bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cleavage of α-1,4-Glycosidic Linkages by the Glycosylphosphatidylinositol-Anchored α-Amylase AgtA Decreases the Molecular Weight of Cell Wall α-1,3-Glucan inAspergillus oryzae
Authors: Koizumi Ami , Miyazawa Ken , Ogata Makoto , Takahashi Yuzuru , Yano Shigekazu , Yoshimi Akira , Sano Motoaki , Hidaka Masafumi , Nihira Takanori , Nakai Hiroyuki , Kimura Satoshi , Iwata Tadahisa , Abe Keietsu 
Published: October 2022

AbstractAspergillusfungi contain α-1,3-glucan with a low proportion of α-1,4-glucan as a major cell wall polysaccharide. Glycosylphosphatidylinositol (GPI)-anchored α-amylases are conserved inAspergillusfungi. The GPI-anchored α-amylase AmyD inAspergillus nidulanshas been reported to directly suppress the biosynthesis of cell wall α-1,3-glucan but not to degrade itin vivo. However, the detailed mechanism of cell wall α-1,3-glucan biosynthesis regulation by AmyD remains unclear. Here we focused on AoAgtA, which is encoded by theAspergillus oryzae agtAgene, an ortholog of theA. nidulans amyDgene. Similar to findings inA. nidulans, agtAoverexpression inA. oryzaegrown in submerged culture decreased the amount of cell wall α-1,3-glucan and led to the formation of smaller hyphal pellets in comparison with the wild-type strain. We analyzed the enzymatic properties of recombinant (r)AoAgtA produced inPichia pastorisand found that it degraded soluble starch, but not linear bacterial α-1,3-glucan. Furthermore, rAoAgtA cleaved 3-α-maltotetraosylglucose with a structure similar to the predicted boundary structure between the α-1,3-glucan main chain and a short spacer composed of α-1,4-linked glucose residues in cell wall α-1,3-glucan. Interestingly, rAoAgtA randomly cleaved only the α-1,4-glycosidic bonds of 3-α-maltotetraosylglucose, indicating that AoAgtA may cleave the spacer in cell wall α-1,3-glucan. Consistent with this hypothesis, heterologous overexpression ofagtAinA. nidulansdecreased the molecular weight (MW) of cell wall α-1,3-glucan. Thesein vitroandin vivoproperties of AoAgtA suggest that GPI-anchored α-amylases can degrade the spacer α-1,4-glycosidic linkages in cell wall α-1,3-glucan before its insolubilization, and this spacer cleavage decreases the MW of cell wall α-1,3-glucanin vivo.
A pluripotent stem cell atlas of multilineage differentiation revealsTMEM88as a developmental regulator of mammalian blood pressure
Authors: Shen Sophie , Werner Tessa , Lukowski Samuel W. , Andersen Stacey , Sun Yuliangzi , Shim Woo Jun , Mizikovsky Dalia , Kobayashi Sakurako , Outhwaite Jennifer , Chiu Han Sheng , Chen Xiaoli , Chapman Gavin , Martin Ella M. M. A. , Xia Di , Pham Duy , Su Zezhuo , Kim Daniel , Yang Pengyi , Tan Men Chee , Sinniah Enakshi , Zhao Qiongyi , Negi Sumedha , Redd Meredith A. , Powell Joseph E. , Dunwoodie Sally L. , Tam Patrick P. L. , Bodén Mikael , Ho Joshua W. K. , Nguyen Quan , Palpant Nathan J. 
Published: October 2022

SUMMARYPluripotent stem cells provide a scalable approach to analyse molecular regulation of cell differentiation across multiple developmental lineage trajectories. In this study, we engineered barcoded iPSCs to generate an atlas of multilineage differentiation from pluripotency, encompassing a time-course of WNT-induced differentiation perturbed using modulators of WNT, BMP, and VEGF signalling. Computational mapping ofin vitrocell types toin vivodevelopmental lineages revealed a diversity of iPSC-derived cell types comprising mesendoderm lineage cell types including lateral plate and paraxial mesoderm, neural crest, and primitive gut. Coupling this atlas ofin vitrodifferentiation with Summary data-based Mendelian Randomisation analysis of human complex traits, we identify the WNT-inhibitor proteinTMEM88as a putative regulator of mesendodermal cell types governing development of diverse cardiovascular and anthropometric traits. Using genetic loss of function models, we show thatTMEM88is required for differentiation of diverse endoderm and mesoderm cell lineagesin vitroand thatTMEM88knockoutin vivoresults in a significant dysregulation of arterial blood pressure. This study provides an atlas of multilineage iPSC differentiation coupled with new molecular, computational, and statistical genetic tools to dissect genetic determinants of mammalian developmental physiology.
An RNA modification enzyme directly senses reactive oxygen species for translational regulation inEnterococcus faecalis
Authors: Lee Wei Lin , Sinha Ameya , Lam Ling Ning , Loo Hooi Linn , Liang Jiaqi , Ho Peiying , Cui Liang , Chan Cheryl Siew Choo , Begley Thomas , Kline Kimberly , Dedon Peter 
Published: October 2022

AbstractBacteria possess elaborate systems to manage reactive oxygen and nitrogen species (ROS) arising from exposure to the mammalian immune system and environmental stresses. Here we report the discovery of an ROS-sensing RNA-modifying enzyme that regulates translation of stress-response proteins in the gut commensal and opportunistic pathogenEnterococcus faecalis. We analyzed the tRNA epitranscriptome ofE. faecalisin response to reactive oxygen species (ROS) or sublethal doses of ROS-inducing antibiotics and identified large decreases in N2-methyladenosine (m2A) in both 23S ribosomal RNA and transfer RNA. This we determined to be due to ROS-mediated inactivation of the Fe-S cluster-containing methyltransferase, RlmN. Genetic knockout of RlmN gave rise to a proteome that mimicked the oxidative stress response, with increased levels of superoxide dismutase and decreased virulence proteins. While tRNA modifications are established to be dynamic for fine-tuning translation, here we report the first instance of a dynamically regulated, environmentally responsive rRNA modification. These studies lead to model in which RlmN serves as a redox-sensitive molecular switch, directly relaying oxidative stress to modulating translation through the rRNA and the tRNA epitranscriptome, revealing a new paradigm for understanding direct regulation of the proteome by RNA modifications.
IRE-1α is a key switch of pyroptosis and necroptosis in mice by dominating Gasdermin D
Authors: Zeng Xin , Zheng Qing , Li Deyong , Li Yumeng , Xu Qing , Liu Xiaohong , Zhu Min , Liu Wen , Li Lan , Lu Yanrong , Cheng Jingqiu , Chen Younan 
Published: October 2022

AbstractNecroptosis and pyroptosis are lytic and inflammatory types of programmed cell death that require the membrane destruction predominantly driven by the mixed lineage kinase domain-like (MLKL) and gasdermin D (GSDMD) proteins, respectively. However, the crosstalk between them remains largely unknown. Here, our research discloses that endoplasmic reticulumn transmembrane protein inositol-requiring enzyme-1α (IRE-1α) is a potential modulator of both necroptosis and pyroptosis, paricularly in liver pathology. Interestingly, enhanced expression of IRE-1α triggers hepatic pyroptosis, while defective IRE-1α level activates hepatic necroptosis, and both processes are closed related to the activity of GSDMD in mice. To elucidate unknown crosstalk, by using pharmacological and genetic methods, we first demonstrated that IRE-1α suppresses necroptosis by promoting the expression of GSDMD and cleaves caspase-8 and by inhibiting the expression of receptor-interacting serine/threonine-protein kinase 1 (RIPK1), RIPK3 and MLKL. Unexpectedly, excess IRE-1α initiates pyroptosis by increasing GSDMD and NLRP3 levels. Our work clearly provides insight into the modulation of IRE-1α to dominate necroptosis and pyroptosis and suggests that IRE-1α may be a promising therapeutic target for drug discovery in both types of tissue injuries.
Taxonomy of introns, their evolution, and the role of minor introns in stress response
Authors: Olthof Anouk M , Schwoerer Charles F , Weber Audrey L , Arokiadhas Iswarya , Doggett Karen , Mieruszynski Stephen , Cnaani Avner , Heath Joan K , Biran Jakob , Kanadia Rahul N 
Published: October 2022

AbstractDespite the high conservation of minor introns across eukaryotic supergroups, specific lineages have completely lost minor intron splicing, which has raised questions about their evolution and purpose. Addressing these questions requires identification of the introns that are affected by minor spliceosome inhibition. To this end, we applied principles of Linnaean taxonomy combined with position-weight matrices to produce five intron classes: minor, minor-like, hybrid, major-like and major. We classified introns across the genomes of 263 species of six eukaryotic supergroups, which can be viewed at the Minor Intron Database (MIDB). Transcriptomic analysis revealed that ∼40% of the minor introns are responsive to minor spliceosome inhibition, while an additional 5% of the minor-like and hybrid introns are also affected. We propose that minor-like introns represent an intermediate in the conversion of minor to major introns and uncover the importance of a guanine at the −1 position of the 5’ splice site in facilitating this shift in spliceosome dependence. Finally, we find that minor introns are aberrantly spliced in fish and plants upon cold stress, thereby providing a potential explanation for their high degree of conservation in these lineages.
Expression ofMicrocystisbiosynthetic gene clusters in natural populations suggests temporally dynamic synthesis of novel and known secondary metabolites in western Lake Erie
Authors: Yancey Colleen E. , Yu Fengan , Tripathi Ashootosh , Sherman David H. , Dick Gregory J. 
Published: October 2022

SummaryMicrocystisspp. produces diverse secondary metabolites within freshwater cyanoHABs around the world. In addition to the biosynthetic gene clusters (BGCs) encoding known compounds,Microcystisgenomes harbor numerous BGCs of unknown function, indicating its poorly understood chemical repertoire. While recent studies show thatMicrocystisproduces several metabolites in the lab and field, little work has focused on analyzing the abundance and expression of its broader suite of BGCs during cyanoHAB events. Here, we use metagenomic and metatranscriptomic approaches to track the relative abundance ofMicrocystisBGCs and their transcripts throughout the 2014 western Lake Erie cyanoHAB. Results indicate the presence of several transcriptionally active BGCs that are predicted to synthesize both known and novel secondary metabolites. The abundance and expression of these BGCs shifted throughout the bloom, with transcript abundance levels correlating with temperature, nitrate and phosphorus concentrations, and the abundance of co-occurring predatory and competitive eukaryotic microorganisms, suggesting the importance of both abiotic and biotic controls in regulating expression. This work highlights the need for understanding the chemical ecology and potential risks to human and environmental health posed by secondary metabolites that are produced but unmonitored, as well as the potential discovery of pharmaceutical compounds from cyanoHAB-derived BGCs.Originality-Statement of SignificanceMicrocystis spp. dominate cyanobacterial harmful algal blooms (cyanoHABs) worldwide and pose significant threats to water quality through the production of numerous secondary metabolites, many of which are toxic. While the toxicity and biochemistry of microcystins and several other compounds have been well studied, the broader suite of secondary metabolites produced byMicrocystisremains poorly understood, leaving gaps in our understanding of their impacts on ecology, human and ecosystem health, or potential pharmaceutical application. In this study, we use metagenomic and transcriptomic datasets to examine the diversity of genes encoding synthesis of secondary metabolites in naturalMicrocystispopulations and assess their patterns of transcription in the context of biotic and abiotic conditions in western Lake Erie cyanoHABs. Our results reveal the presence of a large diversity of both known gene clusters that encode toxic secondary metabolites as well as novel ones that encode cryptic compounds. This research highlights the need for targeted studies of the secondary metabolite diversity in western Lake Erie, a vital freshwater source to the United States and Canada.
Reduction in RBD Binding Affinity to Glycosylated ACE2 is Entropic in Origin
Authors: Mugnai Mauro L. , Shin Sucheol , Thirumalai D. 
Published: October 2022

AbstractThe spike protein in the virus SARS-CoV-2 (the causative agent of COVID-19) recognizes the host cell by binding to the peptidase domain (PD) of the extracellular enzyme Angiotensin-converting Enzyme 2 (ACE2). A variety of carbohydrates could be attached to the six asparagines in the PD, resulting in a heterogeneous population of ACE2 glycoforms. Experiments have shown that the binding affinity of glycosylated and deglycosylated ACE2 to the virus is virtually identical. In most cases, the reduction in glycan size correlates with stronger binding, which suggests that volume exclusion, and hence entropic forces, determine the binding affinity. Here, we quantitatively test the entropy-based hypothesis by developing a lattice model for the complex between ACE2 and the SARS-CoV-2 spike protein Receptor-binding Domain (RBD). Glycans are treated as branched polymers with only volume exclusion, which we justify using all atom molecular dynamics simulations in explicit water. We show that the experimentally measured changes in the ACE2-RBD dissociation constants for a variety of engineered ACE2 glycoforms are well accounted for by our theory, thus affirming that ACE2 glycans have only a weak, entropic effect on RBD binding.
In vitroandin vivoevaluation of the biofilm-degradingPseudomonasphage Motto, as a candidate for phage therapy
Authors: Manohar Prasanth , Loh Belinda , Turner Dann , Tamizhselvi Ramasamy , Mathankumar Marimuthu , Elangovan Namasivayam , Nachimuthu Ramesh , Leptihn Sebastian 
Published: October 2022

AbstractInfections caused byPseudomonas aeruginosaare becoming increasingly difficult to treat due to the emergence of strains that have acquired multidrug resistance. Therefore, phage therapy has gained attention as an alternative to the treatment of pseudomonal infections. Phages are not only bactericidal but occasionally show activity against biofilm as well. Here, we describe thePseudomonasphage Motto which has the ability to clearP. aeruginosainfections in an animal model and also exhibited biofilm-degrading properties. The phage has substantial antibiofilm activity against strong biofilm-producing isolates (n = 10), with at least a 2-fold reduction within 24 hours. To demonstrate the safety of using phage Motto, we performed cytotoxicity studies with human cell lines (HEK 293 and RAW 264.7 macrophages). Using a previously establishedin vivomodel, we demonstrated the efficacy of Motto inC. elegans, with a 90% survival rate when treated with the phage at an MOI of 10.ImportancePhages are often evaluated mainly on their ability to kill bacterial hosts. One important aspect, however, is often neglected: Their ability to degrade biofilms. Not all phages are able to disintegrate biofilms. While phages can kill planktonic cells, it also has often been observed that phages are not able to infect those that are embedded in biofilms. Phage Motto appears to have highly efficient enzymes that degrade biofilms, and might therefore be a highly valuable therapeutic candidate.
Molecular Profiling of the Hippocampus of Children with Autism Spectrum Disorder
Authors: Rexrode Lindsay , Hartley Joshua , Showmaker Kurt C , Challagundla Lavanya , Vandewege Michael W. , Martin Brigitte E. , Blair Estelle , Bollavarapu Ratna , Antonyraj Rhenius B. , Hilton Keauna , Gardiner Alex , Valeri Jake , Gisabella Barbara , Garrett Michael , Theoharides Theoharis C. , Pantazopoulos Harry 
Published: October 2022

AbstractSeveral lines of evidence point to a key role of the hippocampus in Autism Spectrum Disorders (ASD). Altered hippocampal volume and deficits in memory for person and emotion related stimuli have been reported, along with enhanced ability for declarative memories. Mouse models have demonstrated a critical role of the hippocampus in social memory dysfunction, associated with ASD, together with decreased synaptic plasticity. Chondroitin sulphate proteoglycans (CSPGs), a family of extracellular matrix molecules, represent a potential key link between neurodevelopment, synaptic plasticity, and immune system signaling. There is a lack of information regarding the molecular pathology of the hippocampus in ASD. We conducted RNAseq profiling on postmortem human brain samples containing the hippocampus from male children with ASD (n=7) and normal male children (3-14 yrs old), (n=6) from the NIH NeuroBioBank. Gene expression profiling analysis implicated molecular pathways involved in extracellular matrix organization, neurodevelopment, synaptic regulation, and immune system signaling. qRT-PCR and Western blotting were used to confirm several of the top markers identified. The CSPG protein BCAN was examined with multiplex immunofluorescence to analyze cell-type specific expression of BCAN and astrocyte morphology. We observed decreased expression of synaptic proteins PSD95 (p<0.02) and SYN1 (p<0.02), increased expression of the extracellular matrix (ECM) protease MMP9 (p<0.03), and decreased expression of MEF2C (p<0.03). We also observed increased BCAN expression with astrocytes in children with ASD, together with altered astrocyte morphology. Our results point to alterations in immune system signaling, glia cell differentiation, and synaptic signaling in the hippocampus of children with ASD, together with alterations in extracellular matrix molecules. Furthermore, our results demonstrate altered expression of genes implicated in genetic studies of ASD including SYN1 and MEF2C.
Therapeutic modulation of the blood-brain barrier and ischemic stroke by a bioengineered FZD4-selective WNT surrogate
Authors: Ding Jie , Lee Sung-Jin , Vlahos Lukas , Yuki Kanako , Rada Cara C. , van Unen Vincent , Vuppalapaty Meghah , Chen Hui , Sura Asmiti , McCormick Aaron K. , Tomaske Madeline , Alwahabi Samira , Nguyen Huy , Nowatzke William , Kim Lily , Kelly Lisa , Vollrath Douglas , Califano Andrea J. , Yeh Wen-Chen , Li Yang , Kuo Calvin J. 
Published: October 2022

AbstractDerangements of the blood-brain barrier (BBB) or blood-retinal barrier (BRB) occur in disorders ranging from stroke, cancer, diabetic retinopathy, and Alzheimer’s disease. The Norrin/FZD4/TSPAN12 pathway activates WNT/β-catenin signaling, which is essential for BBB and BRB function. However, systemic pharmacologic FZD4stimulation is hindered by obligate palmitoylation and insolubility of native WNTs and suboptimal properties of the FZD4-selective ligand Norrin. Here, we developed L6-F4-2, a non-lipidated, FZD4-specific surrogate with significantly improved sub-picomolar affinity versus native Norrin. In Norrin knockout (NdpKO) mice, L6-F4-2 not only potently reversed neonatal retinal angiogenesis deficits, but also restored BRB and BBB function. In adult C57Bl/6J mice, post-stroke systemic delivery of L6-F4-2 strongly reduced BBB permeability, infarction, and edema, while improving neurologic score and capillary pericyte coverage. Our findings reveal systemic efficacy of a bioengineered FZD4-selective WNT surrogate during ischemic BBB dysfunction, with general applicability to adult CNS disorders characterized by an aberrant blood-brain barrier.
Host association and intracellularity evolved multiple times independently in theRickettsiales
Authors: Castelli Michele , Nardi Tiago , Gammuto Leandro , Bellinzona Greta , Sabaneyeva Elena , Potekhin Alexey , Serra Valentina , Petroni Giulio , Sassera Davide 
Published: October 2022

AbstractThe order Rickettsiales (Alphaproteobacteria) encompasses multiple diverse lineages of host-associated bacteria, including pathogens, reproductive manipulators, and mutualists. In order to understand how intracellularity and host association originated in this order, and whether they are ancestral or convergently evolved characteristics, we built an unprecedentedly large and phylogenetically-balanced dataset that includesde novosequenced genomes and an accurate selection of published genomic and metagenomic assemblies. We performed detailed functional reconstructions that clearly indicated “late” and parallel evolution of obligate host-association and intracellularity in differentRickettsialeslineages. According to the depicted scenario, multiple independent series of horizontal acquisitions of transporters led to the progressive loss of biosynthesis of nucleotides, amino acids and other metabolites, producing distinct conditions of host-dependence. Coherently, each clade experienced a different pattern of evolution of the ancestral arsenal of interaction apparatuses, including development of specialised effectors involved in the lineage-specific mechanisms of host cell adhesion/invasion and intracellularity.
Kinase Inhibitor Pulldown Assay (KiP) for Clinical Proteomics
Authors: Saltzman Alexander B , Chan Doug W , Holt Matthew V , Wang Junkai , Jaehnig Eric J , Anurag Meenakshi , Singh Purba , Malovannaya Anna , Kim Beom-Jun , Ellis Matthew J 
Published: October 2022

ABSTRACTProtein kinases are frequently dysregulated and/or mutated in cancer and represent essential targets for therapy. Accurate quantification is essential, but current approaches are inadequate. For breast cancer treatment for example, the identification and quantification of the protein kinase ERBB2 is critical for therapeutic decisions. While immunohistochemistry (IHC) is the current clinical diagnostic approach, it is only semiquantitative. Mass spectrometry-based proteomics offers more quantitative assays that, unlike IHC, can be used to accurately evaluate hundreds of kinases simultaneously. The enrichment of less abundant kinase targets for quantification, along with depletion of interfering proteins, improves sensitivity and thus promotes more effective downstream analyses. Multiple kinase inhibitors were therefore deployed as a capture matrix for kinase inhibitor pulldown (KiP) assays designed to profile the human protein kinome as broadly as possible. Optimized assays were initially evaluated in 16 patient derived xenograft models (PDX) where KiP identified multiple differentially expressed and biologically relevant kinases. From these analyses, an optimized single-shot parallel reaction monitoring (PRM) method was developed to improve quantitative fidelity. The PRM KiP approach was then reapplied to low quantities of proteins typical of protein yields from core needle biopsies of human cancers. The initial prototype targeting 100 kinases recapitulated intrinsic subtyping of PDX models obtained from comprehensive proteomic and transcriptomic profiling. Luminal and HER2 enriched OCT-frozen patient biopsies subsequently analyzed through KiP-PRM also clustered by subtype. Finally, stable isotope labeled peptide standards were developed to define a prototype clinical method.