bluuebunny/crossref_metadata_2025_split huggingface dataset:
Oxidation of hemoproteins byStreptococcus pneumoniaecollapses the cell cytoskeleton and disrupts mitochondrial respiration leading to cytotoxicity of human lung cells
Authors: Scasny Anna , Alibayov Babek , Khan Faidad , Rao Shambavi J. , Murin Landon , Jop Vidal Ana G. , Smith Perriann , Wei Li , Edwards Kristin , Warncke Kurt , Vidal Jorge E. 
Published: June 2023

AbstractStreptococcus pneumoniae(Spn) causes pneumonia that kills millions through acute toxicity and invasion of the lung parenchyma. During aerobic respiration, Spn releases hydrogen peroxide (Spn-H2O2), as a by-product of enzymes SpxB and LctO, and causes cell death with signs of both apoptosis and pyroptosis by oxidizing unknown cell targets. Hemoproteins are molecules essential for life and prone to oxidation by H2O2. We recently demonstrated that during infection-mimicking conditions, Spn-H2O2oxidizes the hemoprotein hemoglobin (Hb), releasing toxic heme. In this study, we investigated details of the molecular mechanism(s) by which the oxidation of hemoproteins by Spn-H2O2causes human lung cell death. Spn strains, but not H2O2-deficient SpnΔspxBΔlctOstrains caused time-dependent cell cytotoxicity characterized by the rearrangement of the actin, the loss of the microtubule cytoskeleton and nuclear contraction. Disruption of the cell cytoskeleton correlated with the presence of invasive pneumococci and an increase of intracellular reactive oxygen species. In cell culture, the oxidation of Hb or cytochrome c (Cytc) caused DNA degradation and mitochondrial dysfunction from inhibition of complex I-driven respiration, which was cytotoxic to human alveolar cells. Oxidation of hemoproteins resulted in the creation of a radical, which was identified as a protein derived side chain tyrosyl radical by using electron paramagnetic resonance (EPR). Thus, we demonstrate that Spn invades lung cells, releasing H2O2that oxidizes hemoproteins, including Cytc, catalyzing the formation of a tyrosyl side chain radical on Hb and causing mitochondrial disruption, that ultimately leads to the collapse of the cell cytoskeleton.
Anoctamin-1 is induced by TGF-beta and contributes to lung myofibroblast differentiation
Authors: Reed Eleanor B. , Orbeta Shaina , Miao Bernadette A. , Sitikov Albert , Chen Bohao , Levitan Irena , Solway Julian , Mutlu Gökhan M. , Fang Yun , Mongin Alexander A. , Dulin Nickolai O. 
Published: June 2023

ABSTRACTIdiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by progressive scarring of the lungs and resulting in deterioration in lung function. Transforming growth factor-beta (TGF-β) is one of the most established drivers of fibrotic processes. TGF-β promotes transformation of tissue fibroblasts to myofibroblasts, a key finding in the pathogenesis of pulmonary fibrosis. We report here that TGF-β robustly upregulates the expression of the calcium-activated chloride channel Anoctamin-1 (ANO1) in human lung fibroblasts (HLF) at mRNA and protein levels. ANO1 is readily detected in fibrotic areas of IPF lungs in the same area with smooth muscle alpha-actin (SMA)-positive myofibroblasts. TGF-β-induced myofibroblast differentiation (determined by the expression of SMA, collagen-1 and fibronectin) is significantly inhibited by a specific ANO1 inhibitor, T16Ainh-A01, or by siRNA-mediated ANO1 knockdown. T16Ainh-A01 and ANO1 siRNA attenuate pro-fibrotic TGF-β signaling, including activation of RhoA pathway and AKT, without affecting initial Smad2 phosphorylation. Mechanistically, TGF-β treatment of HLF results in a significant increase in intracellular chloride levels, which is prevented by T16Ainh-A01 or by ANO1 knockdown. The downstream mechanism involves the chloride-sensing “with-no-lysine (K)” kinase (WNK1). WNK1 siRNA significantly attenuates TGF-β-induced myofibroblast differentiation and signaling (RhoA pathway and AKT), whereas the WNK1 kinase inhibitor WNK463 is largely ineffective. Together, these data demonstrate that (i) ANO1 is a TGF-β-inducible chloride channel that contributes to increased intracellular chloride concentration in response to TGF-β; and (ii) ANO1 mediates TGF-β-induced myofibroblast differentiation and fibrotic signaling in a manner dependent on WNK1 protein, but independent of WNK1 kinase activity.NEW & NOTEWORTHYThis study describes a novel mechanism of differentiation of human lung fibroblasts (HLF) to myofibroblasts – the key process in the pathogenesis of pulmonary fibrosis. TGF-β drives the expression of calcium-activated chloride channel anoctmin-1 (ANO1) leading to an increase in intracellular levels of chloride. The latter recruits chloride-sensitive With-No-Lysine (K) kinase (WNK1) to activate pro-fibrotic RhoA and AKT signaling pathways, possibly through activation of mammalian target of rapamycin complex-2 (mTORC2), altogether promoting myofibroblast differentiation.
Identification and functional analysis of novel stress-resistance genes from metagenomes of extreme environments
Authors: Juarez Joshelin Huanca , Nascimento Silva Edson do , Silva Ninna Hirata , Silva-Rocha Rafael , Guazzaroni María-Eugenia 
Published: June 2023

AbstractCurrently, industrial bioproducts are less competitive than chemically produced goods due to the shortcomings of conventional microbial hosts. Metagenomic approaches from extreme environments can provide useful biological parts to improve bacterial robustness to process-specific parameters. Here, in order to build synthetic genetic circuits that increase bacterial resistance to diverse stress conditions, we mined novel stress tolerance genes from metagenomic databases using anin silicoapproach based on Hidden-Markov-Model profiles. For this purpose, we used metagenomic shotgun sequencing data from microbial communities of extreme environments to identify genes encoding chaperones and other proteins that confer resistance to stress conditions. We identified and characterized ten novel protein-encoding sequences related to the DNA-binding protein HU, the ATP-dependent protease ClpP, and the chaperone protein DnaJ. By expressing these genes inEscherichia coliunder several stress conditions (including high temperature, acidity, oxidative and osmotic stress, and UV radiation), we identified five genes conferring resistance to at least two stress conditions when expressed inE. coli. Moreover, one of the identified HU coding-genes which was retrieved from an acidic soil metagenome increasedE. colitolerance to four different stress conditions, implying its suitability for the construction of a synthetic circuit directed to expand broad bacterial resistance.
Aspergillus fumigatuscytochrome c impacts conidial survival during sterilizing immunity
Authors: James Matthew R , Aufiero Mariano A , Vesely Elisa M , Dhingra Sourabh , Liu Ko-Wei , Hohl Tobias M , Cramer Robert A 
Published: June 2023

AbstractInvasive pulmonary aspergillosis (IPA) is a life-threatening infection caused by species in the ubiquitous fungal genusAspergillus. While leukocyte-generated reactive oxygen species (ROS) are critical for the clearance of fungal conidia from the lung and resistance to IPA, the processes that govern ROS-dependent fungal cell death remain poorly defined. Using a flow cytometric approach that monitors two independent cell death markers, an endogenous histone H2A:mRFP nuclear integrity reporter and Sytox Blue cell impermeable (live/dead) stain, we observed that loss ofA. fumigatuscytochrome c (cycA) results in reduced susceptibility to cell death from hydrogen peroxide (H2O2) treatment. Consistent with these observationsin vitro, loss ofcycAconfers resistance to both NADPH-oxidase -dependent and -independent killing by host leukocytes. Fungal ROS resistance is partly mediated in part by Bir1, a homolog to survivin in humans, as Bir1 overexpression results in decreased ROS-induced conidial cell death and reduced killing by innate immune cellsin vivo. We further report that overexpression of the Bir1 N-terminal BIR domain inA. fumigatusconidia results in altered expression of metabolic genes that functionally converge on mitochondrial function and cytochrome c (cycA) activity. Together, these studies demonstrate thatcycAinA. fumigatuscontributes to cell death responses that are induced by exogenous H2O2and by host leukocytes.ImportanceAspergillus fumigatuscan cause a life-threatening infection known as invasive pulmonary aspergillosis (IPA), which is marked by fungus-attributable mortality rates of 20%-30%. Individuals at risk of IPA harbor genetic mutations or incur pharmacologic defects that impair myeloid cell numbers and/or function, exemplified by bone marrow transplant recipients, patients that receive corticosteroid therapy, or patients with Chronic Granulomatous Disease (CGD). However, treatments forAspergillusinfections remains limited, and resistance to the few existing drug classes is emerging. Recently, the World Health Organization (WHO) classifiedA. fumigatusas a critical priority fungal pathogen. Our research identifies an important aspect of fungal biology that impacts susceptibility to leukocyte killing. Furthering our understanding of mechanisms that mediate the outcome of fungal-leukocyte interactions will increase our understanding of both the underlying fungal biology governing cell death and innate immune evasion strategies utilized during mammalian infection pathogenesis. Consequently, our studies are a critical step toward leveraging these mechanisms for novel therapeutic advances.
A dual-fluorescent recombinant for live observation of Herpes simplex-type 1 infection outcomes
Authors: Domanico Luke F. , Dunn G. P. , Kobiler O. , Taylor M.P. 
Published: June 2023

AbstractCritical stages of lytic Herpes simplex type 1 (HSV-1) replication are marked by the sequential expression of immediate early (IE) to early (E), then late (L) viral genes. HSV-1 also persists in neuronal tissues via a non-replicative, transcriptionally repressed infection called latency. Understanding the regulation of lytic and latent transcriptional profiles provides focused insight into HSV-1 infection and disease. We sought a fluorescence-based approach to observe temporal progression of HSV-1 infection at the single-cell level. We constructed and characterized a novel HSV-1 recombinant that reports IE and L gene expression by fluorescent protein detection. The dual-reporter HSV-1 visualizes IE gene expression by a CMV promotor-driven YFP, and L gene expression by an endogenous mCherry-VP26 fusion. We confirmed that viral gene expression, replication and spread of infection in epithelial cells is not altered by the incorporation of the fluorescent reporters. Interference with viral DNA polymerase activity abolishes VP26-mCherry detection late in HSV-1 infection, visually reporting the failure to complete viral replication. Viral replication in primary neurons is not altered, but retrograde-directed inoculation of the dual-reporter HSV-1 exhibits a modest reduction in titer, compared to unlabeled HSV-1. Low-dose axonal inoculation in the presence of small molecule modulation of neuronal signaling results in divergent outcomes of YFP and mCherry detection, suggesting different states of latent and lytic replication. Rigorous characterization of this dual-reporter HSV-1 recombinant has demonstrated the utility of temporal observation of HSV-1 replication in live cells and the potential for further insight into the dynamics of infection.ImportanceHerpes simplex virus-type 1 (HSV-1) is a prevalent human pathogen that infects approximately 67% of the global population. HSV-1 invades the peripheral nervous system, where latent HSV-1 infection persists within the host for life. Immunological evasion, viral persistence, and herpetic pathologies are determined by regulation of HSV-1 gene expression. Studying HSV-1 gene expression during neuronal infection is challenging but essential for the development of antiviral therapeutics and interventions. We constructed and characterized a dual-fluorescent HSV-1 recombinant that enables visualization of IE and L gene expression. The recombinant HSV-1 is used to observe the progression and outcome of infection. We demonstrate that drug treatments targeting cellular pathways can manipulate latent HSV-1 infection in neurons to achieve divergent outcomes of infection.
Chitin degradation bySynechococcusWH7803
Authors: Capovilla Giovanna , Castro Kurt G. , Collani Silvio , Kearney Sean M. , Kehoe David M. , Chisholm Sallie W. 
Published: June 2023

AbstractChitin is an abundant, carbon-rich polymer in the marine environment. Chitinase activity has been detected in spent media ofSynechococcusWH7803 cultures – yet it was unclear which specific enzymes were involved. Here we delivered a CRISPR tool into the cells via electroporation to generate loss-of-function mutants of putative candidates and identified ChiA as the enzyme required for the activity detected in the wild type.
Understanding the General Principles of T Cell Engagement by Multiscale Computational Simulations
Authors: Su Zhaoqian , Almo Steven C. , Wu Yinghao 
Published: June 2023

ABSTRACTThe use of bispecific antibodies as T cell engagers can bypass the normal TCR-MHC interaction, redirect the cytotoxic activity of T-cells, and lead to highly efficient tumor cell killing. However, this immunotherapy also causes significant on-target off-tumor toxicologic effects, especially when they were used to treat solid tumors. In order to avoid these adverse events, it is necessary to understand the fundamental mechanisms during the physical process of T cell engagement. We developed a multiscale computational framework to reach this goal. The framework combines simulations on the intercellular and multicellular levels. On the intercellular level, we simulated the spatial-temporal dynamics of three-body interactions among bispecific antibodies, CD3 and TAA. The derived number of intercellular bonds formed between CD3 and TAA were further transferred into the multicellular simulations as the input parameter of adhesive density between cells. Through the simulations under various molecular and cellular conditions, we were able to gain new insights of how to adopt the most appropriate strategy to maximize the drug efficacy and avoid the off-target effect. For instance, we discovered that the low antibody binding affinity resulted in the formation of large clusters at the cell-cell interface, which could be important to control the downstream signaling pathways. We also tested different molecular architectures of the bispecific antibody and suggested the existence of an optimal length in regulating the T cell engagement. Overall, the current multiscale simulations serve as a prove-of-concept study to help the future design of new biological therapeutics.SIGNIFICANCET-cell engagers are a class of anti-cancer drugs that can directly kill tumor cells by bringing T cells next to them. However, current treatments using T-cell engagers can cause serious side-effects. In order to reduce these effects, it is necessary to understand how T cells and tumor cells interact together through the connection of T-cell engagers. Unfortunately, this process is not well studied due to the limitations in current experimental techniques. We developed computational models on two different scales to simulate the physical process of T cell engagement. Our simulation results provide new insights into the general properties of T cell engagers. The new simulation methods can therefore serve as a useful tool to design novel antibodies for cancer immunotherapy.
Circular viral copy DNA of Dengue virus (DENV-2) isolated from infected mosquito cell cultures
Authors: Gangnonngiw Warachin , Thongda Wilawan , Kanthong Nipaporn , Flegel T.W. 
Published: June 2023

ABSTRACTDengue virus serotype 2 (DENV-2) is a mosquito-borne disease in the familyFlaviviridae. We have previously shown that DENV-2 can infect C6/36 mosquito cells and cause initial cytopathic effects that dissipate upon serial split-passage to yield persistently infected cultures with normal growth and morphology. In other words, the cell line accommodated persistent DENV-2 infections. It has recently been found that insect viral infections induce production of viral copy DNA (vcDNA) fragments via host reverse transcriptase (RT). The vcDNA occurs in both linear (lvcDNA) and circular (cvcDNA) forms and produces small interfering RNA (siRNA) transcripts that can result in an immediate protective RNA interference (RNAi) response. The vcDNA can also result in host acquisition of endogenous viral elements (EVE) in genomic DNA. Thus, we hypothesized that DENV-2 cvcDNA and DENV-2-EVE would arise in C6/36 insect cells challenged with DENV-2 virusin vitro. Here we describe the successful isolation and characterization of cvcDNA constructs homologous to DENV-2 from laboratory challenges with C6/36 cells. At least 1 of these appeared to arise from a DENV-2-EVE. We also show that a linear vcDNA preparation derived from the DENV-2-EVE sequence by PCR significantly reduced DENV-2 replication when applied to naive C6/36 cells prior to DENV-2 challenge. This is preliminary work designed to lay the groundwork for further studies on use of the C6/36 cell model for screening and characterization of protective EVE against both insect and shrimp viruses.
Presence of vitamin B12metabolism in the last common ancestor of land plants
Authors: Dorrell Richard G. , Nef Charlotte , Altan-Ochir Setsen , Bowler Chris , Smith Alison G. 
Published: June 2023

AbstractVitamin B12, or cobalamin, (hereinafter B12) is an essential organic micronutrient, required by humans as a cofactor for methionine synthase (METH) and for methylmalonyl CoA mutase (MCM), involved in the propionate shunt. B12is a complex corrinoid molecule made only by a subset of bacteria. Plants and fungi have an alternative methionine synthase (METE) that does not need a B12cofactor, so these organisms are typically considered to neither synthesise nor utilise B12. In contrast many algal species utilise B12if it is available, because they encode both METE and METH. Moreover, a large proportion of algal species encode METH only, and so are like animals in being dependent on an external source of the vitamin. Here, we performed a detailed phylogenetic analysis of the distribution of METE, METH and eleven further proteins implicated in B12metabolism in eukaryotic cells across an exhaustive library of over 1,500 plant and algal genomes and transcriptomes. The results reveal the hitherto undetected existence of B12-associated metabolism deep into the streptophytes. The B12-dependent synthase METH, and the accessory proteins MTRR, CblB, CblC, CblD and CblJ were detected in the basally divergent plant lineage of hornworts, and CblB and CblJ were further identified in liverworts. Using phylogenetic and PFAM analysis we demonstrate this is due to retention of ancestral B12-metabolism pathways in the last common ancestor of land plants, followed by at least two independent complete losses in mosses and vascular plants. We further show more limited distributions of genes encoding B12-related proteins across the algal tree of life, including MCM and type II ribonucleotide reductase, alongside an obligate B12-dependency across several major marine algal orders. Finally, by considering the functional biology of early-diverging land plants, together with the collection sites of ten further algal species inferred to have lost B12-dependent metabolism, we propose freshwater-to-land transitions and symbiotic associations to have been major constraining factors in B12availability in early plant evolution.
What levels of informality tackle poverty in Africa? Evidence from dynamic panel threshold analysis
Authors: Bolarinwa Segun Thompson , Simatele Munacinga 
Published: June 2023

PurposeThe paper validates the threshold argument in the informality–poverty nexus. Recent literature and policy have argued the existence of a threshold in the relationship.Design/methodology/approachThe study adopts dynamic panel threshold analysis, estimated within the framework of system Generalized Method of Moments (SGMM) to control for endogeneity and simultaneity. Data from 40 selected sub-Saharan African countries between 1991 and 2018 are used for the study.FindingsEmpirical results confirm the existence of an average threshold of 31% share of informality in GDP. Also, the paper finds that threshold of informality that addresses mild and severe poverty varies between 24.32 and 36.75%.Research limitations/implicationsThe work is limited to African economies. Evidence from other emerging and developed economies is suggested for further research.Practical implicationsOverall, the empirical results indicate a threshold in the informality–poverty nexus. Therefore, an excessive informality level does not benefit the African growth process. Policymakers and governments are advised to operate within the bounds of the threshold of informality that reduces poverty and improve the African economic growth process.Originality/valueThe paper is the first study to provide empirical findings on the nonlinear and threshold argument in the informality–poverty nexus, as far as the authors know.
Spillover effects of crash and jump events: evidence from Chinese market
Authors: Usman Muhammad , Akhter Waheed , Haque Abdul 
Published: June 2023

PurposeThis paper aims to investigate the spillover effects of jump and crash events among Chinese nonfinancial firms.Design/methodology/approachThis sample consists of more than 1.5 million weekly observations of over 3,000 Chinese listed firms over the period 1991–2015. The authors utilize univariate tests to compare the post-event performance of matched peer and non-peer control firms and cross-sectional regressions of their abnormal returns/cumulative abnormal returns (ARs/CARs) and returns on assets (ROAs).FindingsThe authors find that extreme risk-adjusted abnormal stock returns (stock price crashes and jumps) generate statistically significant ARs/CARs in the same directions in industry, size, leverage, and geographical location matched peer firms in Chinese stock market. Further tests reveal that peer firms' response to the crash event is pronounced more in the group of firms about which the information asymmetry is high between investors and firms.Research limitations/implicationsPortfolio investors can adjust their portfolios accordingly by selling stocks of the matching rival firms during a crash period. Policymakers may develop policies so as to protect the interests of small investors in the events of crashes in the markets. They can reduce the information asymmetry between the firms and the investors by making information about the firms more transparent, so as to reduce the contagion in case of crash event.Practical implicationsThis study has important implications for portfolio investment managers and policymakers.Originality/valueTo the best of authors' knowledge, this is the first study that combines the jump and crash events and attempts to assess their spillover effects on other firms in Chinese stock market.