bluuebunny/crossref_metadata_2025_split huggingface dataset:
Rosetta’s Predictive Ability for Low-Affinity Ligand Binding in Fragment-Based Drug Discovery
Authors: Okwei Elleansar , Smith Shannon T. , Bender Brian J. , Allison Brittany , Ganguly Soumya , Geanes Alexander , Zhang Xuan , Ledwitch Kaitlyn , Meiler Jens 
Published: October 2022

AbstractFragment-based drug discovery begins with the identification of small molecules with a molecular weight of usually less than 250 Da that weakly bind to the protein of interest. This technique is challenging for computational docking methods as binding is determined by only a few specific interactions. Inaccuracies in the energy function or slight deviations in the docking pose can lead to the prediction of incorrect binding or difficulties in ranking fragments inin silicoscreening. Here we test RosettaLigand by docking a series of fragments to a cysteine-depleted variant of the TIM-barrel protein, HisF. We compare the computational results with experimental NMR spectroscopy screens. NMR spectroscopy gives details on binding affinities of individual ligands, which allows assessment of the ligand-ranking ability by RosettaLigand, and also provides feedback on the location of the binding pocket, which serves as a reliable test of RosettaLigand’s ability to identify plausible binding poses. From a library screen of 3456 fragments, we identified a set of 31 ligands with intrinsic affinities to HisF with dissociation constants as low as 400 µM. The same library of fragments was blindly screenedin silico. RosettaLigand was able to rank binders before non-binders with an area under the curve (AUC) of the receiver operating characteristics (ROC) of 0.74. The docking poses observed for binders agreed with the binding pocket identified by NMR chemical shift perturbations for all fragments. Taken together, these results provide a baseline performance of RosettaLigand in a fragment-based drug discovery setting.
A microtubule associated protein is essential for malaria parasite transmission
Authors: Wichers-Misterek Jan Stephan , Binder Annika M. , Mesén-Ramírez Paolo , Dorner Lilian Patrick , Safavi Soraya , Fuchs Gwendolin , Lenz Tobias L. , Bachmann Anna , Wilson Danny , Frischknecht Friedrich , Gilberger Tim-Wolf 
Published: October 2022

ABSTRACTMature gametocytes ofPlasmodium(P.)falciparumdisplay a banana (falciform) shape conferred by a complex array of subpellicular microtubules (SPMT) associated to the inner membrane complex (IMC). Microtubule associated proteins (MAPs) define MT populations and modulate interaction to pellicular components. Several MAPs have been identified inToxoplasma gondiiand homologues can be found in the genome ofPlasmodiumspecies, but the function of these proteins for asexual and sexual development of malaria parasites is still unknown. Here we identified a novel subpellicular MAP, termed SPM3, that is conserved within the genusPlasmodium., especially within theLaveraniasubgenus, but absent in other Apicomplexa. Conditional knockdown and targeted gene disruption ofPfspm3inP. falciparumcause severe morphological defects during gametocytogenesis leading to round, non-falciform gametocytes with an aberrant SPMT pattern. In contrast,Pbspm3knockout inP. berghei, a species with round gametocytes, caused no defect in gametocytogenesis, but sporozoites displayed an aberrant motility and a dramatic defect in sporozoite invasion of salivary glands leading to a decreased efficiency in transmission. Electron microscopy revealed a dissociation of the SPMT from the IMC inPbspm3knockout parasites suggesting a function of SPM3 in anchoring MTs to the IMC. Overall, our results highlight SPM3 as a pellicular component with essential functions for malaria parasite transmission.IMPORTANCEA key structural feature driving the transition between different life cycle stages of the malaria parasite is the unique three membrane “pellicle”, consisting of the parasite plasma membrane (PPM) and a double membrane structure underlying the PPM termed the “inner membrane complex” (IMC). Additionally, there are numerous linearly arranged intramembranous particles (IMPs) linked to the IMC, which likely link the IMC to the subpellicular microtubule cytoskeleton. Here we identify, localize and characterize a novel subpellicular microtubule associated protein unique to the genusPlasmodium(P.). The knockout of this protein in the human infectingP. falciparumspecies result in malformed gametocytes and aberrant microtubules. We confirmed the microtubule association in theP. bergheirodent malaria homologue and show that its knockout results in a perturbated microtubule architecture, aberrant sporozoite motility and decreased transmission efficiency.
Noncanonical CDK4 signaling rescues diabetes in a mouse model by promoting beta cell differentiation
Authors: Stamateris Rachel E. , Sharma Rohit B. , Landa-Galván Huguet , Darko Christine , Redmond David , Rane Sushil G. , Alonso Laura C. 
Published: October 2022

ABSTRACTExpanding beta cell mass is a critical goal in the fight against diabetes. CDK4, an extensively characterized cell cycle activator, is required to establish and maintain beta cell number. Beta cell failure in the IRS2-deletion mouse type 2 diabetes model is in part due to loss of CDK4 regulator Cyclin D2. We set out to determine whether replacement of endogenous CDK4 with the inhibitor-resistant mutant CDK4-R24C rescued the loss of beta cell mass in Irs2-deficient mice. Surprisingly, not only beta cell mass but also beta cell dedifferentiation status was effectively rescued, despite no improvement in insulin sensitivity.Ex vivostudies in primary islet cells revealed a novel mechanism in which CDK4 intervened downstream in the insulin signaling pathway to prevent FOXO1-mediated transcriptional repression of critical beta cell transcription factorPdx1. FOXO1 inhibition was not related to E2F1 activity, to FOXO1 phosphorylation, or even to FOXO1 subcellular localization, but rather was related to deacetylation of FOXO1 and reduced FOXO1 abundance. Taken together, these results demonstrate a novel differentiation-promoting activity of the classical cell cycle activator CDK4 and support the concept that beta cell mass can be expanded without compromising function.
ResFinderFG v2.0: a database of antibiotic resistance genes obtained by functional metagenomics
Authors: Gschwind Rémi , Perovic Svetlana Ugarcina , Weiss Maja , Petitjean Marie , Lao Julie , Coelho Luis Pedro , Ruppé Etienne 
Published: October 2022

ABSTRACTMetagenomics can be used to monitor the spread of antibiotic resistance genes (ARGs). ARGs found in databases such as ResFinder and CARD primarily originate from culturable and pathogenic bacteria, while ARGs from non-culturable and non-pathogenic bacteria remain understudied. Functional metagenomics is based on phenotypic gene selection and can identify ARGs from non-culturable bacteria with a potentially low identity shared with known ARGs. In 2016, the ResFinderFG v1.0 database was created to collect ARGs from functional metagenomics studies. Here, we present the second version of the database, ResFinderFG v2.0, which is available on the Center of Genomic Epidemiology web server (https://cge.food.dtu.dk/services/ResFinderFG/). It comprises 3,913 ARGs identified by functional metagenomics from 50 carefully curated datasets. We assessed its potential to detect ARGs in comparison to other popular databases in gut, soil and water (marine + freshwater) Global Microbial Gene Catalogues (https://gmgc.embl.de). ResFinderFG v2.0 allowed for the detection of ARGs that were not detected using other databases. These included ARGs conferring resistance to beta-lactams, cycline, phenicol, glycopeptide/cycloserine and trimethoprim/sulfonamide. Thus, ResFinderFG v2.0 can be used to identify ARGs differing from those found in conventional databases and therefore improve the description of resistomes.GRAPHICAL ABSTRACTCaption: Additional use of ResFinderFG v2.0 database (composed of antibiotic resistance genes obtained with functional metagenomics) on the Center of Genomic Epidemiology webserver (https://cge.food.dtu.dk/services/ResFinderFG/), allows for more exhaustive resistome descriptions.
RyR2 regulates store-operated Ca2+entry, phospholipase C activity, and electrical excitability in the insulinoma cell line INS-1
Authors: Harvey Kyle E. , Tang Shiqi , LaVigne Emily K. , Pratt Evan P.S. , Hockerman Gregory H. 
Published: October 2022

AbstractThe ER Ca2+channel ryanodine receptor 2 (RyR2) is required for maintenance of insulin content and glucose-stimulated insulin secretion, in part, via regulation of the protein IRBIT in the insulinoma cell line INS-1. Here, we examined store-operated and depolarization-dependent Ca2+entry using INS-1 cells in which either RyR2 or IRBIT were deleted. Store-operated Ca2+entry (SOCE) stimulated with thapsigargin was reduced in RyR2KOcells compared to controls, but was unchanged in IRBITKOcells. STIM1 protein levels were not different between the three cell lines. Basal and stimulated (500 μM carbachol) phospholipase C (PLC) activity was also reduced specifically in RyR2KOcells. Insulin secretion stimulated by tolbutamide was reduced in RyR2KOand IRBITKOcells compared to controls, but was potentiated by an EPAC-selective cAMP analog in all three cell lines. Cellular PIP2levels were increased and cortical f-actin levels were reduced in RyR2KOcells compared to controls. Whole-cell Cavchannel current density was increased by 65% in RyR2KOcells compared to controls, and barium current was reduced by acute activation of the lipid phosphatase pseudojanin preferentially in RyR2KOcells over control INS-1 cells. Action potentials stimulated by 18 mM glucose were more frequent in RyR2KOcells compared to controls, and insensitive to the SK channel inhibitor apamin. Taken together, these results suggest that RyR2 plays a critical role in regulating PLC activity and PIP2levels via regulation of SOCE. RyR2 also regulates β-cell electrical activity by controlling Cavcurrent density, via regulation of PIP2levels, and SK channel activation.
Cardiolipin synthesis inPseudomonas fluorescensUM270 plays a relevant role in stimulating plant growth under salt stress
Authors: Rojas-Solis Daniel , Vences-Guzman Miguel A. , Sohlenkamp Christian , Santoyo Gustavo 
Published: October 2022

AbstractMembrane cardiolipin (CL) phospholipids play a fundamental role in the adaptation of bacteria to various environmental conditions, including saline stress. Here, we constructed deletion mutants of two CL synthetase genes,clsAandclsB, in the rhizobacteriumPseudomonas fluorescensUM270, and evaluated their role in plant growth promotion under salt stress.P. fluorescensUM270 ΔclsAand ΔclsBmutants showed a significant reduction in CL synthesis compared to the UM270 wild-type strain (58% ΔclsAand 53% ΔclsB), and their growth rate was not affected, except when grown at 100 and 200 mM NaCl. Additionally, the root colonization capacity of both mutant strains was impaired compared with that of the wild type. Concomitant with the deletion ofclsAandclsB, some physiological changes were observed in the UM270 ΔclsAand ΔclsBmutants, such as a reduction in indole acetic acid and biofilm production. By contrast, an increase in siderophore biosynthesis was observed. Further, inoculation of the UM270 wild-type strain in tomato plants (Lycopersicon esculentumSaladette) grown under salt stress conditions (100 and 200 mM NaCl) resulted in an increase in root and shoot length, chlorophyll content, and dry weight. On the contrary, when each of the mutants(ΔclsAand ΔclsB) were inoculated in tomato plants, a reduction in root length was observed when grown at 200 mM NaCl, but the shoot length, chlorophyll content, and total plant dry weight parameters were significantly reduced under normal or saline conditions (100 and 200 mM NaCl), compared to UM270 wild-type-inoculated plants. In conclusion, these results suggest that CL synthesis inP. fluorescensUM270 plays an important role in the promotion of tomato plant growth under normal conditions, but to a greater extent, under salt-stress conditions.
Timing and magnitude of the type-I interferon response are determinants of disease tolerance in arbovirus infection
Authors: Hardy Alexandra , Bakshi Siddharth , Furnon Wilhelm , MacLean Oscar , Gu Quan , Varjak Margus , Varela Mariana , Aziz Muhamad A , Shaw Andrew , Pinto Rute Maria , Ruiz Natalia Cameron , Mullan Catrina , Taggart Aislynn , Filipe Ana , Randall Richard E. , Wilson Sam , Stewart Meredith E. , Palmarini Massimo 
Published: October 2022

ABSTRACTInfected hosts possess two alternative strategies to protect themselves against the negative impact of virus infections: (i) “resistance”, directed to abrogate virus replication, or (ii) “disease tolerance”, aimed to avoid organ and tissue damage without overly controlling viral burden. The overall principles governing pathogen resistance are well understood, while less is known about those involved in disease tolerance. Here, we studied bluetongue virus (BTV), the cause of a major disease of ruminants, bluetongue, as a model system to investigate the mechanisms of disease tolerance. BTV induces clinical disease mainly in sheep, while cattle are considered reservoirs of infection, rarely exhibiting clinical symptoms despite sustained viremia. Here, we show that BTV consistently reaches higher titres in ovine primary cells, compared to cells derived from cattle. The variable replication kinetics of BTV in sheep and cattle cells were mostly abolished by abrogating the cell type-I interferon (IFN) response. By screening a library of bovine interferon stimulated genes (ISGs), we identified restriction factors blocking BTV replication, however both sheep and cattle orthologues of these antiviral genes possess anti-BTV properties. Importantly, we demonstrate that BTV induces a faster host cell protein synthesis shutoff in primary sheep cells, compared to cattle cells, which results in an earlier downregulation of antiviral proteins. Moreover, by RNAseq we also show a more pronounced expression of ISGs in BTV infected cattle cells compared to sheep cells. Our data provide a new perspective on how the type-I IFN response in reservoir species can have overall positive effects on both virus and host evolution.
L-Aspartate oxidase provides new insights into fumarate reduction in anaerobic darkness inSynechocystissp. PCC6803
Authors: Kukil Kateryna , Hawkes Jeffrey A. , Blikstad Cecilia , Lindberg Pia 
Published: October 2022

ABSTRACTCyanobacteria are promising microbial hosts for production of various industrially relevant compounds, such as succinate, a central metabolite of the tricarboxylic acid cycle (TCA). Cyanobacteria have been engineered to produce succinate during photoautotrophic growth, and are also able to secrete it during anoxic fermentation conditions. It has been assumed that under anoxic darkness, succinate can be formed by reduction of fumarate catalyzed by the succinate dehydrogenase complex (SDH), however, no characterization of SDH regarding this activity has been performed. In this study, we address this issue by generating strains of the unicellular cyanobacteriumSynechocystisPCC 6803 (Synechocystis) deficient in one or several subunits of SDH, and investigating succinate accumulation in these strains during dark anaerobic fermentation. The results showed higher succinate accumulation in SDH deletion strains than in the wild type, indicating a succinate dehydrogenase activity of SDH rather than fumarate reduction under these conditions. We further explored the possibility of another potential route for succinate formation from fumarate via L-aspartate oxidase (Laspo). The gene encoding Laspo inSynechocystiscould not be inactivated, indicating an essential function for this enzyme. Using purifiedSynLaspo, we could demonstratein vitrothat in addition to L-aspartate oxidation the enzyme exhibits an L-aspartate-fumarate oxidoreductase activity. We therefore suggest that reduction of fumarate to succinate during anoxic darkness can be a byproduct of the Laspo reaction, which is the first step in biosynthesis of NAD cofactors. This work contributes to the understanding of cyanobacterial TCA cycle for future engineering and sustainable production of dicarboxylic acids.
ATP modulates self-perpetuating conformational conversion generating structurally distinct yeast prion amyloids that limit autocatalytic amplification
Authors: Mahapatra Sayanta , Sarbahi Anusha , Punia Neha , Joshi Ashish , Avni Anamika , Walimbe Anuja , Mukhopadhyay Samrat 
Published: October 2022

AbstractPrion-like self-perpetuating conformational conversion of proteins into amyloid aggregates is associated with both transmissible neurodegenerative diseases and non-Mendelian inheritance. Here, we demonstrate that ATP modulates the formation and dissolution of amyloids from a yeast prion domain (NM domain ofSaccharomyces cerevisiaeSup35) and restricts autocatalytic amplification by controlling the amount of fragmentable and seeding-competent aggregates. ATP, at (high) physiological concentrations in the presence of Mg2+, kinetically accelerates NM aggregation. Interestingly, ATP also promotes phase-separation-mediated aggregation of a human protein harboring a yeast prion-like domain. We also show that ATP dose independently disaggregates preformed NM fibrils. Furthermore, high concentrations of ATP delimited the number of seeds by generating compact, ATP-bound NM fibrils that exhibited nominal fragmentation by either free ATP or Hsp104 disaggregase. Additionally, (low) pathological ATP concentrations restricted autocatalytic amplification by forming structurally distinct seeding-inefficient amyloids. Our results provide mechanistic underpinnings of concentration-dependent chemical chaperoning by ATP against prion-like transmissions.
PP2A-B55α controls keratinocyte adhesion through dephosphorylation of the Desmoplakin C-terminus
Authors: Perl Abbey L , Koetsier Jennifer L , Green Kathleen J 
Published: October 2022

ABSTRACTCritical for the maintenance of epidermal integrity and function are attachments between intermediate filaments (IF) and intercellular junctions called desmosomes. The desmosomal cytoplasmic plaque protein desmoplakin (DP) is essential for anchoring IF to the junction. DP-IF interactions are regulated by a phospho-regulatory motif within the DP C-terminus controlling keratinocyte intercellular adhesion. Here we identify the protein phosphatase 2A (PP2A)-B55α holoenzyme as the major serine/threonine phosphatase regulating DP’s C-terminus and consequent intercellular adhesion. Using a combination of chemical and genetic approaches, we show that the PP2A-B55α holoenzyme interacts with DP at intercellular membranes in 2D- and 3D- epidermal models and human skin samples. Our experiments demonstrate that PP2A-B55α regulates the phosphorylation status of junctional DP and is required for maintaining strong desmosome-mediated intercellular adhesion. These data identify PP2A-B55α as part of a regulatory module capable of tuning intercellular adhesion strength and a candidate disease target in desmosome-related disorders of the skin and heart.
Patterning of phase-separated condensates by Dnd1 controls cell fate
Authors: Westerich Kim Joana , Tarbashevich Katsiaryna , Gupta Antra , Zhu Mingzhao , Hull Kenneth , Romo Daniel , Gross-Thebing Theresa , Raz Erez 
Published: October 2022

SUMMARYGerm granules, condensates of phase-separated RNA and protein, are essential for germline development, but how these molecules are organized within the granules and whether such an organization is relevant for germ cell fate is unclear. Combining three-dimensionalin vivostructural and functional analyses, we study the dynamic spatial organization of molecules within zebrafish germ granules. We find that the vertebrate-specific Dead end protein is essential for positioningnanos3RNA at the condensates’ periphery, where ribosomes are located. Without Dead end, or when translation is inhibited,nanos3RNA translocates into granule interiors, far from the ribosomes’ location. These findings reveal the molecular mechanisms controlling the spatial organization of RNA within the phase-separated organelle and the importance of subgranule RNA localization for preserving germ cell totipotency.
Extracellular matrix stiffness regulates fibroblast differentiation by influencing DNA methyltransferase 1 expression through microtubule polymerization
Authors: Zhao Zhihan , Huang Guotao , He Yong , Zuo Xiaohu , Han Wuyue , Li Hong 
Published: October 2022

AbstractCells sense physical cues, such as changes in extracellular matrix (ECM) stiffness, and translate these stimuli into biochemical signals that control various aspects of cellular behavior, thereby facilitating physiological and pathological processes in various organs. Evidence from multiple studies suggests that the anterior vaginal wall stiffness is higher in POP patients than in non-POP patients. Our experiments found that the expression of α-smooth muscle actin (α-SMA) in the anterior vaginal wall of patients with POP was increased, and the expression of DNMT1 was decreased. We used polyacrylamide gel to simulate matrix stiffening in vitro, and substrate stiffening induced the high expression of myofibroblast markers α-SMA and CTGF in L929 cells. Inhibition of DNMT1 promotes fibroblast differentiation into myofibroblasts in vitro. The results of bioinformatics analysis showed that the expression of DNMT1 was significantly correlated with microtubule polymerization-related proteins. The experiment showed that the microtubule polymerization inhibitor nocodazole could eliminate the decrease of DNMT1 expression in fibroblasts induced by high stiffness. We conclude that fibroblasts sense an increase in the stiffness of the surrounding matrix and regulate fibroblast differentiation by regulating the expression of DNA methyltransferase 1 (DNMT1) through the regulation of microtubule polymerization. This study may help to elucidate the complex crosstalk between vaginal fibroblasts and their surrounding matrix in both healthy and pathological conditions, and provide new insights into the implications of potentially targeted phenotypic regulation mechanisms in material-related therapeutic applications.
Subcritical fluid extraction of antioxidant compounds from soybean seed coats (Glycine max (L.) Merr.)
Authors: Bergesse Antonella Estefania , Velez Alexis Rafael , Ryan Liliana Cecilia , Nepote Valeria 
Published: October 2022

PurposeThe aim of this work was to evaluate the efficiency of subcritical conditions using different water–ethanol mixtures to recover antioxidant compounds from soybean seed coats (SSCs).Design/methodology/approachSSCs were subjected to high temperature and pressure conditions, using ethanol–water mixtures as extractive solvent, to obtain phenolic and flavonoid compounds with antioxidant activity. A mathematical model, namely one-site desorption kinetic model, was used to describe the extraction kinetics.FindingsTemperature, solvent mass flow rate and solvent composition were studied, and the best extraction conditions were defined by a screening design. The maximum concentration of phenolics was obtained at 220 °C, 50% of ethanol and 2.5 g/min of solvent mass flow rate and a high antioxidant capacity toward different techniques was achieved. The one-site desorption kinetic model showed that before 30 min under optimal conditions, more than 90% of phenolics and flavonoids were recovered, a shorter extraction time than the commonly used at normal pressure and room temperature.Originality/valueThe seed coat is a major by-product of soybean processing, and it only markets as a low value ruminant feed. To date, there are no reports on the extract phenolics from SSCs by means of this methodology. The extraction technique described in this study provides a potential alternative for extraction of bioactive compounds from SSCs. This study contributes to adding value to this industrial waste and, ultimately, to optimize the postharvest production chain of soybean grains.