferation), molecular functions (cytokine and growth factor receptors binding and cytokine activity), and other important pathways (cytokine-cytokine receptor interaction, JAK-STAT signaling pathway, IL-17 signaling pathway, NF-kappa B signaling pathway, Th17 cell differentiation, and intestinal immune network for IgA production), all of which might be involved in the pathology of GO. CONCLUSION: Our cytokine protein microarray analysis indicated that several proteins were differentially expressed in GO patients, which provides potential targets for GO prevention.