bluuebunny/crossref_metadata_2025_split huggingface dataset:
The K2: Open-source simultaneous triple-color TIRF microscope for live-cell and single-molecule imaging
Authors: Niederauer Christian , Seynen Marco , Zomerdijk Jan , Kamp Marko , Ganzinger Kristina A. 
Published: December 2022

AbstractImaging the dynamics and interactions of biomolecules at the single-molecule level in live cells and reconstituted systems has generated unprecedented knowledge about the biomolecular processes underlying many cellular functions. To achieve the speed and sensitivity needed to detect and follow individual molecules, these experiments typically require custom-built microscopes or custom modifications of commercial systems. The costs of such single-molecule microscopes, their technical complexity and the lack of open-source documentation on how to build custom setups therefore limit the accessibility of single-molecule imaging techniques. To advance the adaptation of dynamic single-molecule imaging by a wider community, we present the “K2”: an open-source, simultaneous triple-color total internal reflection fluorescence (TIRF) microscope specifically designed for live-cell and single-molecule imaging. We explain our design considerations and provide step-by-step building instructions, parts list and full CAD models. The flexible design of this TIRF microscope allows users to customize it to their scientific and financial needs, or to re-use parts of our design to improve the capabilities of their existing setups without necessarily having to build a full copy of the K2 microscope.
Non-equilibrium processes prevail in shaping species richness and functional diversity of terrestrial vertebrates in a global hotspot
Authors: Moroti Matheus de Toledo , Skeels Alexander , Da Silva Fernando R. , Provete Diogo B. 
Published: December 2022

AbstractAimThe effects of equilibrium and nonequilibrium processes are generally investigated using species richness on a single biological group. However, little is known about how these two classes of processes also affect trait diversity, considering multiple taxa within the same geographical template. Here, we evaluated which variables representing equilibrium (topography, climate, and primary productivity) and nonequilibrium (diversification rate and evolutionary time) processes best explain species richness and trait diversity of four clades of vertebrates within the same global hotspot. We also investigated how trait disparity has accumulated over time and whether there are congruent spatial patterns between groups.LocationAtlantic RainforestTime periodContemporary.Major taxa studiedTerrestrial vertebrates.MethodsWe tested whether the spatial pattern of Functional Dispersion (FDis), richness, diversification rate, and evolutionary time of each group are correlated. We used a spatially explicit structural equation model to test how species richness and functional dispersion are influenced by variables representing equilibrium and nonequilibrium mechanisms. Additionally, we explored how trait disparity accumulated over time in the four groups.ResultsWe found that non-equilibrium proxies, evolutionary time and diversification rate, played a primary role in driving species richness and trait diversity, with elevation and climate variables having only an indirect effect species and trait diversity via diversification rate and evolutionary time. We found a congruent pattern of species richness among all groups, except among ectotherms. In contrast, the spatial distribution of evolutionary time was distinct for each group.Main conclusionsDespite nonequilibrium processes were more important for generating large-scale diversity patterns within the same geographical template, the interplay between evolutionary time and dispersal ability have disparately determined the assembly of communities.
HBM-CITEseq: a uniform CITE-seq processing pipeline for the HuBMAP Consortium
Authors: Lu Xinyue , Ruffalo Matthew 
Published: December 2022

AbstractAs part of the HuBMAP Consortium we have been developing methods for uniformly processing and indexing multiple single-cell datasets, which enable efficient integration of data from different platforms. HuBMAP computational pipelines have so far focused on unimodal data types such as single-cell/nucleus RNA sequencing and single nucleus ATAC-seq. Here we present HBM-CITEseq, a processing pipeline for Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) datasets, the first multimodal sequencing processing pipeline for the HuBMAP Consortium, with transcriptomic outputs from HBM-CITEseq matching those of the HuBMAP RNA-seq pipeline.HBM-CITEseq is a CWL workflow wrapping command-line tools encapsulated in Docker images. It is freely available on GitHub athttps://github.com/hubmapconsortium/citeseq-pipeline.
Different B cell activation patterns in asymptomatic and symptomatic COVID-19 patients
Authors: Pham Nhung , Talih Nuray , Ehrhart Friederike , Evelo Chris T , Kutmon Martina 
Published: December 2022

AbstractEarly and persistent defects in B cell subsets such as memory B cells were shown to be correlated with poor outcomes in COVID-19 patients. This research aimed to develop a molecular pathway model to understand the B cell development in COVID-19. A B cell transcriptomics dataset, obtained from COVID-19 patients, was analyzed on the resulting pathway model to study B cell activation. The pathway showed two distinct gene expression profiles between asymptomatic and symptomatic patients. In asymptomatic patients, there is an increase in transcript levels of antiviral interferon-stimulated genes such as ISG15, IFITM1, and NEAT1 and a driving gene for the extrafollicular pathway CXCR4 indicating a formation of plasmablast. In symptomatic patients, the results suggest an inhibition occurring at the germinal center hinting at a reduction in memory B cell production. Transcripts of driver gene CXCR5 involved in germinal center development is one of the most downregulated genes. This could contribute to the shortage in the formation of memory B cells in COVID-19. Concluding, in SARS-CoV-2 infection, B cells follow different activation routes in asymptomatic and symptomatic patients. In this study, we constructed a pathway that allowed us to analyze and interpret activation patterns of B cells in COVID-19 patients and their link to disease severity. Importantly, the pathway and approach can be reused for further research in COVID-19 or other diseases.
Genomic diversity ofStreptococcus uberisisolated from clinical mastitis of cattle in selected areas of Bangladesh
Authors: Hassan Jayedul , Bag Md. Abdus Sattar , Kabir Ajran , Ali Md. Wohab , Hossain Maqsud , Rahman Md. Tanvir , Islam Md. Shafiqul , Khan Md. Shahidur Rahman 
Published: December 2022

ABSTRACTStreptococci are the major etiology in mastitis, a cause of huge economic losses in the dairy industries.Streptococcus(S.)agalactiae, S. dysagalactiaeandS. uberisare mostly encountered in bovine mastitis; however, data on the diversity and characteristics of Streptococcus in clinical mastitis of cattle in Bangladesh is lacking. Thus, the present study was aimed to determine the diversity and antimicrobial resistance pattern ofStreptococcusspp. isolated from clinical mastitis of cattle reared in Bangladesh. A total of 105 milk samples comprising eighty (80) from cattle with clinical mastitis (CCM) and twenty-five (25) from apparently healthy cattle (AHC) in four prominent dairy farms and one dairy community were purposively collected and examined in this study. Milk samples were enriched in Luria Bertani broth (LB) andStreptococcusspp. was isolated on Modified Edwards Medium and identified by 16S rRNA gene sequencing. Among eighty (80) clinical samples, eighteen (18) were positive forStreptococcusspp. while none of the milk from AHC revealed Streptococcus by cultural and molecular examination. Sequencing and phylogenetic analysis identified 55.6%, 33.3%, 5.6% and 5.6% of the Streptococcus isolates asS. uberis, S. agalactiae, S. hyovaginalisandS. urinalis, respectively. Antibiotic sensitivity testing with antimicrobials commonly used to treat clinical mastitis revealed 100%, 100%, and 30% of theS. agalactiae,S. hyovaginalis, andS. uberisas multidrug-resistant, respectively. Molecular characterization through whole genome sequencing of five (5)S. uberisisolates identified at least two novel ST types ofS. uberiscirculating in the study areas with one ST (4/5 isolates) clustered with the isolates from China, India and Thailand, and the other (1/5) with UK, Ireland and Australia. Pan-genome analysis and phylogeny of the core genome sequences also clustered the isolates into two sub-clusters, indicating the presence of at least two different subtypes ofS. uberisin the study area. On virulence profiling, all the isolates of this study were found to harbor at least 35 virulence and putative virulence genes probably associated with intramammary infection (IMI) indicating all theS. uberisisolated in this study as potential pathogen. From the overall findings it was evident that Strepococcus occurring in bovine mastitis are diverse andS. uberisgenome carries an array of putative virulence factors which need to be investigated genotypically and phenotypically to identify a specific trait or determinant governing the virulence and fitness of this bacterium. Moreover, Streptococcus isolated in this study carried multidrug resistance which needs careful consideration during the selection of a treatment regimen for mastitis.
How to tuna fish: constraint, convergence and integration in the neurocranium of pelagiarian fishes
Authors: Knapp Andrew , Rangel-de Lázaro Gizéh , Goswami Anjali , Friedman Matt , Evans Kory M , Giles Sam , Beckett Hermione T , Johanson Zerina 
Published: December 2022

AbstractMorphological evolution of the vertebrate skull has been explored across a wide range of tetrapod clades, but teleost fishes, accounting for roughly half of all vertebrate species, have largely been overlooked. Here we present the results of a study investigating three-dimensional morphological evolution across 114 species of Pelagiaria, a morphologically diverse clade of open-ocean teleost fishes that includes tuna and mackerel. Despite showing high shape disparity, the majority of taxa are concentrated in fairly restricted regions of morphospace, with taxa from all families falling into three distinct clusters. Phylogenetic signal in shape data is significant but low (Kmult= 0.27,p= 0.001) and a single-rate Ornstein-Uhlenbeck model of evolution is supported, revealing convergence in shape within and between families. Shape is significantly correlated with body elongation (R2= 0.35,p=0.001), but correlation with size, diet, and habitat depth is weak. Integration of the neurocranium is high, supporting the hypothesis that high integration may promote the evolution of more extreme morphologies. Taken together, these results suggest that shape evolution in the pelagiarian neurocranium is constrained by a number of factors, resulting in the repeated evolution of a restricted range of morphologies.
Hippocampal ripples coincide with “up-state” and cortical spindles in Retrosplenial Cortex
Authors: Pedrosa Rafael , Nazari Mojtaba , Kergoat Loig , Bernard Christophe , Mohajerani Majid , Stella Federico , Battaglia Francesco 
Published: December 2022

AbstractDuring NREM sleep hippocampal Sharp-wave ripples (SWR) events are thought to stabilize memory traces for long-term storage in downstream neocortical structures. Within the neocortex, Default Mode Network (DMN) areas interact preferentially with the hippocampus purportedly to consolidate those traces. Transient bouts of slow oscillations and sleep spindles in DMN areas are often observed around SWRs, suggesting that these two activities are related and that their interplay possibly contributes to memory consolidation. To investigate how SWRs interact with the DMN and spindles, we combined cortical wide-field voltage imaging, ECoG, and hippocampal LFP recordings in anesthetized and sleeping mice. Here we show that, during SWR, “up-states” and spindles reliably co-occur in a cortical subnetwork centered around the Retrosplenial cortex. Furthermore, Retrosplenial transient activations and spindles predict Slow Gamma oscillations in CA1 during SWRs. Together, our results suggest that Retrosplenial-hippocampal interaction may be a central source of information exchange between cortex and hippocampus.
Calcium flux through ER-TGN contact sites facilitates cargo export
Authors: Ramazanov Bulat R. , Di Martino Rosaria , Kumar Abhishek , Parchure Anup , Kim Yeongho , Griesbeck Oliver , Schwartz Martin A. , Luini Alberto , von Blume Julia 
Published: December 2022

AbstractCa2+influx into the trans-Golgi Network (TGN) promotes secretory cargo sorting by the Ca2+-ATPase SPCA1 and the luminal Ca2+binding protein Cab45. Cab45 oligomerizes upon a local Ca2+influx, and Cab45 oligomers sequester and separate soluble secretory cargo from the bulk flow of proteins in the TGN. However, how this Ca2+flux into the lumen of the TGN is achieved remains elusive, as the cytosol has a very low steady-state Ca2+concentration. The TGN forms membrane contact sites (MCS) with the Endoplasmic Reticulum (ER), whereby the close apposition of the two organelles allows the protein-mediated exchange of molecular species such as lipids. Here we show that the TGN export of Cab45 clients requires the integrity of ER-TGN MCS and IP3R-dependent Ca2+fluxes in the MCS, suggesting Ca2+transfer between these organelles. Using an MCS-targeted Ca2+FRET sensor module, we measure the Ca2+flow in these sites in real-time. These data show for the first time that ER-TGN MCS facilitates Ca2+transfer required for SPCA1-dependent cargo sorting and export from the TGN, thus solving a fundamental question in cell biology.SummaryThe current study demonstrates that the trafficking of COMP and LyzC relies on Ca2+flux between the endoplasmic reticulum (ER) and trans-Golgi Network (TGN). This process requires the activity of IP3 receptors, present in ER membranes, and depends on the integrity of the membrane contact site between these two organelles.
Cas phosphorylation regulates focal adhesion assembly
Authors: Kumar Saurav , Stainer Amanda , Dubrulle Julien , Simpkins Christopher , Cooper Jonathan A 
Published: December 2022

AbstractIntegrin-mediated cell attachment rapidly induces tyrosine kinase signaling. Despite years of research, the role of this signaling in integrin activation and focal adhesion assembly is unclear. We provide evidence that the Src-family kinase (SFK) substrate Cas (Crk-associated substrate, p130Cas, BCAR1) is phosphorylated, and associated with its effectors, Crk/CrkL, in clusters that are precursors of focal adhesions. The initial phospho-Cas clusters contain integrin β1 in its inactive, bent closed, conformation. Later, phospho-Cas and total Cas levels decrease as integrin β1 is activated and core focal adhesion proteins including vinculin, talin, kindlin and paxillin are recruited. Cas is required for cell spreading and focal adhesion assembly in epithelial and fibroblast cells on collagen and fibronectin. Cas cluster formation requires Cas, Crk/CrkL, SFKs and Rac1 but not vinculin. Rac1 provides positive feedback onto Cas through reactive oxygen, opposed by negative feedback from the ubiquitin proteasome system. The results suggest a two-step model for focal adhesion assembly in which clusters of phospho-Cas, effectors and inactive integrin β1 grow through positive feedback prior to integrin activation and recruitment of core focal adhesion proteins.
Dynamic Interplay of Autophagy and Membrane Repair DuringMycobacterium tuberculosisInfection
Authors: Augenstreich Jacques , Phan Anna T. , Allen Charles N.S. , Poddar Anushka , Srinivasan Lalitha , Briken Volker 
Published: December 2022

AbstractAutophagy plays a crucial role in the host response to Mycobacterium tuberculosis (Mtb) infection, yet the dynamics and regulation of autophagy induction on mycobacterial phagosomes remain partially understood. In this study, we employed time-lapse confocal microscopy to investigate in real time the recruitment of LC3B (LC3), a key autophagy marker, to Mtb-containing vacuoles (MCVs) at the single cell level with our newly developed workflow for single cell and single MCV tracking and fluorescence quantification. The results reveal that approximately 70% of MCVs exhibited LC3 recruitment but was lost in about 40% of those MCVs. The LC3 recruitment to MCVs displayed a high variability in timing that was independent of the size of the MCV or the bacterial burden. Most notably, the LC3-positive MCVs did not acidify, indicating that LC3 recruitment does not necessarily lead to the formation of mature autophagolysosomes. In addition, interferon-gamma (IFN-γ) pre-treatment did not affect LC3 recruitment frequency or autophagosome maturation, but increased the susceptibility of the macrophage to Mtb-induced cell death. Instead, LC3 recruitment and lysotracker staining were mutually exclusive events alternating on some MCVs multiple times showing a new reversible aspect of this autophagy response. It also suggested a role of autophagy in membrane repair of the MCV. Consistently, LC3 recruitment was strongly associated with galectin-3 and oxysterol-binding protein 1 staining, indicating a correlation with membrane damage and repair mechanisms. However, knockdown of ATG7 did not impact membrane repair, suggesting that autophagy is not directly involved in this process but is coregulated by the membrane damage of MCVs.In summary, our findings provide novel insights into the dynamic and variable nature of LC3 recruitment and autophagy to the MCVs over time during Mtb infection. Our data suggests that there is no major role of autophagy in cell autonomous defense against Mtb nor membrane repair of the MCV in human macrophages. However, the combined dynamics of LC3 recruitment and Lysoview staining emerged as promising markers for future research focused on directly investigating the damage and repair processes of phagosomal membranes.
CMA-ES-Rosetta: Blackbox optimization algorithm traverses rugged peptide docking energy landscapes
Authors: Claussen Erin R. , Renfrew P. Douglas , Müller Christian L. , Drew Kevin 
Published: December 2022

AbstractEnergy minimization is necessary for virtually all modeling and design tasks and involves traversing extremely rugged energy landscapes. Although the gradient descent based minimization routines in Rosetta have fast runtimes, due to these rugged landscapes, minimization often converges into high-energy local minima. Alternative numerical optimization techniques, such as evolution strategies, are more robust to rugged landscapes and have been shown to be highly successful on a diverse set of problems. Here we explore the Covariance Matrix Adaptation Evolution Strategy (CMA-ES), a state-of-the-art derivative-free optimization algorithm, as a complementary approach to the default minimizer in Rosetta. We used a benchmark of 26 peptides, from the FlexPepDock Benchmark, to assess the performance of three algorithms in Rosetta, specifically, CMA-ES, Rosetta’s default minimizer, and a Monte Carlo protocol of small backbone perturbations. We test the algorithms’ performance on their ability to dock an idealized peptide to a series of hotspots residues (i.e. constraints) along a native peptide. Of the three methods, CMA-ES was able to find the lowest energy conformation for 23 out of 26 benchmark peptides. The application of CMA-ES allows for an alternative optimization method for macromolecular modeling problems with rough energy landscapes.
Transcriptome guided metabolic network analysis reveals rearrangements of carbon flux distribution inNeisseria gonorrhoeaeduring neutrophil co-culture
Authors: Potter Aimee D. , Baiocco Christopher M. , Papin Jason A. , Criss Alison K. 
Published: December 2022

AbstractThe ability of bacterial pathogens to metabolically adapt to the environmental conditions of their hosts is critical to both colonization and invasive disease. Infection withNeisseria gonorrhoeae(the gonococcus, Gc) is characterized by the influx of neutrophils (PMNs), which fail to clear the bacteria and make antimicrobial products that can exacerbate tissue damage. The inability of the human host to clear Gc infection is particularly concerning in light of the emergence of strains that are resistant to all clinically recommended antibiotics. Bacterial metabolism represents a promising target for the development of new therapeutics against Gc. Here, we generated a curated genome-scale metabolic network reconstruction (GENRE) of Gc strain FA1090. This GENRE links genetic information to metabolic phenotypes and predicts Gc biomass synthesis and energy consumption. We validated this model with published data and in new results reported here. Contextualization of this model using the transcriptional profile of Gc exposed to PMNs revealed substantial rearrangements of Gc central metabolism and induction of Gc nutrient acquisition strategies for alternate carbon source use. These features enhanced the growth of Gc in the presence of neutrophils. From these results we conclude that the metabolic interplay between Gc and PMNs helps define infection outcomes. The use of transcriptional profiling and metabolic modeling to reveal new mechanisms by which Gc persists in the presence of PMNs uncovers unique aspects of metabolism in this fastidious bacterium, which could be targeted to block infection and thereby reduce the burden of gonorrhea in the human population.ImportanceThe World Health Organization (WHO) designatedNeisseria gonorrhoeae(Gc) as a high priority pathogen for research and development of new antimicrobials. Bacterial metabolism is a promising target for new antimicrobials, as metabolic enzymes are widely conserved among bacterial strains and are critical for nutrient acquisition and survival within the human host. Here we used genome-scale metabolic modeling to characterize the core metabolic pathways of this fastidious bacterium, and to uncover the pathways used by Gc during culture with primary human immune cells. These analyses revealed that Gc relies on different metabolic pathways during co-culture with human neutrophils than in rich media. Conditionally essential genes emerging from these analyses were validated experimentally. These results show that metabolic adaptation in the context of innate immunity is important to Gc pathogenesis. Identifying the metabolic pathways used by Gc during infection can highlight new therapeutic targets for drug-resistant gonorrhea.