Protein Function Research Articles

Insulin oxidation and oxidative modifications alter glucose uptake, cell metabolism, and inflammatory secretion profiles

Insulin participates in glucose homeostasis in the body and regulates glucose, protein, and lipid metabolism. Chronic hyperglycemia triggers oxidative stress and the generation of reactive oxygen species (ROS), leading to oxidized insulin variants. Oxidative protein modifications can cause functional changes or altered immunogenicity as known from the context of autoimmune disorders. However, studies on the biological function of native and oxidized insulin on glucose homeostasis and cellular function are lacking. Native insulin showed heterogenous effects on metabolic activity, proliferation, glucose carrier transporter (GLUT) 4, and insulin receptor (INSR) expression, as well as glucose uptake in cell lines of five different human tissues. Diverse ROS compositions produced by different gas plasma approaches enabled the investigations of variously modified insulin (oxIns) with individual oxidative post-translational modification (oxPTM) patterns as identified using high-resolution mass spectrometric analysis. Specific oxIns variants promoted cellular metabolism and proliferation in several cell lines investigated, and nitrogen plasma emission lines could be linked to insulin nitration and elevated glucose uptake. In addition, insulin oxidation modified blood glucose levels in the chicken embryos (in ovo), underlining the importance of assessing protein oxidation and function in health and disease.

Histopathologic, genomic, transcriptomic, and functional characteristics of eight melanocytic tumors with BRAF fusions showing stronger MAPK pathway activation compared to BRAF V600E tumors.

Activating BRAF gene alterations are central to melanocytic tumor pathogenesis. A small, emerging subset of melanocytic tumors driven by BRAF fusions has distinct therapeutic implications and has been described to have Spitzoid morphology patterns. However, such morphological patterns do not encompass all cases, and little is known about the functional molecular events. We conducted a retrospective search through our molecular archives to identify melanocytic tumors with BRAF fusions. We reviewed clinical, histopathological, and genomic features. We further explored transcriptomic and protein-level findings. Histopathologic patterns varied, with many cases without a distinctive pattern. We identified novel and diverse BRAF gene fusion partners. Differential transcriptomic analysis between low-risk BRAF fusion tumors and reference BRAF V600E tumors showed no differentially expressed genes. However, quantitatively stronger MAPK pathway activation of BRAF fusion tumors over BRAF V600E tumors was demonstrated by statistically significant stronger staining of p-ERK immunohistochemistry. Gene-specific RNA analysis shows comparable BRAF transcript levels between the two groups. The quantitatively stronger activation of the MAPK pathway of BRAF fusion tumors, instead of qualitatively different transcriptomes, may account for the morphology difference from conventional BRAF V600E tumors. BRAF fusions likely act through dysregulated protein function rather than RNA upregulation related to the characteristics of the fusion partners.

Elliptic geometry-based kernel matrix for improved biological sequence classification

Protein sequence classification plays a pivotal role in bioinformatics as it enables the comprehension of protein functions and their involvement in diverse biological processes. While numerous machine learning models have been proposed to tackle this challenge, traditional approaches face limitations in capturing the intricate relationships and hierarchical structures inherent in genomic sequences. These limitations stem from operating within high-dimensional non-Euclidean spaces. To address this issue, we introduce the application of the elliptic geometry-based approach for protein sequence classification. First, we transform the problem in elliptic geometry and integrate it with the Gaussian kernel to map the problem into the Mercer kernel. The Gaussian-Elliptic approach allows for the implicit mapping of data into a higher-dimensional feature space, enabling the capture of complex nonlinear relationships. This feature becomes particularly advantageous when dealing with hierarchical or tree-like structures commonly encountered in biological sequences. Experimental results highlight the effectiveness of the proposed model in protein sequence classification, showcasing the advantages of utilizing elliptic geometry in bioinformatics analyses. It outperforms state-of-the-art methods by achieving 76% and 84% accuracies for DNA and Protein datasets, respectively. Furthermore, we provide theoretical justifications for the proposed model. This study contributes to the burgeoning field of geometric deep learning, offering insights into the potential applications of elliptic representations in the analysis of biological data.

Characterization of mitochondrial DNA mutations in colorectal cancer progression by in silico approach and use as potential biomarkers for diagnosis and prognosis

BackgroundMitochondrial DNA variants are significant contributors to cancer progression, as evidenced by numerous findings. This study focuses on characterizing mitochondrial DNA mutations in colorectal cancer progression and their potential as biomarkers.MethodologyNext generation sequencing technology was employed to analyze mitochondrial DNA variants in tumor and adjacent normal tissues from 25 patients with colon/rectal cancer. In silico prediction tools (SIFT, Polyphen2, Mutation Assessor, and SNP&GO) were utilized to assess the pathogenicity of these variants. Additionally, homology modeling of mutated protein structures was conducted, and molecular dynamic simulations were performed to assess the impact of mutation on protein function.ResultsEighteen variants were identified across most tumor tissue samples, located in genes from Complex I, IV, and V. Among the identified variants, the V302M and S461 mutations in the MT-ND5 gene and L137F and L220P mutations in the ATP6 gene were predicted to be deleterious, potentially affecting protein function. 3D structural analysis of both wild-type and mutant proteins of MT-ND5 revealed changes in flexibility for the V302M and S461G mutations. The MT-ATP6 mutations L135F and L220P disrupt the interactions with surrounding residues and affect the overall function of protein. Further changes in protein dynamics of the mutated proteins by molecular dynamic simulations also indicate the effects; the mutations have on protein function.ConclusionMT-ND5 and MT-ATP6 variants could serve as potential biomarkers and drug targets in colorectal cancer. This study underscores the significance of mitochondrial DNA variants in cancer progression.

Loss of IDH1 and IDH2 in macrophages induces pathological phenotypes in cardiomyocytes

Abstract Introduction The clonal expansion of hematopoietic stem cells can be induced by a variety of age-related somatic mutations, a phenomenon known as "clonal hematopoiesis of indeterminate potential" (CHIP). CHIP is associated with an increased incidence and poor outcomes in patients with cardiovascular disease. The mechanisms underlying the crosstalk between CHIP cells and cardiac tissue are yet to be elucidated. Rare mutations in the metabolism-regulating isocitrate dehydrogenases encoding genes IDH1 and IDH2 have been identified as AML-like and cardiovascular high-risk CHIP-driving mutations in patient cohorts. However, the potential paracrine effects of these mutations in immune cells on cardiac tissue are unknown. Therefore, we assessed the impact of IDH1 and IDH2 mutations on the paracrine activities of macrophages. Methods and Results To gain first insights into the functional consequences of the IDH mutations, we used the AlphaMissense algorithm, which predicts the impact of mutations on protein function. IDH1 and IDH2 mutations identified in our patient cohorts were predicted to induce pathological alterations of protein function with a score >0.99. Therefore, we assessed the effects of silencing of the two genes in primary human CD14+ M0 macrophages on the paracrine activity on neonatal rat cardiomyocytes. Transfection with siRNA pools targeting IDH1 and IDH2 to mimic CHIP-driving conditions reduced mRNA abundance by 78% and 73%, respectively (both p<0.05). To assess the impact of IDH-silencing on cardiovascular cells, cardiomyocytes were treated with the supernatant of the macrophages. Interestingly, supernatants of IDH1 silenced macrophages significantly increased apoptosis of cardiomyocytes (8.6-fold increased expression of cleaved caspase-3; p<0.05) and reduced beating frequency. In addition, supernatants of IDH1 silenced macrophages induced a 1.5-fold increase in cardiomyocyte hypertrophy (p<0.01), whereas silencing of IDH2 showed no effect on cardiomyocyte hypertrophy and apoptosis. However, supernatants of both IDH1 and IDH2 silenced macrophages resulted in significantly increased numbers of bi- and multinucleated cardiomyocytes. Conclusion: Loss of isocitrate dehydrogenases in human macrophages affects cardiomyocyte functions in a paracrine fashion. Particularly silencing of IDH1 in macrophages introduced cardiomyocyte hypertrophy, apoptosis, and bi- and multinucleation, and reduced contractile function. Ongoing studies will address how IDH1 and IDH2 silencing-induced alterations in macrophages control their paracrine activity on cardiomyocytes.

Developmental cues are encoded by the combinatorial phosphorylation of Arabidopsis RETINOBLASTOMA-RELATED protein RBR1.

RETINOBLASTOMA-RELATED (RBR) proteins orchestrate cell division, differentiation, and survival in response to environmental and developmental cues through protein-protein interactions that are governed by multisite phosphorylation. Here we explore, using a large collection of transgenic RBR phosphovariants to complement protein function in Arabidopsis thaliana, whether differences in the number and position of RBR phosphorylation events cause a diversification of the protein's function. While the number of point mutations influence phenotypic strength, phosphosites contribute differentially to distinct phenotypes. RBR pocket domain mutations associate primarily with cell proliferation, while mutations in the C-region are linked to stem cell maintenance. Both phospho-mimetic and a phospho-defective variants promote cell death, suggesting that distinct mechanisms can lead to similar cell fates. We observed combinatorial effects between phosphorylated T406 and phosphosites in different protein domains, suggesting that specific, additive, and combinatorial phosphorylation events fine-tune RBR function. Suppression of dominant phospho-defective RBR phenotypes with a mutation that inhibits RBR interacting with LXCXE motifs, and an exhaustive protein-protein interaction assay, not only revealed the importance of DREAM complex members in phosphorylation-regulated RBR function but also pointed to phosphorylation-independent RBR roles in environmental responses. Thus, combinatorial phosphorylation defined and separated developmental, but not environmental, functions of RBR.

Suppressing Expression of SERPINE1/PAI1 Through Activation of GPER1 Reduces Progression of Vulvar Carcinoma.

The serine proteinase inhibitor 1 (SERPINE1) gene codes for the plasminogen activator inhibitor 1 (PAI1) protein and is thought to play a tumor supportive role in various cancers. In this work we aimed to uncover the role PAI1 plays in the proliferation, migration, and invasion of vulvar cancer (VC), and define the protein's function as an oncogene or tumor suppressor. Through treatment with an agonist (G1) and antagonist (G36) of G-coupled estrogen receptor 1 (GPER1), an upstream regulator of SERPINE1 expression, and a forward transfection knockdown protocol, the expression of SERPINE1/PAI1 in VC cells was altered. The effects these altered SERPINE1/PAI1 levels had on tumor cell functions were then examined. Proliferation was analyzed using the resazurin assay, while migration was studied via the gap closure assay. Through colony- and tumor sphere- formation assays clonogenicity was tested, and western blots showed protein expression. In A431 VC cells, when the levels of PAI1 were reduced via knockdown or treatment with G1, migration, proliferation, and colony growth was reduced. Treatment with G36 increased expression of PAI1 and increased migration and colony size in CAL39 cells. Based on the findings in this study, suppressing PAI1 expression in VC cells appears to reduce their progression and tumorigenic potential. Therefore, PAI1 could possibly function as an oncogene in VC. GPER1 appears to be a suitable target for suppressing PAI1 in VC.

LncRNA BRE-AS1 regulates the JAK2/STAT3-mediated inflammatory activation via the miR-30b-5p/SOC3 axis in THP-1 cells

Long non-coding RNAs (lncRNAs) have emerged as pivotal regulators in numerous biological processes, including macrophage-mediated inflammatory responses, which play a critical role in the progress of diverse diseases. This study focuses on the regulatory function of lncRNA brain and reproductive organ-expressed protein (BRE) antisense RNA 1 (BRE-AS1) in modulating the inflammatory activation of monocytes/macrophages. Employing the THP-1 cell line as a model, we demonstrate that lipopolysaccharide (LPS) treatment significantly upregulates BRE-AS1 expression. Notably, specific knockdown of BRE-AS1 via siRNA transfection enhances LPS-induced expression of interleukin (IL)-6 and IL-1β, while not affecting tumor necrosis factor (TNF)-α levels. This selective augmentation of pro-inflammatory cytokine production coincides with increased phosphorylation of Janus kinase (JAK)2 and signal transducer and activator of transcription (STAT)3. Furthermore, BRE-AS1 suppression results in the downregulation of suppressor of cytokine signaling (SOCS)3, an established inhibitor of the JAK2/STAT3 pathway. Bioinformatics analysis identified binding sites for miR-30b-5p on both BRE-AS1 and SOCS3 mRNA. Intervention with a miR-30b-5p inhibitor and a synthetic RNA fragment that represents the miR-30b-5p binding site on BRE-AS1 attenuates the pro-inflammatory effects of BRE-AS1 knockdown. Conversely, a miR-30b-5p mimic replicated the BRE-AS1 attenuation outcomes. Our findings elucidate the role of lncRNA BRE-AS1 in modulating inflammatory activation in THP-1 cells via the miR-30b-5p/SOCS3/JAK2/STAT3 signaling pathway, proposing that manipulation of macrophage BRE-AS1 activity may offer a novel therapeutic avenue in diseases characterized by macrophage-driven pathogenesis.

Protein language models learn evolutionary statistics of interacting sequence motifs

Protein language models (pLMs) have emerged as potent tools for predicting and designing protein structure and function, and the degree to which these models fundamentally understand the inherent biophysics of protein structure stands as an open question. Motivated by a finding that pLM-based structure predictors erroneously predict nonphysical structures for protein isoforms, we investigated the nature of sequence context needed for contact predictions in the pLM Evolutionary Scale Modeling (ESM-2). We demonstrate by use of a "categorical Jacobian" calculation that ESM-2 stores statistics of coevolving residues, analogously to simpler modeling approaches like Markov Random Fields and Multivariate Gaussian models. We further investigated how ESM-2 "stores" information needed to predict contacts by comparing sequence masking strategies, and found that providing local windows of sequence information allowed ESM-2 to best recover predicted contacts. This suggests that pLMs predict contacts by storing motifs of pairwise contacts. Our investigation highlights the limitations of current pLMs and underscores the importance of understanding the underlying mechanisms of these models.

Differential amino acid usage leads to ubiquitous edge effect in proteomes across domains of life that can be explained by amino acid secondary structure propensities

Amino acids are the building blocks of proteins and enzymes which are essential for life. Understanding amino acid usage offers insights into protein function and molecular mechanisms underlying life histories. However, genome-wide patterns of amino acid usage across domains of life remain poorly understood. Here, we analysed the proteomes of 5590 species across four domains and found that only a few amino acids are consistently the most and least used. This differential usage results in lower amino acid usage diversity at the most and least frequent ranks, creating a ubiquitous inverted U-shape pattern of amino acid diversity and rank which we call an ‘edge effect’ across proteomes and domains of life. This effect likely stems from protein secondary structural constraints, not the evolutionary chronology of amino acid incorporation into the genetic code, highlighting the functional rather than evolutionary influences on amino acid usage. We also tested other contemporary hypotheses regarding amino acid usage in proteomes and found that amino acid usage varies across life’s domains and is only weakly influenced by growth temperature. Our findings reveal a novel and pervasive amino acid usage pattern across genomes with the potential to help us probe deep evolutionary relationships and advance synthetic biology.

Clonal hematopoiesis in cardiovascular aging: Insights from the verona heart study.

Clonal hematopoiesis of indeterminate potential (CHIP), marked by the accumulation of somatic mutations in hematopoietic stem cells, significantly elevates the risk of all-cause mortality, mainly due to cardiovascular events. Therefore, investigating this pathophysiological phenomenon is crucial for understanding cardiovascular aging and enhancing both health span and lifespan. In the present study, we examined samples of subjects enrolled within the angiographically controlled Verona Heart Study (VHS), which provides a robust model for cardiovascular aging, particularly regarding coronary artery disease (CAD). We analyzed 44 older subjects diagnosed with coronary artery disease (CAD) and 42 healthy, sex- and age-matched controls (CAD-FREE). Employing deep sequencing and an amplicon-based approach, we focused on 11 key genetic regions in ASXL1, DNMT3A, IDH1, IDH2, JAK2, PPM1D, SF3B1, SRSF2, TET2, TP53, and U2AF1 genes to investigate clonal hematopoiesis. Subjects in the CAD group exhibited a significantly higher variant burden than those in the CAD-FREE group, both in terms of the total number of somatic variants and disruptive variants affecting protein function. This increased mutational load was notably influenced by six specific genetic regions: ASXL1, DNMT3A, IDH2, JAK2, TET2, and U2AF1, which displayed elevated variant rates in the CAD subjects. Moreover, ASXL1, DNMT3A, IDH2, JAK2, SF3B1, TET2, and TP53 exhibited substantially higher levels of disruptive variants in the CAD group. In summary, our findings highlight a correlation between clonal hematopoiesis and the accumulation of disruptive variants in specific genomic regions in the VHS cohort, thereby shedding light on their potential role in cardiovascular aging.

Lactylation in cancer: Mechanisms in tumour biology and therapeutic potentials.

Lactylation, a recently identified form of protein post-translational modification (PTM), has emerged as a key player in cancer biology. The Warburg effect, a hallmark of tumour metabolism, underscores the significance of lactylation in cancer progression. By regulating gene transcription and protein function, lactylation facilitates metabolic reprogramming, enabling tumours to adapt to nutrient limitations and sustain rapid growth. Over the past decade, extensive research has revealed the intricate regulatory network underlying lactylation in tumours. Large-scale sequencing and machine learning have confirmed the widespread occurrence of lactylation sites across the tumour proteome. Targeting lactylation enzymes or metabolic pathways has demonstrated promising anti-tumour effects, highlighting the therapeutic potential of this modification. This review comprehensively explores the mechanisms of lactylation in cancer cells and the tumour microenvironment. We expound on the application of advanced omics technologies for target identification and data modelling within the lactylation field. Additionally, we summarise existing anti-lactylation drugs and discuss their clinical implications. By providing a comprehensive overview of recent advancements, this review aims to stimulate innovative research and accelerate the translation of lactylation-based therapies into clinical practice. KEY POINTS: Lactylation significantly influences tumour metabolism and gene regulation, contributing to cancer progression. Advanced sequencing and machine learning reveal widespread lactylation sites in tumours. Targeting lactylation enzymes shows promise in enhancing anti-tumour drug efficacy and overcoming chemotherapy resistance. This review outlines the clinical implications and future research directions of lactylation in oncology.

Prokaryotic expression of wheat TaABI5-D3 gene and polyclonal antibody preparation

Abscisic acid insensitive 5 (ABI5) is a basic leucine zipper transcription factor regulating ABA-mediated seed germination, and TaABI5 is closely related to the pre-harvest sprouting in wheat. Studies have shown that TaABI5-D3, one of multiple copies of TaABI5 gene, encodes the intact TaABI5 protein. In this study, we constructed the prokaryotic expression vector pET-28a-TaABI5-D3 for expression of TaABI5-D3 in Escherichia coli and obtained the purified recombinant protein His-TaABI5-D3. This protein existed in the form of inclusion bodies, with the best expression induced by incubation with 0.6 mmol/L IPTG at 16 ℃, 150 r/min overnight (for 12 h). Subsequently, the purified His-TaABI5-D3 was injected into Balb/C mice for the preparation of polyclonal antibodies. Western blotting analysis indicated that the polyclonal antibody had relatively high specificity, laying foundations for clarification of the function of TaABI5 protein in wheat.

The secreted protein FonCHRD is essential for vegetative growth, asexual reproduction, and pathogenicity in watermelon Fusarium wilt fungus

Fungal pathogens often secrete numerous effectors to interfere with and/or suppress plant immunity to promote their infection. Watermelon Fusarium wilt, caused by the soil-borne fungus Fusarium oxysporum f. sp. niveum (Fon), is one of the devastating diseases that severely affect the watermelon industry. Here, we report the function of a candidate effector protein, FonCHRD, in Fon. FonCHRD harbors a chordin (CHRD) domain of unknown function and has a signal peptide with secretion activity. FonCHRD shows a relatively high expression level in Fon marcoconidia and is inducible by watermelon root tissues. Phenotypic analysis of the targeted deletion mutant revealed that FonCHRD plays roles in vegetative growth, asexual reproduction, and conidial morphology of Fon, while it is not involved in spore germination as well as cell wall, oxidative and salt stress responses. Deletion of FonCHRD impaired the ability to colonize and spread within host plants, significantly reducing its virulence on watermelon. FonCHRD is distributed across multiple compartments of plant cells but can target to the apoplast space in plants. FonCHRD inhibits the INF1- and Bcl2-associated X protein-triggered cell death and defense gene expression in transiently expressed Nicotiana benthamiana leaves. These findings suggest that FonCHRD is essential for Fon pathogenicity by modulating invasive growth and spreading abilities as well as by suppressing plant immune responses.

Uncovering ASO-Targetable Deep Intronic AIRE Variants: Insights and Therapeutic Implications.

High-throughput DNA sequencing has accelerated the discovery of disease-causing genetic variants, yet only in 10-40% of cases yield a genetic diagnosis. Increased implementation of genome sequencing has enabled a deeper exploration of the noncoding genome and recognition of noncoding variants as major contributors to disease. In a recent study, we identified a deep intronic variant in the AutoImmune REgulator (AIRE) gene (c.1504-818 G>A) as the cause of autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), a life-threatening monogenic autoimmune disorder most often caused by biallelic AIRE defects. This deep intronic variant disrupts normal splicing AIRE , causing pseudoexon inclusion and altered protein function. By developing an antisense oligonucleotide (ASO) targeting the pseudoexon sequence, we restored normal AIRE transcript in vitro, thereby revealing a potential genotype-specific candidate treatment. Our study illustrates key aspects of intronic variant detection, validation, and candidate ASO development. Herein, we briefly highlight the growing potential of ASO-based therapies for deep intronic variants, addressing the unmet need of personalized, genotype-specific therapies in diseases lacking curative options.

Separation and purification of antimicrobial substances from Paenibacillus polymyxa KH-19 and analysis of its physicochemical characterization.

Soft rot is one of the top ten most dangerous plant pathogens in agricultural production, storage, and transport, and the use of microorganisms and their metabolites to control soft rot is a current research hotspot. In this study, we identified the antimicrobial substance in the metabolite of Paenibacillus polymyxa KH-19, and determined that the antimicrobial substance of this strain was an active protein. The protein was completely precipitated at 40-60% ammonium sulphate saturation and showed good inhibitory effects against seven pathogenic bacteria including Pectobacterium carotovorum BC2 and seven pathogenic fungi including Pyricularia oryzae. The MIC of the protein was 51.563µg/mL, temperature acid-base UV and light stability insensitive to protease, with high-temperature resistance. The antimicrobial protein was isolated and purified by DEAE-anion exchange column and Sephadex G-75 gel filtration chromatography, and the LC-MS/MS assay identified the protein as lysophosphatidyl esterase with a molecular weight of 25.255kDa. The purified antimicrobial protein increased the inhibitory effect against P. carotovorum BC2, with the diameter of the circle of inhibition being 26.50 ± 0.915mm. Bioinformatics analysis showed that the protein has the molecular formula of C1117H1732N316O338S5, encodes 224 amino acids, has an aliphatic index of 88.39, and belongs to the category of hydrophilic unstable proteins. The present study is the first report of an active protein with extreme thermoplastic and resistance to P. carotovorum BC2, which provides a reference for the preparation and application of the antimicrobial substances of P. polymyxa KH-19, as well as a theoretical basis for the study of the function of lysophosphodiesterase protein and its use as a microbial preparation.

Frameshift mutations in the mmpR5 gene can have a bedaquiline-susceptible phenotype by retaining a protein structure and function similar to wild-type Mycobacterium tuberculosis.

Bedaquiline (BDQ) is crucial for the treatment of rifampicin-resistant tuberculosis, yet resistance threatens its effectiveness, mainly linked to mutations in the mmpR5 (Rv0678) gene. While frameshift mutations are thought to produce non-functional proteins, we hypothesize that they can result in conserved proteins through late-stop codons or alternative reading frames and remain BDQ susceptible. We extracted 512 isolates harboring frameshift mutations in mmpR5 from the World Health Organization (WHO) catalog and 68 isolates with minimum inhibitory concentration (MIC) in mycobacterial growth indicator tube (MGIT) through a literature review. Using BioPython and AlphaFold2 we computed open (ORF) and alternative reading frames (ARFs) sequences and protein structures and assessed similarity to the wild type using an alignment and template modeling (TM)-score. Among the WHO 512 isolates, 24.8% were BDQ-sensitive. Out of 184 unique frameshift mutations with available nucleotide information, a late-stop codon in the ORF occurred for 32% of the mutations. Also, 40.7% resulted in a conserved sequence, through the ORF or one of the forward ARFs. In 68 isolates with available MGIT MIC data, the presence of late-stop codons in the ORF (OR 4.71, 95% CI 1.36-19.3) or a conserved reading frame (OR 10.4, 95% CI 2.07-102.9) were associated with BDQ sensitivity. Protein structures from the conserved sequences showed high similarity (TM > 0.8). We show that frameshift mutations may retain BDQ susceptibility through late-stop codons in the ORF or conserved ARFs. These findings could improve the prediction of the BDQ phenotype from genomic data and have important implications for treatment decisions. Research Foundation-Flanders, Academy of Medical Sciences, the Wellcome Trust, the Government Department of Business, Energy and Industrial Strategy, the British Heart Foundation and Diabetes UK, and the Global Challenges Research Fund.IMPORTANCETuberculosis (TB), caused by Mycobacterium tuberculosis, remains the deadliest infectious disease and is particularly challenging to treat when it becomes drug-resistant. Bedaquiline (BDQ) is a recently recommended core drug for treating drug-resistant TB. However, resistance to bedaquiline is already emerging, primarily due to mutations in the mmpR5 gene. Identifying which mutations cause resistance and which do not is a critical knowledge gap. In particular, little is known about the effect of frameshift mutations, typically thought to make TB bacteria resistant to bedaquiline by producing non-functional proteins. Yet, one-quarter of isolates with a frameshift mutation are still susceptible to bedaquiline. How the bacteria produce a functional protein despite the frameshift mutation is unknown. We analyzed over 500 frameshift mutations using computational methods to model their effects on protein structure and bedaquiline resistance. Our findings revealed that some frameshift mutations can still produce functional proteins, allowing bacteria to remain sensitive to bedaquiline. Specifically, bacteria can produce a functional protein despite frameshift mutations if the mutation occurs near the end of the protein or if an alternative reading frame is available. These insights improve our ability to interpret mutations associated with bedaquiline, the most important drug for drug-resistant TB, allowing more accurate and effective treatment decisions.

Histone Methylation-Mediated Reproductive Toxicity to Consumer Product Chemicals in Caenorhabditis elegans: An Epigenetic Adverse Outcome Pathway (AOP).

The significance of histone methylation in epigenetic inheritance underscores its relevance to disease and the chronic effects of environmental chemicals. However, limited evidence of the causal relationships between chemically induced epigenetic changes and organismal-level effects hinders the application of epigenetic markers in ecotoxicological assessments. This study explored the contribution of repressive histone marks to reproductive toxicity induced by chemicals in consumer products in Caenorhabditis elegans, applying the adverse outcome pathway (AOP) framework. Triclosan (TCS) and tetrabromobisphenol A (TBBPA) exposures caused reproductive toxicity and altered histone methyltransferase (HMT) and histone demethylase (HDM) activities, increasing the level of trimethylation of H3K9 and H3K27. Notably, treatment with an H3K27-specific HMT inhibitor alleviated reproductive defects and the transcriptional response of genes related to vitellogenin, xenobiotic metabolism, and oxidative stress. Comparison of points of departure (PODs) based on calculated benchmark concentrations (BMCs) revealed the sensitivity of histone-modifying enzyme activities to these chemicals. Our findings suggest that the 'disturbance of HMT and HDM' can serve as the molecular initiating event (MIE) leading to reproductive toxicity in the epigenetic AOP for TCS and TBBPA. The study extended the biological applicability of these enzymes by identifying model species with analogous protein sequences and functions. This combined approach enhances the essentiality, empirical support, and taxonomic domain of applicability (tDOA), which are crucial considerations for ecotoxicological AOPs. Given the widespread use and environmental distribution of chemicals in consumer products, this study proposes histone-modifying enzyme activity as an effective screening tool for reproductive toxicants and emphasizes the integration of epigenetic mechanisms into a prospective ERA.

T lymphocyte-dependent IL-10 down-regulates a cytokine storm driven by Toxoplasma gondii GRA24.

As a model organism in the study of immunity to infection, Toxoplasma gondii has been instrumental in establishing key principles of host anti-microbial defense and its regulation. Here, we employed an attenuated uracil auxotroph strain of Type I Toxoplasma designated OMP to further untangle the early immune response to this parasitic pathogen. Experiments using αβ T cell-deficient Tcrb-/- mice unexpectedly revealed that an intact αβ T lymphocyte compartment was essential to survive infection with OMP. Subsequent antibody depletion and knockout mouse experiments demonstrated contributions from CD4+ T cells and most predominantly CD8+ T cells in resistance. Using transgenic knockout mice, we found only a partial requirement for IFN-γ and a lack of requirement for Toll-like receptor (TLR) adaptor MyD88 in resistance. In contrast to other studies on Toxoplasma, the ability to survive OMP infection did not require IL-12p40. Surprisingly, T cell-dependent IL-10 was found to be critical for survival, and deficiency of this cytokine triggered an abnormally high systemic inflammatory response. We also found that parasite molecule GRA24, a dense granule protein that triggers TLR-independent IL-12 production, acts as a virulence factor contributing to death of OMP-infected Tcrb-/- and IL-10-/- mice. Furthermore, resistance against OMP was restored in Tcrb-/- mice via monoclonal depletion of IL-12p40, suggesting that GRA24-induced IL-12 underlies the fatal immunopathology observed. Collectively, our studies provide insight into a novel and rapidly arising T lymphocyte-dependent anti-inflammatory response to T. gondii which operates independently of MyD88 and IL-12 and that depends on the function of parasite-dense granule protein GRA24.IMPORTANCEAs a model infectious microbe and an important human pathogen, the apicomplexan Toxoplasma gondii has provided many important insights into innate and adaptive immunity to infection. We show here that a low virulence uracil auxotrophic Toxoplasma strain emerges as a virulent parasite in the absence of an intact T cell compartment. Both CD4+ and CD8+ T lymphocytes are required for optimal protection, in line with previous findings in other models of Toxoplasma infection. Nevertheless, several novel aspects of the response were identified in our study. Protection occurs independently of IL-12 and MyD88 and only partially requires IFN-γ. This is noteworthy particularly because the cytokines IL-12 and IFN-γ have previously been regarded as essential for protective immunity to T. gondii. Instead, we identified the anti-inflammatory effects of T cell-dependent IL-10 as the critical factor enabling host survival. The parasite dense granule protein GRA24, a host-directed mitogen-activated protein kinase activator, was identified as a major virulence factor in T cell-deficient hosts. Collectively, our results provide new and unexpected insights into host resistance to Toxoplasma.

Generative AI Models for the Protein Scaffold Filling Problem.

De novo protein sequencing is an important problem in proteomics, playing a crucial role in understanding protein functions, drug discovery, design and evolutionary studies, etc. Top-down and bottom-up tandem mass spectrometry are popular approaches used in the field of mass spectrometry to analyze and sequence proteins. However, these approaches often produce incomplete protein sequences with gaps, namely scaffolds. The protein scaffold filling problem refers to filling the missing amino acids in the gaps of a scaffold to infer the complete protein sequence. In this article, we tackle the protein scaffold filling problem based on generative AI techniques, such as convolutional denoising autoencoder, transformer, and generative pretrained transformer (GPT) models, to complete the protein sequences and compare our results with recently developed convolutional long short-term memory-based sequence model. We evaluate the model performance both on a real dataset and generated datasets. All proposed models show outstanding prediction accuracy. Notably, the GPT-2 model achieves 100% gap-filling accuracy and 100% full sequence accuracy on the MabCampth protein scaffold, which outperforms the other models.