bluuebunny/crossref_metadata_2025_split huggingface dataset:
Targeting YAP mechanosignaling to ameliorate stiffness-induced Schlemm’s canal cell pathobiology
Authors: Li Haiyan , Kuhn Megan , Kelly Ruth A. , Singh Ayushi , Palanivel Kavipriya Kovai , Salama Izzy , De Ieso Michael L. , Stamer W. Daniel , Ganapathy Preethi S. , Herberg Samuel 
Published: September 2023

AbstractPathologic alterations in the biomechanical properties of the Schlemm’s canal (SC) inner wall endothelium and its immediate vicinity are strongly associated with ocular hypertension in glaucoma due to decreased outflow facility. Specifically, the underlying trabecular meshwork is substantially stiffer in glaucomatous eyes compared to that from normal eyes. This raises the possibility of a critical involvement of mechanotransduction processes in driving SC cell dysfunction. Yes-associated protein (YAP) has emerged as a key contributor to glaucoma pathogenesis. However, the molecular underpinnings of SC cell YAP mechanosignaling in response to glaucomatous extracellular matrix (ECM) stiffening are not well understood. Using a novel biopolymer hydrogel that facilitates dynamic and reversible stiffness tuning, we investigated how ECM stiffening modulates YAP activity in primary human SC cells, and whether disruption of YAP mechanosignaling attenuates SC cell pathobiology and increasesex vivooutflow facility. We demonstrated that ECM stiffening drives pathologic YAP activation and cytoskeletal reorganization in SC cells, which was fully reversible by matrix softening in a distinct time-dependent manner. Furthermore, we showed that pharmacologic or genetic disruption of YAP mechanosignaling abrogates stiffness-induced SC cell dysfunction involving altered cytoskeletal and ECM remodeling. Lastly, we found that perfusion of the clinically-used, small molecule YAP inhibitor verteporfin (without light activation) increasesex vivooutflow facility in normal mouse eyes. Collectively, our data provide new evidence for a pathologic role of aberrant YAP mechanosignaling in SC cell dysfunction and suggest that YAP inhibition has therapeutic value for treating ocular hypertension in glaucoma.
RNA Binding of Syntaxin 5 Synergises Membrane Fusion to Accelerate miRNA Export, Activate Macrophage and Clear Pathogens
Authors: Hom Choudhury Sourav , Bhattacharjee Shreya , Mukherjee Kamalika , Bhattacharyya Suvendra N. 
Published: September 2023

AbstractIntercellular miRNA exchange acts as a key mechanism to control gene expression post-transcriptionally in mammalian cells. Regulated export of repressive miRNAs allows the expression of inflammatory cytokines in activated macrophages. Intracellular trafficking of miRNAs from endoplasmic reticulum to endosomes is a rate determining step in miRNA export process and plays an important role in controlling cellular miRNA level and inflammatory processes in macrophages. We have identified the SNARE protein Syntaxin5 to show a synchronized expression pattern with miRNA activity loss in activated mammalian macrophage cells. Syntaxin 5 is both necessary and sufficient for macrophage activation and clearance of the intracellular pathogenLeishmania donovanifrom infected macrophages. Exploring the mechanism of how Syntaxin5 acts as an immunostimulant, we have identified thede novoRNA binding property of this SNARE protein that binds specific miRNAs and facilitates their accumulation in endosomes in a cooperative manner with human ELAV protein HuR to ensure export of miRNAs and allows the expression of miRNA-repressed cytokines. Conversely, in its dual role in miRNA export, this SNARE protein prevents lysosomal targeting of endosomes by enhancing the fusion of miRNA-loaded endosomes with plasma membrane to ensure accelerated release of extracellular vesicles and associated miRNAs.Graphical AbstractHuR transfers miRNAs to STX5 for exportSTX5 utilizes its N-terminal disordered domain to bind and target miRNAs to endosomes.In its dual role, STX5 uses the SNARE domain to export miRNA by promoting EV biogenesis and release.STX5-mediated miRNA export activates macrophage to clear internalized parasites.
Yerba mate (Ilex paraguariensis) genome provides new insights into convergent evolution of caffeine biosynthesis
Authors: Vignale Federico A. , Hernandez Garcia Andrea , Modenutti Carlos P. , Sosa Ezequiel J. , Defelipe Lucas A. , Oliveira Renato R.M. , Nunes Gisele L. , Acevedo Raúl M. , Burguener German F. , Rossi Maximiliano , Zapata Pedro D. , Marti Dardo A. , Sansberro Pedro A. , Oliveira Guilherme , Smith Madeline N. , Dubs Nicole M. , Nair Satish , Barkman Todd J. , Turjanski Adrian G. 
Published: September 2023

AbstractYerba mate (Ilex paraguariensis) is an economically important crop marketed for the elaboration of mate, the third-most widely consumed caffeine-containing infusion worldwide. Here we report the first genome assembly of this species, which has a total length of 1.06 Gb and contains 53,390 protein-coding genes. Comparative analyses revealed that the large yerba mate genome size is partly due to a whole-genome duplication (Ip-α) during the early evolutionary history ofIlex, in addition to the hexaploidization event (γ) shared by core eudicots. Characterization of the genome allowed us to clone the genes encoding methyltransferase enzymes that catalyse multiple reactions required for caffeine production. To our surprise, this species has converged upon a different biochemical pathway compared to that of its relatives, coffee and tea. In order to gain insight into the structural basis for the convergent enzyme activities, we obtained a crystal structure for the terminal enzyme in the pathway that forms caffeine. The structure reveals that convergent solutions have evolved for substrate positioning because different amino acid residues facilitate a different substrate orientation such that efficient methylation occurs in the independently evolved enzymes in yerba mate and coffee. While our results show phylogenomic constraint limits the genes coopted for convergence of caffeine biosynthesis, the x-ray diffraction data suggests structural constraints are minimal for the convergent evolution of individual reactions.
A General Method to Accurately Count Molecular Complexes and Determine the Degree of Labelling in Cells Using Protein Tags
Authors: Tashev Stanimir Asenov , Euchner Jonas , Yserentant Klaus , Hänselmann Siegfried , Hild Felix , Chmielewicz Wioleta , Hummert Johan , Schwörer Florian , Tsopoulidis Nikolaos , Germer Stefan , Saßmannshausen Zoe , Fackler Oliver T. , Klingmüller Ursula , Herten Dirk-Peter 
Published: September 2023

AbstractDetermining the label to target ratio, also known as degree of labelling (DOL), is crucial for quantitative fluorescence microscopy and a high DOL with minimal unspecific labelling is beneficial for fluorescence microscopy in general. Yet, robust, versatile, and easy-to-use tools for measuring cell-specific labelling efficiencies are not available. This study presents a novel DOL determination technique named Protein-tag DOL (ProDOL), which enables fast DOL measurements and optimisation of protein-tag labelling. With ProDOL various factors affecting labelling efficiency, including substrate type, incubation time, and concentration, as well as sample fixation and cell type can be easily assessed. We applied ProDOL to investigate how HIV-1 pathogenesis factor Nef modulates CD4 T cell activation measuring total and activated copy numbers of the adaptor protein SLP-76 in signalling microclusters. ProDOL proved to be a versatile and robust tool for labelling calibration, enabling determination of labelling efficiencies, optimisation of strategies, and quantification of protein stoichiometry.
Tradeoffs of increasing temperatures for the spread of antimicrobial resistance in river biofilms
Authors: Bagra Kenyum , Kneis David , Padfield Daniel , Szekeres Edina , Teban-Man Adela , Coman Cristian , Singh Gargi , Berendonk Thomas U. , Klümper Uli 
Published: September 2023

AbstractRiver microbial communities regularly act as the first defense barrier against the spread of antimicrobial resistance genes (ARG) that enter environmental microbiomes through wastewaters. However, how the invasion dynamics of wastewater-born ARGs into river biofilm communities will shift due to increasing average and peak temperatures worldwide through climate change remains unknown. Here we aimed at elucidating the effects of increasing temperatures on both, the natural river biofilm resistome, as well as the river biofilms invadability by foreign, wastewater-born ARGs. To achieve this, natural biofilms were grown in a pristine German river and transferred to artificial laboratory recirculation flume systems at three different temperatures (20°C, 25°C, 30°C). Already after one week of acclimatization to the temperatures, significant increases in the abundance of most naturally occurring ARGs were detected in the biofilms exposed to the highest temperature. Thereafter, biofilms were exposed to a single pulse of wastewater and the invasion dynamics of wastewater-born ARGs were analyzed over a period of two weeks. While initially after one day ARGs were able to invade all biofilms successfully and in equal proportions, the foreign invading ARGs were lost at a far increased rate at 30°C over time. ARG levels dropped to the initial natural levels at 30°C after 14 days. Contrary at the lower temperatures ARGs remained far elevated and certain ARGs were able to establish themselves in the biofilms. Overall, we here demonstrate tradeoffs of increasing temperature between increases in naturally occurring and faster loss dynamics of invading ARGs.
Comparative transcriptomics reveal a novel tardigrade specific DNA binding protein induced in response to ionizing radiation
Authors: Anoud M. , Delagoutte E. , Helleu Q. , Brion A. , Duvernois-Berthet E. , As M. , Marques X. , Lamribet K. , Senamaud C. , Jourdren L. , Adrait A. , Heinrich S. , Toutirais G. , Hamlaoui S. , Gropplero G. , Giovannini I. , Ponger L. , Gèze M. , Blugeon C. , Coute Y. , Guidetti R. , Rebecchi L , Giovannangeli C. , De Cian A. , Concordet J-P. 
Published: September 2023

ABSTRACTTardigrades, microscopic animals found in virtually all ecosystems, are renowned for their remarkable ability to withstand extreme conditions. Recent studies have identified novel tardigrade specific protein families that aid in resistance to desiccation and ionizing radiation (IR). Notably, a tardigrade specific DNA binding protein called Dsup (for DNA damage suppressor) has been found to protect from X-ray damage in human cells and from hydroxyl radicalsin vitro. However, Dsup has only been found in two species within the Hypsibioidea superfamily.To better understand mechanisms underlying radio-resistance in the Tardigrada phylum, we first characterized DNA damage and repair in response to IR in the model speciesHypsibius exemplaris. By analysis of phosphorylated H2AX, we demonstrated the induction and repair of DNA double-strand breaks after IR exposure. Importantly, the rate of single-strand breaks induced was roughly equivalent to that in human cells, suggesting that DNA repair plays a predominant role in the remarkable radio-resistance of tardigrades. In order to identify novel tardigrade specific genes involved, we next conducted a comparative transcriptomics across three species,H. exemplaris,Acutuncus antarcticusandParamacrobiotus fairbanksi, the latter belonging to the Macrobiotoidea superfamily known to lack Dsup homologs. In all three species, many genes of DNA repair were among the most strongly overexpressed genes alongside a novel tardigrade specific gene, namedTardigradeDNA damageResponse protein1(TDR1). We found that TDR1 protein interacts with DNA and forms aggregates at high concentration suggesting it may condensate DNA and act by preserving chromosome organization until DNA repair is accomplished. Remarkably, when expressed in human cells, TDR1 improved resistance to Bleomycin, a radiomimetic drug. Based on these findings, we propose that TDR1 is a novel tardigrade specific gene responsible for conferring resistance to IR. Our study sheds light on mechanisms of DNA repair helping to cope with high levels of DNA damage. Furthermore, it suggests that at least two tardigrade specific genes, respectively for Dsup and TDR1, have independently evolved DNA-binding functions that contribute to radio-resistance in the Tardigrada phylum.
Development of evolutionarily conserved viral integration sites as safe harbors for human gene therapy
Authors: Quezada-Ramírez Marco A. , Loncar Shannon , Campbell Matthew A. , Parsi Krishna M. , Gifford Robert J. , Kotin Robert M. 
Published: September 2023

AbstractGene transfer into CD34+hematopoietic stem and progenitor cells (HSPCs) involving integrating viral vectors has unpredictable outcomes including potential adverse events like leukemogenesis, resulting from insertional mutagenesis. Therefore, identifying and characterizing genome safe harbor (GSH) sites where exogenous gene material can be safely integrated into adult progenitor and stem cells is critically important for therapeutic gene addition. Here, we present a novel approach to identify new GSH sites based on a proven system of stable transgene insertion: the evolutionarily conserved integration of parvovirus DNA into the germlines of host species. From a dataset of 199 unique endogenous parvovirus (EPV) integration events identified in host species genomes, 102 loci were mapped to the human genome with 17 being experimentally evaluated as GSHs in primary human CD34+HSPCs. Nine promising GSHs resulted in cells edited using nucleofection alone or in combination with rAAV transduction. Of the nine GSH sites, six edited loci displayed sustained transgene expression in both erythroid and immune phenotypes while three clearly exhibited immune branch specific-regulation. Following this approach, additional GSH sites are likely to emerge from the remaining mapped loci for gene addition in hematopoietic stem and progenitor cells. Since it is unlikely that the GSH-lineage-restricted transgene expression is exclusive to hematopoietic stem cells, this approach extends the options for gene knock-ins while reducing the risks of insertional mutagenesis, unpredictable expression profiles, effects on differentiation, and increasing therapeutic effects.
Top-down biological motion perception does not differ between adults scoring high versus low on autism traits
Authors: Oomen Danna , Wiersema Jan R. , Orgs Guido , Cracco Emiel 
Published: September 2023

AbstractThe perception of biological motion is an important social cognitive ability. Models of biological motion perception recognize two processes that contribute to the perception of biological motion: a bottom-up process that binds optic-flow patterns into a coherent percept of biological motion and a top-down process that binds sequences of body-posture ‘snapshots’ over time into a fluent percept of biological motion. The vast majority of studies on autism and biological motion perception have used point-light figure stimuli, which elicit biological motion perception predominantly via bottom-up processes. Here, we investigated whether autism is associated with deviances in the top-down processing of biological motion. For this, we tested a sample of adults scoring lowvshigh on autism traits on a recently validated EEG paradigm in which apparent biological motion is combined with frequency tagging (Cracco et al., 2022) to dissociate between two percepts: 1) the representation of individual body postures, and 2) their temporal integration into movements. In contrast to our hypothesis, we found no evidence for a diminished temporal body posture integration in the high-scoring group. We did, however, find a group difference that suggests that adults scoring high on autism traits have a visual processing style that focuses more on a single percept (i.e. either body postures or movements, contingent on saliency) compared to adults scoring low on autism traits who instead seemed to represent the two percepts included in the paradigm in a more balanced manner. Although unexpected, this finding aligns well with the autism literature on perceptual stability(/rigidity).
Mechanosensitive recruitment of Vinculin maintains junction integrity and barrier function at epithelial tricellular junctions
Authors: van den Goor Lotte , Iseler Jolene , Koning Katherine , Miller Ann L. 
Published: September 2023

SummaryApical cell-cell junctions, including adherens junctions (AJs) and tight junctions (TJs), adhere epithelial cells to one another and regulate selective permeability at both bicellular junctions (BCJs) and tricellular junctions (TCJs). Although several specialized proteins are known to localize at TCJs, it remains unclear how actomyosin-mediated tension transmission at TCJs contributes to the maintenance of junction integrity and barrier function at these sites. Here, utilizing gastrula-stageXenopus laevisembryos as a model system, we describe a mechanism by which Vinculin, a mechanosensitive protein, anchors the actomyosin network at TCJs, thus maintaining TJ stability and barrier function. Using an optogenetic approach, we found that acutely increasing junctional tension results in robust recruitment of Vinculin to apical junctions immediately surrounding TCJs. In Vinculin knockdown (KD) embryos, junctional actomyosin intensity is decreased and becomes disorganized at TCJs. Using fluorescence recovery after photobleaching (FRAP), we show that loss of Vinculin results in reduced Actin stability at TCJs. Vinculin knockdown also destabilizes Angulin-1, a key protein involved in regulating barrier function at TCJs. When Vinculin KD embryos are subjected to increased tension, TCJs cannot maintain their proper morphology. Finally, using a live imaging barrier assay, we detect increased barrier leaks at TCJs in Vinculin KD embryos. Together, our findings show that Vinculin-mediated actomyosin organization is required to maintain junction integrity and barrier function at TCJs and reveal new information about the interplay between adhesion and barrier function at TCJs.HighlightsVinculin is mechanosensitively recruited to tricellular junctionsVinculin’s actin-binding function is needed for tricellular actomyosin organizationTricellular tight junctions are unstable when Vinculin is knocked downVinculin is required to maintain barrier function at tricellular junctions
Inhibition of human immunodeficiency virus (HIV-1) infectivity by expression of poorly or broadly neutralizing antibodies against Env in virus-producing cells
Authors: Wang Qian , Zhang Shijian , Nguyen Hanh T. , Sodroski Joseph 
Published: September 2023

ABSTRACTThe human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) precursor (gp160) trimerizes, is modified by high-mannose glycans in the endoplasmic reticulum, and is transported via Golgi and non-Golgi secretory pathways to the infected cell surface. In the Golgi, gp160 is partially modified by complex carbohydrates and proteolytically cleaved to produce the mature functional Env trimer, which is preferentially incorporated into virions. Broadly neutralizing antibodies (bNAbs) generally recognize the cleaved Env trimer, whereas poorly neutralizing antibodies (pNAbs) bind the conformationally flexible gp160. We found that expression of bNAbs, pNAbs or soluble/membrane forms of the receptor, CD4, in cells producing HIV-1 all decreased viral infectivity. Four patterns of co-expressed ligand:Env were observed: 1) Ligands (CD4, soluble CD4-Ig and some pNAbs) that specifically recognize the CD4-bound Env conformation resulted in uncleaved Envs lacking complex glycans that were not incorporated into virions; 2) Other pNAbs produced Envs with some complex carbohydrates and severe defects in cleavage, which were relieved by brefeldin A treatment; 3) bNAbs that recognize gp160 as well as mature Envs resulted in Envs with some complex carbohydrates and moderate decreases in virion Env cleavage; and 4) bNAbs that preferentially recognize mature Envs produced cleaved Envs with complex glycans in cells and on virions. The low infectivity observed upon co-expression of pNAbs or CD4 could be explained by disruption of Env trafficking, reducing the level of Env and/or increasing the fraction of uncleaved Env on virions. In addition to bNAb effects on virion Env cleavage, the secreted bNAbs neutralized the co-expressed viruses.IMPORTANCEThe envelope glycoprotein (Env) trimers on the human immunodeficiency virus (HIV-1) mediate virus entry into host cells. Env is synthesized in infected cells, modified by complex sugars and cleaved to form a mature, functional Env, which is incorporated into virus particles. Env elicits antibodies in infected individuals, some of which can neutralize the virus. We found that antibodies co-expressed in the virus-producing cell can disrupt Env transit to the proper compartment for cleavage and sugar modification and, in some cases, block incorporation into viruses. These studies provide insights into the processes by which Env becomes functional in the virus-producing cell and may assist attempts to interfere with these events to inhibit HIV-1 infection.
Service evaluation of “GP at Door” of Accident and Emergency Services in Eastern England
Authors: Brainard Julii , Rice Aiden , Hughes Gareth , Everden Paul 
Published: September 2023

AbstractAimsTo describe activity and outcomes after streaming low urgency attenders to general practice services at door of Accident and Emergency departments (GDAE), including possible benefits to co-located emergency departments.MethodsAs a service evaluation, we describe GDAE users, their reasons for presentation, wait times, outcomes and co-located emergency department performance metrics at two hospitals in eastern England.FindingsEach GDAE saw about 928 patients per month. Wait times for usual A&E care relatively shortened at only one site. Reattendances were common (about 10% of attenders), 75% of GDAE attenders were seen within 1 hour of arrival, 7% of patients initially allocated to GDAE were referred back to A&E for further investigations, 59% of GDAE patients were treated and discharged with no further treatment or referral required. Pain, injury, infection or feeling generally unwell each comprised > 10% of primary reasons for attendance. Referrals to specialist health services were outcome for 4% and 16% at respective sites.ConclusionsAbout 26,000 A&E attendances appear to have been prevented. Patients were seen quickly at both GDAE sites, while there were more specialist referrals or shorter wait times for usual A&E services at only one site. Process evaluation could illuminate reasons for these differences.