bluuebunny/crossref_metadata_2025_split huggingface dataset:
Efficacy and safety of a patch containing adipose-derived stem cells for skin wound healing – results form a comprehensive pre-clinical evaluation program
Authors: Brembilla Nicolo C. , Modarressi Ali , André-Lévigne Dominik , Brioudes Estelle , Lanza Florian , Vuagnat Hubert , Durual Stéphane , Marger Laurine , Boehncke Wolf-Henning , Krause Karl-Heinz , Preynat-Seauve Olivier 
Published: March 2022

AbstractMesenchymal stem cell-based therapies are emerging as innovative approaches to treat chronic wounds. A common administration route used in clinical trials consists of local injections leading to uncontrolled/sub-optimal delivery. This study reports a comprehensive pre-clinical evaluation program on the mechanism of action, efficacy and safety of an easy-to-use patch that concentrates Adipose-derived Stem Cells (ASCs) in a clinical-grade sponge of porcine crosslinked-gelatin. ASCs were prepared from the fat of ischemic patients. Transcriptome and proteome of ASC-patches and ASC monolayers were assessed by microarrays, bio-arrays and mass spectrometry. Tumorigenesis was investigated in immunosuppressed mice according to the European Pharmacopeia. Angiogenesis was assessed in vivo in the chick chorioallantoic membrane model. Efficacy of the ASC-patch was tested in a rat model of ischemic full-thickness skin defect. Cell stability was assessed by luminescence using ASC-patches generated from ASCs stably transduced with firefly luciferase. ASCs from ischemic patients upregulated the transcription of multiple genes involved in skin wound healing when cultured within the ASC-patch formulation. The patch was not only a concentrator, but also a reservoir of both ASC-derived regenerative factors and sponge-derived soluble fragments having healing capacity. The secretome of the ASC-patch promoted dermal fibroblast survival and epidermal epithelialization. No tumor formation was observed in immunodeficient Nude mice subcutaneously transplanted with the ASC-patch. Transplanted patches were early invaded by new vessels in vivo, and a marked angiogenesis was confirmed in two independent animal models. Finally, ASC-patches prepared from syngeneic rats promoted faster healing and re-vascularization of full-thickness skin defects in a rat animal model. Of note, ASCs were viable and locally stable for at least two weeks in vivo. We provide here compelling pre-clinical evidence that patches concentrating ASCs within crosslinked gelatin may represent a convenient and effective tool for the management of chronic wounds.
Temporin B forms hetero-oligomers with Temporin L, modifies its membrane activity and increases the cooperativity of its antibacterial pharmacodynamic profile
Authors: Ferguson Philip M. , Clarke Maria , Manzo Giorgia , Hind Charlotte K. , Clifford Melanie , Sutton J. Mark , Lorenz Christian D. , Phoenix David A. , Mason A. James 
Published: March 2022

ABSTRACTThe pharmacodynamic profile of antimicrobial peptides (AMPs) and their in vivo synergy are two factors that are thought to restrict resistance evolution and ensure their conservation. The frog Rana temporaria secretes a family of closely related AMPs, temporins A-L, as an effective chemical dermal defence. The antibacterial potency of temporin L has been shown to increase synergistically in combination with both temporins B and A but this is modest. Here we show that the less potent temporin B enhances the cooperativity of the in vitro antibacterial activity of the more potent temporin L against EMRSA-15 and that this may be associated with an altered interaction with the bacterial plasma membrane, a feature critical for the antibacterial activity of most AMPs. Addition of buforin II, a histone H2A fragment, can further increase the cooperativity. Molecular dynamics simulations indicate temporins B and L readily form hetero-oligomers in models of Gram-positive bacterial plasma membranes. Patch-clamp studies show transmembrane ion conductance is triggered with lower amounts of both peptides and more quickly, when used in combination, but conductance is of a lower amplitude and pores are smaller. Temporin B may therefore act by forming temporin L/B hetero-oligomers that are more effective than temporin L homo-oligomers at bacterial killing and/or by reducing the probability of the latter forming until a threshold concentration is reached. Exploration of the mechanism of synergy between AMPs isolated from the same organism may therefore yield antibiotic combinations with advantageous pharmacodynamic properties.
Unblocking genome editing of major animal mycoplasmas using CRISPR/Cas9 base editor systems
Authors: Ipoutcha Thomas , Rideau Fabien , Gourgues Geraldine , Arfi Yonathan , Lartigue Carole , Blanchard Alain , Sirand-Pugnet Pascal 
Published: March 2022

AbstractMycoplasmas are minimal bacteria that infect humans, wildlife, and most economically important livestock species. Mycoplasma infections cause a large range of chronic inflammatory diseases, eventually leading to death in some animals. Due to the lack of efficient recombination and genome engineering tools, the production of mutant strains for the identification of virulence factors and the development of improved vaccine strains is still a bottleneck for many mycoplasma species. Here, we demonstrate the efficacy of a CRISPR-derived genetic tool to introduce targeted mutations in three major pathogenic species that span the phylogenetic diversity of these bacteria: the avian pathogen Mycoplasma gallisepticum and the two most important bovine mycoplasmas, Mycoplasma bovis and Mycoplasma mycoides subsp. mycoides. As a proof of concept, we successfully used an inducible dCas9-cytidine deaminase system to disrupt several major virulence factors in these pathogens. Various induction times and inducer concentrations were evaluated to optimize editing efficiency. The optimized system was sufficiently powerful to disrupt 54 of 55 insertion sequence transposases in a single step. Whole genome sequencing showed that off-target mutations were limited and suggest that most variations detected in the edited genomes are Cas9-independent. This effective, rapid, and easy-to-use genetic tool opens a new avenue for the study of these important animal pathogens and, most likely, the entire class Mollicutes.SignificanceMycoplasmas are minimal wall-less pathogenic bacteria that infect a wide range of hosts, including humans, livestock, and wild animals. Major pathogenic species cause acute to chronic infections involving still poorly characterized virulence factors. The lack of precise genome editing tools has hampered functional studies for many species, leaving multiple questions about the molecular basis of their pathogenicity unanswered. We developed a CRISPR-derived base editor for three major pathogenic species, Mycoplasma gallisepticum, Mycoplasma bovis, and Mycoplasma mycoides subsp. mycoides. Several virulence factors were successfully targeted and we were able to edit up to 54 target sites in a single step. The availability of this efficient and easy-to-use genetic tool will greatly facilitate functional studies in these economically important bacteria.
Comparative thermophysiology of marine Synechococcus CRD1 strains isolated from different thermal niches in iron-depleted areas
Authors: Ferrieux Mathilde , Dufour Louison , Doré Hugo , Ratin Morgane , Guéneuguès Audrey , Chasselin Léo , Marie Dominique , Rigaut-Jalabert Fabienne , Le Gall Florence , Sciandra Théo , Monier Garance , Hoebeke Mark , Corre Erwan , Xia Xiaomin , Liu Hongbin , Scanlan David J. , Partensky Frédéric , Garczarek Laurence 
Published: March 2022

AbstractMarine Synechococcus cyanobacteria are ubiquitous in the ocean, a feature likely related to their extensive genetic diversity. Amongst the major lineages, clades I and IV preferentially thrive in temperate and cold, nutrient-rich waters, whilst clades II and III prefer warm, nitrogen or phosphorus-depleted waters. The existence of such cold (I/IV) and warm (II/III) thermotypes is corroborated by physiological characterization of representative strains. A fifth clade, CRD1, was recently shown to dominate the Synechococcus community in iron-depleted areas of the world ocean and to encompass three distinct ecologically significant taxonomic units (ESTUs CRD1A-C) occupying different thermal niches, suggesting that distinct thermotypes could also occur within this clade.Here, using comparative thermophysiology of strains representative of these three CRD1 ESTUs we show that the CRD1A strain MITS9220 is a warm thermotype, the CRD1B strain BIOS-U3-1 a cold temperate thermotype, and the CRD1C strain BIOS-E4-1 a warm temperate stenotherm. Curiously, the CRD1B thermotype lacks traits and/or genomic features typical of cold thermotypes. In contrast, we found specific physiological traits of the CRD1 strains compared to their clade I, II, III and IV counterparts, including a lower growth rate and photosystem II maximal quantum yield at most temperatures and a higher turnover rate of the D1 protein. Together, our data suggests that the CRD1 clade prioritizes adaptation to low-iron conditions over temperature adaptation, even though the occurrence of several CRD1 thermotypes likely explains why the CRD1 clade as a whole occupies most iron-limited waters.
Constitutive 5-HT2C receptor knock-out facilitates fear extinction through altered activity of a dorsal raphe-bed nucleus of the stria terminalis pathway
Authors: Süß Sandra T. , Kremer Daniel , Olbricht Linda M. , Herlitze Stefan , Spoida Katharina 
Published: March 2022

AbstractSerotonin 2C receptors (5-HT2CRs) are widely distributed throughout the brain and are strongly implicated in the pathophysiology of anxiety disorders such as post-traumatic stress disorder (PTSD). Although in recent years, a considerable amount of evidence supports 5-HT2CRs facilitating effect on anxiety behavior, the involvement in learned fear responses and fear extinction is rather unexplored. Here we used a 5-HT2CR knock-out mouse line (2CKO) to gain new insights into the involvement of 5-HT2CRs in the neuronal fear circuitry. Using a cued fear conditioning paradigm, our results revealed that global loss of 5-HT2CRs exclusively accelerates fear extinction, without affecting fear acquisition and fear expression. To investigate the neuronal substrates underlying the extinction enhancing effect, we mapped the immediate-early gene product cFos, a marker for neuronal activity, in the dorsal raphe nucleus (DRN), amygdala and bed nucleus of the stria terminalis (BNST). Surprisingly, besides extinction-associated changes, our results revealed alterations in neuronal activity even under basal home cage conditions in specific subregions of the DRN and the BNST in 2CKO mice. Neuronal activity in the dorsal BNST was shifted in an extinction-supporting direction due to 5-HT2CR knock-out. Finally, the assessment of DRN-BNST connectivity using antero- and retrograde tracing techniques uncovered a discrete serotonergic DRC-BNSTad pathway showing increased activity in 2CKO mice. Thus, our results provide new insights for the fear extinction network by revealing a specific serotonergic DRC-BNSTad pathway underlying a 5-HT2CR-sensitive mechanism with high significance in the treatment of PTSD.
DiSCERN - Deep Single Cell Expression ReconstructioN for improved cell clustering and cell subtype and state detection
Authors: Hausmann Fabian , Ergen-Behr Can , Khatri Robin , Marouf Mohamed , Hänzelmann Sonja , Gagliani Nicola , Huber Samuel , Machart Pierre , Bonn Stefan 
Published: March 2022

AbstractSingle cell sequencing provides detailed insights into biological processes including cell differentiation and identity. While providing deep cell-specific information, the method suffers from technical constraints, most notably a limited number of expressed genes per cell, which leads to suboptimal clustering and cell type identification. Here we present DISCERN, a novel deep generative network that reconstructs missing single cell gene expression using a reference dataset. DISCERN outperforms competing algorithms in expression inference resulting in greatly improved cell clustering, cell type and activity detection, and insights into the cellular regulation of disease. We used DISCERN to detect two unseen COVID-19-associated T cell types, cytotoxic CD4+and CD8+Tc2 T helper cells, with a potential role in adverse disease outcome. We utilized T cell fraction information of patient blood to classify mild or severe COVID-19 with an AUROC of 81% that can serve as a biomarker of disease stage. DISCERN can be easily integrated into existing single cell sequencing workflows and readily adapted to enhance various other biomedical data types.
Diet suppresses tumour initiation by maintaining quiescence of mutation-bearing neural stem cells
Authors: Amodeo Valeria , Davies Timothy , Martinez-Segura Amalia , Clements Melanie , Ragdale Holly Simpson , Bailey Andrew , Silva Dos Santos Mariana , MacRae James I. , Mokochinski Joao , Kramer Holger , Garcia-Diaz Claudia , Gould Alex P. , Marguerat Samuel , Parrinello Simona 
Published: March 2022

SummaryGlioblastoma is thought to originate from neural stem cells (NSCs) of the subventricular zone that acquire genetic alterations. In the adult brain, NSCs are largely quiescent, suggesting that deregulation of quiescence maintenance may be a pre-requisite for tumour initiation. Although inactivation of the tumour suppressor p53 is a frequent event in gliomagenesis, whether, or how, it affects quiescent NSCs (qNSCs) remains unclear. Here we show that p53 maintains quiescence by inducing fatty acid oxidation (FAO) and that acute p53 deletion in qNSCs results in their premature activation to a proliferative state. Mechanistically, this occurs through direct transcriptional induction of PPARGC1a, which in turn activates PPARα to upregulate FAO genes. Strikingly, dietary supplementation with fish oil containing omega-3 fatty acids, natural PPARα ligands, fully restores quiescence of p53-deficient NSCs and delays tumour initiation in a glioblastoma mouse model. Thus, diet can silence glioblastoma driver mutations, with important implications for cancer prevention.
NAK associated protein 1/NAP1 is required for mitosis and cytokinesis by activating TBK1
Authors: Paul Swagatika , Sarraf Shireen A. , Nam Ki Hong , Zavar Leila , Biswas Sahitya Ranjan , Fritsch Lauren E. , DeFoor Nicole , Yaron Tomer M. , Johnson Jared L. , Huntsman Emily M. , Cantley Lewis C. , Ordureau Alban , Pickrell Alicia M. 
Published: March 2022

AbstractSubcellular location and activation of Tank Binding Kinase 1 (TBK1) govern precise progression through mitosis. Either loss of activated TBK1 or its sequestration from the centrosomes causes error in mitosis and growth defects. Yet, what regulates its recruitment and activation on the centrosomes is unknown. We identified that NAK Associated Protein 1 (NAP1) is essential for mitosis which binds to TBK1 on the centrosomes to activate it. Loss of NAP1 causes several mitotic and cytokinetic defects due to inactivation of TBK1. Our quantitative phosphoproteomics identified numerous TBK1 substrates that are not only confined to the centrosomes but also are associated with microtubules. Substrate motifs analysis indicates that TBK1 acts upstream of other essential cell cycle kinases like Aurora and PAK kinases. We also identified NAP1 as a TBK1 substrate at S318 promoting its degradation by ubiquitin proteasomal system acting as a negative regulatory step. These data uncover an important distinct function for the NAP1-TBK1 complex during cell division.
Three functionally distinct classes of cGAS proteins in nature revealed by self-DNA-induced interferon responses
Authors: Mosallanejad Kenta , Zhou Wen , Govande Apurva A. , Hancks Dustin C. , Kranzusch Philip J. , Kagan Jonathan C. 
Published: March 2022

AbstractInnate immune pattern recognition receptors (PRRs) emerged early in evolution. It is generally assumed that structurally homologous proteins in distinct species will operate via similar mechanisms. We tested this prediction through the study of interferon responses to self-DNA by the enzymatic PRR cyclic GMP-AMP synthase (cGAS). Contrary to expectations, we identified three functional classes of this PRR in mammals. Class 1 proteins (including human) contained a catalytic domain that was intrinsically self-DNA reactive and stimulated interferon responses in diverse cell types. This reactivity was prevented by an upstream N-terminal domain. Class 2 and 3 proteins were either not self-DNA reactive (including chimpanzee) or included proteins whose N-terminal domain promoted self-DNA reactivity (mouse). While self-DNA reactivity of Class 1 cGAS was linked to an ability to access intra-mitochondrial DNA, mitochondrial localization was not associated with other classes. These studies reveal unexpected diversity in the mechanisms of self-DNA reactivity of a PRR.One Sentence SummaryThe regulation of self-DNA reactivity of cGAS is evolutionarily diverse in mammals.
Development of a Novel Bruton’s Tyrosine Kinase Inhibitor that exerts Anti-Cancer Activities Potentiates Response of Chemotherapeutic Agents In Multiple Myeloma Stem Cell-Like Cells
Authors: Elbezanti Weam Othman , Al-Odat Omar S. , Chitren Robert , Singh Jaikee Kumar , Srivastava Sandeep Kumar , Gowda Krishne , Amin Shantu , Robertson Gavin P. , Jonnalagadda Subash C. , Budak-Alpdogan Tulin , Pandey Manoj K. 
Published: March 2022

ABSTRACTDespite recent improvements in multiple myeloma (MM) treatment, MM remains an incurable disease and most patients experience a relapse. The major reason for myeloma recurrence is the persistent stem cell-like population. It has been demonstrated that overexpression of Bruton’s tyrosine kinase (BTK) in MM stem cell-like cells is correlated with drug resistance and poor prognosis. We have developed a novel small BTK inhibitor, KS151, which is unique compared to other BTK inhibitors. Unlike ibrutinib, and the other BTK inhibitors such as acalabrutinib, orelabrutinib, and zanubrutinib that covalently bind to the C481 residue in the BTK kinase domain, KS151 can inhibit BTK activities without binding to C481. This feature of KS151 is important because C481 becomes mutated in many patients and causes drug resistance. We demonstrated that KS151 inhibits in vitro BTK kinase activities and is more potent than ibrutinib. Furthermore, by performing a semi-quantitative, sandwich-based array for 71-tyrosine kinase phosphorylation, we found that KS151 specifically inhibits BTK. Our western blotting data showed that KS151 inhibits BTK signaling pathways and is effective against bortezomib-resistant cells as well as MM stem cell-like cells. Moreover, KS151 potentiates the apoptotic response of bortezomib, lenalidomide, and panobinostat in both MM and stem cell-like cells. Interestingly, KS151 inhibits stemness markers and is efficient in inhibiting Nanog and Gli1 stemness markers even when MM cells were co-cultured with bone marrow stromal cells (BMSCs). Overall, our results show that we have developed a novel BTK inhibitor effective against the stem cell-like population, and potentiates the response of chemotherapeutic agents.
Voltage-Sensing Phosphatase (VSP) Regulates Endocytosis-Dependent Nutrient Absorption in Chordate Enterocytes
Authors: Ratanayotha Adisorn , Matsuda Makoto , Kimura Yukiko , Takenaga Fumiko , Mizuno Tomoaki , Hossain Md. Israil , Higashijima Shin-ichi , Kawai Takafumi , Ogasawara Michio , Okamura Yasushi 
Published: March 2022

AbstractVoltage-sensing phosphatase (VSP) is a unique membrane protein that translates membrane electrical activities into the changes of phosphoinositide profiles. VSP orthologs from various species have been intensively investigated toward their biophysical properties, primarily using a heterologous expression system. In contrast, the physiological role of VSP in native tissues remains largely unknown. Here we report that zebrafish VSP (Dr-VSP) is functionally expressed on the endomembranes of lysosome-rich enterocytes (LREs) that mediate dietary protein absorption via endocytosis in the zebrafish mid-intestine. Dr-VSP-deficient LREs were remarkably defective in forming endosomal vacuoles after initial uptake of dextran and mCherry. Dr-VSP-deficient zebrafish exhibited growth restriction and higher mortality during the critical period when zebrafish larvae rely primarily on exogenous feeding via intestinal absorption. Furthermore, our comparative study on marine invertebrate Ciona intestinalis VSP (Ci-VSP) revealed co-expression with endocytosis-associated genes in absorptive epithelial cells of the Ciona digestive tract, corresponding to zebrafish LREs. These findings signify a crucial role of VSP in regulating endocytosis-dependent nutrient absorption in specialized enterocytes across animal species.Summary statementVoltage-sensing phosphatase (VSP) is identified in absorptive enterocytes, revealing its crucial role in promoting endocytosis and nutrient absorption during early development.
Structural and Functional Insights into the Action Mode of A Mitochondrial AAA+ Disaggregase CLPB
Authors: Wu Damu , Liu Yan , Dai Yuhao , Wang Guopeng , Lu Guoliang , Chen Yan , Li Ningning , Lin Jinzhong , Gao Ning 
Published: March 2022

AbstractThe human AAA+ ATPase CLPB (SKD3) is a protein disaggregase in the mitochondrial intermembrane space and functions to promote the solubilization of various mitochondrial proteins. CLPB deficiency by mutations is associated with a few human diseases with neutropenia and neurological disorders. Unlike canonical AAA+ proteins, CLPB contains a unique ankyrin repeat domain (ANK) at its N-terminus. The mechanism of CLPB functions as a disaggregase and the role of its ANK domain are currently unclear. Herein, we report a comprehensive structural characterization of human CLPB in both the apo- and substrate-bound states. CLPB assembles into homo- tetradecamers in apo-state and is remodeled into homo-dodecamers upon binding to substrates. Conserved pore- loops on the ATPase domains form a spiral staircase to grip and translocate the substrate in a step-size of two amino acid residues. The ANK domain is not only responsible for maintaining the higher-order assembly but also essential for the disaggregase activity. Interactome analysis suggests that the ANK domain may directly interact with a variety of mitochondrial substrates. These results reveal unique properties of CLPB as a general disaggregase in mitochondria and highlight its potential as a target for the treatment of various mitochondria-related diseases.