bluuebunny/crossref_metadata_2025_split huggingface dataset:
C. perfringens enterotoxin-claudin pore complex: Models for structure, mechanism of pore assembly and cation permeability
Authors: Nagarajan Santhosh Kumar , Weber Joy , Roderer Daniel , Piontek Jörg 
Published: August 2024

AbstractThe pore-formingClostridium perfringensenterotoxin (CPE), a common cause of foodborne diseases, facilitates Ca2+influx in enterocytes, leading to cell damage. Upon binding to certain claudins (e.g. claudin-4), CPE forms oligomeric pores in the cell membrane. While the CPE-claudin interaction mechanism is well known, the structure and assembly of the pore complex remain elusive. Here, using AlphaFold2 complex prediction, structure alignment, and molecular dynamics simulations, we generated models of pre-pore and pore states of the CPE-claudin-4 complex sequentially addressing CPE-claudin, CPE-CPE, and claudin-claudin interactions, along with CPE conformational changes. The CPE pore is a hexameric variation of the typically heptameric pore stem and cap architecture of aerolysin-like β-barrel pore-forming toxins (β-PFT). The pore is lined with three hexa-glutamate rings, which differs from other β-PFTs and confers CPE-specific cation selectivity of the pore. Additionally, the pore center is indicated to be anchored by a dodecameric claudin ring formed by a cis-interaction variant of an interface found in claudin-based tight junction strands. Mutation of an interfacial residue inhibited CPE-mediated cell damagein vitro. We propose that this claudin ring constitutes an anchor for a twisting mechanism that drives extension and membrane insertion of the CPE β-hairpins. Our pore model agrees with previous key experimental data providing insights into the structural mechanisms of CPE-mediated cytotoxic cation influx.
Multiplex genome editing eliminates the Warburg Effect without impacting growth rate in mammalian cells
Authors: Hefzi Hooman , Martínez-Monge Iván , Marin de Mas Igor , Cowie Nicholas Luke , Toledo Alejandro Gomez , Noh Soo Min , Karottki Karen Julie la Cour , Decker Marianne , Arnsdorf Johnny , Camacho-Zaragoza Jose Manuel , Kol Stefan , Schoffelen Sanne , Pristovšek Nuša , Hansen Anders Holmgaard , Miguez Antonio A. , Bjorn Sara Petersen , Brøndum Karen Kathrine , Javidi Elham Maria , Jensen Kristian Lund , Stangl Laura , Kreidl Emanuel , Kallehauge Thomas Beuchert , Ley Daniel , Ménard Patrice , Petersen Helle Munck , Sukhova Zulfiya , Bauer Anton , Casanova Emilio , Barron Niall , Malmström Johan , Nielsen Lars K. , Lee Gyun Min , Kildegaard Helene Faustrup , Voldborg Bjørn G. , Lewis Nathan E. 
Published: August 2024

ABSTRACTThe Warburg effect is ubiquitous in proliferative mammalian cells, including cancer cells, but poses challenges for biopharmaceutical production, as lactate accumulation inhibits cell growth and protein production. Previous efforts to eliminate lactate production via knockout have failed in mammalian bioprocessing since lactate dehydrogenase has proven essential. However, here we eliminated the Warburg effect in Chinese hamster ovary (CHO) and HEK293 cells by simultaneously knocking out lactate dehydrogenase and regulators involved in a negative feedback loop that typically inhibits pyruvate conversion to acetyl-CoA. In contrast to long-standing assumptions about the role of aerobic glycolysis, Warburg-null cells maintain wildtype growth rate while producing negligible lactate. Further characterization of Warburg-null CHO cells showed a compensatory increase in oxygen consumption, a near total reliance on oxidative metabolism, and higher cell densities in fed-batch cell culture. These cells remained amenable for production of diverse biotherapeutic proteins, reaching industrially relevant titers and maintaining product glycosylation. Thus, the ability to eliminate the Warburg effect is an important development for biotherapeutic production and provides a tool for investigating a near-universal metabolic phenomenon.
Computing hematopoietic stem and progenitor cell plasticity in response to genetic mutations and environmental stimulations
Authors: Wen Yuchen , He Hang , Ma Yunxi , Cai Lorie Chen , Wang Huaquan , Li Yanmei , Zhao Baobing , Cai Zhigang 
Published: August 2024

AbstractCell plasticity (CP), describing a dynamic cell state, plays a crucial role in maintaining homeostasis during organ morphogenesis, regeneration and damage-to-repair biological process. Single-cell-omics datasets provide unprecedented resource to empowers analysis on CP. Hematopoiesis offers fertile opportunities to develop quantitative methods for understanding CP with rich supports from experimental ground-truths. In this study we generated high-quality lineage-negative (Lin−) single-cell RNA-sequencing datasets under various conditions and introduced a working pipeline named Snapdragon to interrogate naïve and disturbed plasticity of hematopoietic stem and progenitor cells (HSPCs) with mutational or environmental challenges. Utilizing embedding methods UMAP or FA, a continuum of hematopoietic development is visually observed in wildtype where the pipeline confirms a very low Proportion of hybrid-cells (Phc, with bias range: 0.4-0.6) on a transition trajectory. UponTet2mutation, a driver of leukemia, or treatment of DSS, an inducer of colitis,Phcis increased and plasticity of HSPCs was enhanced. Quantitative analysis indicates thatTet2mutation enhances HSC self-renewal capability while DSS treatment results in an enhanced myeloid-skewing trajectory, suggesting their similar but different consequences. We prioritized several transcription factors (i.e the EGR family) and signaling pathways (i.e. receptors IL1R1 and ADRB, inflammation and sympathy-sensing respectively) which are responsible forPhcalterations. CellOracle-based simulation suggests that knocking-out EGR regulons or pathways of IL1R1 and ADRB partially reversesPhcpromoted byTet2mutation and inflammation. In conclusion, the study provides high-quality datasets with single-cell transcriptomic matrices for diversified hematopoietic simulations and a computational pipeline Snapdragon for quantifying disturbedPhcand CP. (247 words)HighlightsTo guide CP analysis, we introduce a quantizable parameterPhcand a pipeline Snapdragon, which discriminate naive and disturbed hematopoiesis;The Snapdragon pipeline analysis onTet2+/-Lin−cells demonstrates many novel insights, including enhanced HSC plasticity and increased PHC; similar trends are observed in inflammatory Lin−cells;Regulon analysis suggests that transcriptional factor EGR1 is significantly activated to elevated the HSC plasticity and change hematopoietic trajectory;Stress-response-related signaling pathways mediated by receptors IL1R1 or ADRB were obviously activated in the challenged hematopoiesis;CellOracle-based simulation suggests that knocking-out EGR regulons or pathways of IL1R1 and ADRB partially reversesPhcpromoted byTet2mutation and inflammation.
Highly adaptable deep-learning platform for automated detection and analysis of vesicle exocytosis
Authors: Chouaib Abed Alrahman , Chang Hsin-Fang , Khamis Omnia M. , Echeverry Santiago , Demeersseman Lucie , Elizarova Sofia , Daniel James A , Valitutti Salvatore , Barg Sebastian , Pape Constantin , Shaib Ali H. , Becherer Ute 
Published: August 2024

AbstractVesicle exocytosis is a fundamental component of intercellular communication, in all organisms. It has been studied for decades, using various imaging tools. Nevertheless, exocytosis research is still limited by the lack of reliable automated analysis procedures. To address this, we developed the Intelligent Vesicle Exocytosis Analysis Platform (IVEA), a nearly universal solution for analyzing exocytosis acquired with live cell imaging. IVEA is applicable to a wide variety of experimental model systems, microscopes and reporter fluorophores. IVEA combines state-of-the-art deep-learning and computer vision regimes to enable fully automated analysis of large data. IVEA runs as a FIJI plugin and does not require prior training or human intervention. IVEA is 60 times faster than manual analysis and is able to detect rare events often missed by the human eye. Overall, IVEA represents a breakthrough in the analysis of cellular secretory mechanisms and has a transformative potential for the exocytosis imaging field.
Identification of robust RT-qPCR reference genes for studying changes in gene expression in response to hypoxia in breast cancer cell lines
Authors: Malcolm Jodie R. , Bridge Katherine S. , Holding Andrew N. , Brackenbury William J. 
Published: August 2024

AbstractHypoxia is common in breast tumours and is linked to therapy resistance and advanced disease. To understand hypoxia-driven breast cancer progression, RT-qPCR quantifies transcriptional changes important for malignant development. Reference genes (RGs) are endogenous RT-qPCR controls used to normalise mRNA levels, allowing accurate assessment of transcriptional changes. However, hypoxia reprograms transcription and post-transcriptional processing of RNA such that favoured RGs includingGAPDHorPGK1are unsuitable for this purpose. To address the need for robust RGs to study hypoxic breast cancer cell lines, we identified 10 RG candidates by analysing public RNA-seq data of MCF-7, T-47D, MDA-MB-231 and MDA-MB-468 cells cultured in normoxia or hypoxia. RT-qPCR determined RG candidate levels in normoxic breast cancer cells, removingTBPandEPAS1from downstream analysis due to insufficient transcript abundance. Assessing primer efficiency further removedACTB, CCSER2andGUSBfrom consideration. Following culture in normoxia, or acute or chronic hypoxia, we ascertained robust non-variable RGs using RefFinder. Here we presentRPLP1andRPL27 asoptimal RGs for breast cancer cell lines cultured in normoxia or hypoxia. Our result enables accurate evaluation of gene expression in hypoxic breast cancer cell lines and provides an essential resource for assessing hypoxia’s impact in breast cancer progression.Graphical Abstract
ImPaqT - A Golden Gate-based Toolkit for Zebrafish Transgenesis
Authors: Hurst Saskia , Dimmler Christiane , Cronan Mark R. 
Published: August 2024

AbstractTransgenic animals continue to play an essential role in many aspects of zebrafish research, including the development of disease models. The most widely used system for zebrafish transgenesis is the Tol2 transposon system. Here, we have developed ImPaqT (Immunological toolkit forPaqCI-based Golden Gate Assembly of Tol2Transgenes), a new Tol2-based transgenesis system that utilizes Golden Gate assembly to facilitate the production of transgenic zebrafish lines. This system allows for rapid assembly of multiple fragments into a single transgene, facile swapping of individual sequences to generate new transgenes and an easy cloning workflow to incorporate new genetic elements into the existing kit. Within this toolkit framework, we have generated a number of reagents to enable gene expression within immune and non-immune cell types, an array of best-in-class fluorescent proteins to visualize cell populations and transgenes as well as tools to simplify genetic manipulation, purification and ablation of targeted cells. Unlike recombination-based systems, the Golden Gate approach is also expandable, allowing the incorporation of complex designs such as multi-fragment promoters within the established modular framework of ImPaqT. Here, we demonstrate the function of this new system by generating a number of novel transgenic immune reporter lines. While our toolkit is focused on the immune system as an emerging area of study within zebrafish research, the ImPaqT approach can be broadly adapted to the construction of almost any zebrafish transgene, offering new tools for the generation of transgenes within the zebrafish community.
Reduced function of the adaptor SH2B3 promotes T1D via altered gc cytokine-regulated, T cell intrinsic immune tolerance
Authors: Watson Taylor K. , Rosen Aaron B.I. , Drow Travis , Medjo Jacob A. , MacQuivey Matthew A. , Ge Yan , Liggitt H. Denny , Grosvenor Dane A. , Dill-McFarland Kimberly A. , Altman Matthew C. , Concannon Patrick J. , Buckner Jane H. , Rawlings David J. , Allenspach Eric J. 
Published: August 2024

AbstractGenome-wide association studies have identifiedSH2B3as an important non-MHC gene for islet autoimmunity and type 1 diabetes (T1D). In this study, we found a singleSH2B3haplotype significantly associated with increased risk for human T1D, and this haplotype carries the single nucleotide variant rs3184504*T inSH2B3.To better characterize the role of SH2B3 in T1D, we used mouse modeling and found a T cell-intrinsic role for SH2B3 regulating peripheral tolerance. SH2B3 deficiency had minimal effect on TCR signaling or proliferation across antigen doses, yet enhanced cell survival and cytokine signaling including common gamma chain-dependent and interferon-gamma receptor signaling. SH2B3 deficient CD8+T cells showed augmented STAT5-MYC and effector-related gene expression partially reversed with blocking autocrine IL-2 in culture. Using the RIP-mOVA model, we found CD8+ T cells lacking SH2B3 promoted early islet destruction and diabetes without requiring CD4+ T cell help. SH2B3-deficient cells demonstrated increased survival post-transfer compared to control cells despite a similar proliferation profile in the same host. Next, we created a spontaneous NOD.Sh2b3-/-mouse model and found markedly increased incidence and accelerated T1D across sexes. Collectively, these studies identify SH2B3 as a critical mediator of peripheral T cell tolerance limiting the T cell response to self-antigens.Article HighlightsThe rs3184504 polymorphism, encoding a hypomorphic variant of the negative regulator SH2B3, strongly associates with T1D.SH2B3 deficiency results in hypersensitivity to cytokines, including IL-2, in murine CD4+ and CD8+ T cells.SH2B3 deficient CD8+ T cells exhibit a comparable transcriptome to wild-type CD8+ T cells at baseline, but upon antigen stimulation SH2B3 deficient cells upregulate genes characteristic of enhanced JAK/STAT signaling and effector functions.We found a T-cell intrinsic role of SH2B3 leading to severe islet destruction in an adoptive transfer murine T1D model, while global SH2B3 deficiency accelerated spontaneous NOD diabetes across sexes.
A high lipid diet leads to greater pathology and lower tolerance during infection
Authors: Perrine Weston G. , Sauer Erin L. , Love Ashley C. , Morris Ashley , Novotny Johnathon , DuRant Sarah E. 
Published: August 2024

AbstractDespite the clear effect of food resources on wildlife disease dynamics, few studies have investigated how shifts in diet, specifically macronutrient content, shape vertebrate host responses to infection. While each individual macronutrient plays a vital role in physiological processes necessary for pathogen defense, it is often difficult to disentangle the role of an individual macronutrient on disease pathology. We explored the effects of dietary macronutrient composition on disease pathology and feeding behavior of canaries (Serinus canaria domestica)infected withMycoplasma gallisepticum(MG). First, we provided canaries with isocaloric diets comprised of identical ingredients that varied in macronutrient content (high-protein or high-lipid) then MG- or sham-inoculated birds. We then offered both diets to canaries before and after MG- and sham-inoculation. In experiment one, high protein diet birds consumed more food than high lipid diet birds and experienced a more pronounced decrease in food intake after infection. High protein diet birds were more tolerant to MG infection, exhibiting reduced pathology when compared to high lipid diet birds, despite the two treatments having similar levels of MG-specific antibodies and MG loads. When birds had access to both diets, they consumed more of the high protein diet and experienced pathology for less time than lipid or protein restricted birds. These results highlight that macronutrient makeup of the diet can shape host tolerance and pathology and thus affect host-pathogen transmission dynamics.Summary StatementMacronutrient composition of the diet shapes tolerance in an avian host pathogen system. Birds that had greater access to protein exhibited reduced pathology despite similar pathogen loads when compared with birds with greater access to lipids.
Six drivers of aging identified among genes differentially expressed with age
Authors: Coler-Reilly Ariella , Pincus Zachary , Scheller Erica L. , Civitelli Roberto 
Published: August 2024

AbstractMany studies have compared gene expression in young and old samples to gain insights on aging, the primary risk factor for most major chronic diseases. However, these studies only describe associations, failing to distinguish drivers of aging from compensatory geroprotective responses and incidental downstream effects. Here, we introduce a workflow to characterize the causal effects of differentially expressed genes on lifespan. First, we performed a meta-analysis of 25 gene expression datasets comprising samples of various tissues from healthy, untreated adult mammals (humans, dogs, and rodents) at two distinct ages. We ranked each gene according to the number of distinct datasets in which the gene was differentially expressed with age in a consistent direction. The top age-upregulated genes were TMEM176A, EFEMP1, CP, and HLA-A; the top age-downregulated genes were CA4, SIAH, SPARC, and UQCR10. Second, the effects of the top ranked genes on lifespan were measured by applying post-developmental RNA interference of the corresponding ortholog in the nematode C. elegans (two trials, with roughly 100 animals per genotype per trial). Out of 10 age-upregulated and 9 age-downregulated genes that were tested, two age-upregulated genes (csp-3/CASP1 andspch-2/RSRC1) and four age-downregulated genes (C42C1.8/DIRC2,ost-1/SPARC,fzy-1/CDC20, andcah-3/CA4) produced significant and reproducible lifespan extension. Notably, the data do not suggest that the direction of differential expression with age is predictive of the effect on lifespan. Our study provides novel insight into the relationship between differential gene expression and aging phenotypes, pilots an unbiased workflow that can be easily repeated and expanded, and pinpoints six genes with evolutionarily conserved, causal roles in the aging process for further study.Graphical Abstract
scMitoMut for calling mitochondrial lineage–related mutations in single cells
Authors: Sun Wenjie , van Ginneken Daphne , Perié Leïla 
Published: August 2024

AbstractTracing cell lineages has become a valuable tool for studying biological processes. Among the available tools for human data, mitochondria DNA (mtDNA) has a high potential due to its ability to be used in conjunction with single-cell chromatin accessibility data, giving access to the cell phenotype. Nonetheless, the existing mutation calling tools are ill-equipped to deal with the polyploid nature of the mtDNA and lack a robust statistical framework. Here we introduce scMitoMut, an innovative R package that leverages statistical methodologies to accurately identify mitochondrial lineage related mutations at the single-cell level. scMitoMut assigns a mutation quality q-value based on beta-binomial distribution to each mutation at each locus within individual cells, ensuring higher sensitivity and precision of lineage related mutation calling in comparison to current methodologies. We tested scMitoMut using single-cell DNA sequencing, scATAC sequencing and 10× Genomics single cell multiome datasets. Using a single-cell DNA sequencing dataset from a mixed population of cell lines, scMitoMut demonstrated superior sensitivity in identifying small proportion of cancer cell lines compared to existing methods. In a human colorectal cancer scATAC dataset, scMitoMut identified more mutations than state-of-the-art methods. Applied to 10× Genomics multiome datasets, scMitoMut effectively measured the lineage distance in cells from blood or brain tissues. Thus, the scMitoMut is a free available (https://www.bioconductor.org/packages/devel/bioc/html/scMitoMut.html.), well-engineered toolkit for mtDNA mutation calling with high memory and CPU efficiency. Consequently, it will significantly advance the application of single-cell sequencing, facilitating the precise delineation of mitochondrial mutations for lineage tracing purposes in development, tumor and stem cell biology.
Single cell multiome profiling of pancreatic islets reveals physiological changes in cell type-specific regulation associated with diabetes risk
Authors: Mummey Hannah M , Elison Weston , Korgaonkar Katha , Elgamal Ruth M , Kudtarkar Parul , Griffin Emily , Benaglio Paola , Miller Michael , Jha Alokkumar , Manning Fox Jocelyn E , McCarthy Mark I , Preissl Sebastian , Gloyn Anna L , MacDonald Patrick E , Gaulton Kyle J 
Published: August 2024

AbstractPhysiological variability in pancreatic cell type gene regulation and the impact on diabetes risk is poorly understood. In this study we mapped gene regulation in pancreatic cell types using single cell multiomic (joint RNA-seq and ATAC-seq) profiling in 28 non-diabetic donors in combination with single cell data from 35 non-diabetic donors in the Human Pancreas Analysis Program. We identified widespread associations with age, sex, BMI, and HbA1c, where gene regulatory responses were highly cell type- and phenotype-specific. In beta cells, donor age associated with hypoxia, apoptosis, unfolded protein response, and external signal-dependent transcriptional regulators, while HbA1c associated with inflammatory responses and gender with chromatin organization. We identified 10.8K loci where genetic variants were QTLs forcisregulatory element (cRE) accessibility, including 20% with lineage- or cell type-specific effects which disrupted distinct transcription factor motifs. Type 2 diabetes and glycemic trait associated variants were enriched in both phenotype- and QTL-associated beta cell cREs, whereas type 1 diabetes showed limited enrichment. Variants at 226 diabetes and glycemic trait loci were QTLs in beta and other cell types, including 40 that were statistically colocalized, and annotating target genes of colocalized QTLs revealed genes with putatively novel roles in disease. Our findings reveal diverse responses of pancreatic cell types to phenotype and genotype in physiology, and identify pathways, networks, and genes through which physiology impacts diabetes risk.
Comparison of Commercially Available Thermostable DNA Polymerases with Reverse-Transcriptase Activity in Coupled Reverse-Transcription Polymerase Chain Reaction Assays
Authors: Smirnova Evgeniya V. , Blagodatskikh Konstantin A. , Barsova Ekaterina V. , Varlamov Dmitriy A. , Kramarov Vladimir M. , Ignatov Konstantin B. 
Published: August 2024

AbstractReverse-transcription polymerase chain reaction (RT-PCR) is an important tool for the detection of target RNA molecules and the assay of RNA pathogens. Coupled RT-PCR is performed with an enzyme mixture containing a reverse transcriptase and a thermostable DNA polymerase. To date, several biotechnological companies offer artificial thermostable DNA polymerases with a built-in reverse-transcriptase activity for use in the coupled RT-PCR instead of the enzyme mixtures. Here, we compared the artificial DNA polymerases and conventional enzyme mixtures for the RT-PCR by performing end-point and real-time RT-PCR assays using severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV2) RNA and endogenous mRNA molecules as templates. We found that the artificial enzymes were suitable for different RT-PCR applications, including SARS-CoV2 RNA detection, but not for long-fragment RT-PCR amplification.