bluuebunny/crossref_metadata_2025 huggingface dataset:
Transport mechanism of DgoT, a bacterial homolog of SLC17 organic anion transporters
Authors: Dmitrieva Nataliia , Gholami Samira , Alleva Claudia , Carloni Paolo , Alfonso-Prieto Mercedes , Fahlke Christoph
Published: February 2024

The solute carrier 17 (SLC17) family contains anion transporters that accumulate neuro-transmitters in secretory vesicles, remove carboxylated monosaccharides from lysosomes, or extrude organic anions from the kidneys and the liver. We combined classical molecular dynamics simulations, Markov state modeling and hybrid first principles quantum mechani-cal/classical mechanical (QM/MM) simulations with experimental approaches to describe the transport mechanisms of a model bacterial protein, the D-galactonate transporter DgoT, at atomic resolution. We found that protonation of D46 and E133 precedes galactonate binding and that substrate binding induces closure of the extracellular gate, with the conserved R47 coupling substrate binding to transmembrane helix movement. After isomerization to an inward-facing conformation, deprotonation of E133 and subsequent proton transfer from D46 to E133 opens the intracellular gate and permits galactonate dissociation either in its unprotonated form or after proton transfer from E133. After release of the second proton, apo DgoT returns to the outward-facing conformation. Our results provide a framework to understand how various SLC17 transport functions with distinct transport stoichiometries can be attained through subtle variations in proton and substrate binding/unbinding.
Developmental disruption of the mitochondrial fission genedrp-1extends the longevity ofdaf-2insulin/IGF-1 receptor mutant
Authors: Traa Annika , Van Raamsdonk Jeremy M.
Published: February 2024

The dynamic nature of the mitochondrial network is regulated by mitochondrial fission and fusion, allowing for re-organization of mitochondria to adapt to the cell’s ever-changing needs. As organisms age, mitochondrial fission and fusion become dysregulated and mitochondrial networks become increasingly fragmented. Modulation of mitochondrial dynamics has been shown to affect longevity in fungi, yeast,DrosophilaandC. elegans. While disruption of the mitochondrial fission genedrp-1only mildly increases wild-type lifespan, it drastically increases the already long lifespan ofdaf-2insulin/IGF-1 signaling (IIS) mutants. In this work, we determined the conditions required fordrp-1disruption to extenddaf-2longevity and explored the molecular mechanisms involved. We found that knockdown ofdrp-1during development is sufficient to extenddaf-2lifespan, while tissue-specific knockdown ofdrp-1in neurons, intestine or muscle failed to increasedaf-2longevity. Disruption of other genes involved in mitochondrial fission also increaseddaf-2lifespan as did treatment with a number of different RNAi clones that decrease mitochondrial fragmentation. In exploring potential mechanisms involved, we found that deletion ofdrp-1increases resistance to chronic stresses and slows physiologic rates indaf-2worms. In addition, we found that disruption ofdrp-1increased mitochondrial and peroxisomal connectedness indaf-2worms, increased oxidative phosphorylation and ATP levels, and increased mitophagy indaf-2worms, but did not affect their ROS levels or mitochondrial membrane potential. Overall, this work defined the conditions under whichdrp-1disruption increasesdaf-2lifespan and has identified multiple changes indaf-2;drp-1mutants that may contribute to their lifespan extension.
Dimensionality reduction distills complex evolutionary relationships in seasonal influenza and SARS-CoV-2
Authors: Nanduri Sravani , Black Allison , Bedford Trevor , Huddleston John
Published: February 2024

Public health researchers and practitioners commonly infer phylogenies from viral genome sequences to understand transmission dynamics and identify clusters of genetically-related samples. However, viruses that reassort or recombine violate phylogenetic assumptions and require more sophisticated methods. Even when phylogenies are appropriate, they can be unnecessary or difficult to interpret without specialty knowledge. For example, pairwise distances between sequences can be enough to identify clusters of related samples or assign new samples to existing phylogenetic clusters. In this work, we tested whether dimensionality reduction methods could capture known genetic groups within two human pathogenic viruses that cause substantial human morbidity and mortality and frequently reassort or recombine, respectively: seasonal influenza A/H3N2 and SARS-CoV-2. We applied principal component analysis (PCA), multidimensional scaling (MDS), t-distributed stochastic neighbor embedding (t-SNE), and uniform manifold approximation and projection (UMAP) to sequences with well-defined phylogenetic clades and either reassortment (H3N2) or recombination (SARS-CoV-2). For each low-dimensional embedding of sequences, we calculated the correlation between pairwise genetic and Euclidean distances in the embedding and applied a hierarchical clustering method to identify clusters in the embedding. We measured the accuracy of clusters compared to previously defined phylogenetic clades, reassortment clusters, or recombinant lineages. We found that MDS embeddings accurately represented pairwise genetic distances including the intermediate placement of recombinant SARS-CoV-2 lineages between parental lineages. Clusters from t-SNE embeddings accurately recapitulated known phylogenetic clades, H3N2 reassortment groups, and SARS-CoV-2 recombinant lineages. We show that simple statistical methods without a biological model can accurately represent known genetic relationships for relevant human pathogenic viruses. Our open source implementation of these methods for analysis of viral genome sequences can be easily applied when phylogenetic methods are either unnecessary or inappropriate.
Neurons enhance blood-brain barrier function via upregulating claudin-5 and VE-cadherin expression due to GDNF secretion
Authors: Yang Lu , Lin Zijin , Mu Ruijing , Wu Wenhan , Zhi Hao , Liu Xiaodong , Yang Hanyu , Liu Li
Published: February 2024

Blood-brain barrier (BBB) prevents neurotoxins from entering central nervous system. We aimed to establish and characterize an in vitro triple co-culture BBB model consisting of brain endothelial cells hCMEC/D3, astrocytoma U251 cells, and neuroblastoma SH-SY5Y cells. Co-culture of SH-SY5Y and U251 cells markedly enhanced claudin-5 and VE-cadherin expression in hCMEC/D3 cells, accompanied by increased transendothelial electrical resistance and decreased permeability. Conditioned medium (CM) from SH-SY5Y cells (S-CM), U251 cells (U-CM), and co-culture of SH-SY5Y and U251 cells (US-CM) also promoted claudin-5 and VE-cadherin expression. Glial cell line-derived neurotrophic factor (GDNF) levels in S-CM and US-CM were significantly higher than CMs from hCMEC/D3 and U-CM. Both GDNF and US-CM upregulated claudin-5 and VE-cadherin expression, which were attenuated by anti-GDNF antibody and GDNF signaling inhibitors. GDNF increased claudin-5 expression via the PI3K/AKT/FOXO1 and MAPK/ERK pathways. Meanwhile, GDNF promoted VE-cadherin expression by activating PI3K/AKT/ETS1 and MAPK/ERK/ETS1 signaling. The roles of GDNF in BBB integrity were validated using brain-specific Gdnf silencing mice. The developed triple co-culture BBB model was successfully applied to predict BBB permeability. In conclusion, neurons enhance BBB integrity by upregulating claudin-5 and VE-cadherin expression through GDNF secretion and established triple co-culture BBB model may be used to predict drugs's BBB permeability.
Full 16S and 23S rRNA gene-based,strain-level resolution of the microbiota of a mock bacterial microbiome using ONT nanopore sequencing
Authors: Woodruff Christopher J. , Zhang Zhengming , Speed Terence P.
Published: February 2024

Purpose

The feasibility of achieving near-strain taxonomic resolution and reliable microbiota strain-level abundance estimation using amplicon-based nanopore sequencing was investigated.

Methods

Denoising was applied to separate 16S and 23S genes extracted from a metagenomic dataset generated by Sereika et al. [9] using nanopore sequencing. Denoising used Kumar et al.’s [8] Robust Amplicon Denoising (RAD) and generated amplicon sequence variants (ASVs). The Sereika et al. data set was generated from the Zymo [10] D6322 7-bacterial species mock microbiome. A sub-sampling procedure generated additional datasets that allowed sensitivity assessment over multiple orders of relative abundance. Alignment to bespoke 16S and 23S rRNA gene databases, provided both identification and abundance information data for sub-species analyses.

Results

Sub-species identification was clearly achieved, both with the “even” D6322 dataset, and with the 4 sub-sampled datasets in which 3-orders of magnitude relative abundances were present. Multiple ASVs of length approximately 2500 bases were generated which gave perfect alignments to reference 23S rRNA genes. Similarly for the approximately 1500 base long 16S rRNA genes. Despite strain ambiguity for some species, the strain of each species known to be present was identified in all but 1 (of 70) cases examined for all species. A process to merge the 16S and 23S rRNA genes results reduced ambiguity, allowing better sub-species resolution, and better species, and sub-species, relative cellular abundance estimates.

Conclusion

Principled methods for dealing with strain-level ambiguity in microbiota analysis have been developed that are generally applicable. Their application to nanopore sequencing with the current state of Oxford Nanopore Technology, together with state-of-the-art denoising, allows near strain resolution of bacterial microbiota identity, and improved estimates of species, and sub-species, relative abundances.
Predicting COVID-19 booster immunogenicity against future SARS-CoV-2 variants and the benefits of vaccine updates
Authors: Cromer Deborah , Reynaldi Arnold , Hie Ainslie , Schlub Timothy E. , Juno Jennifer A. , Wheatley Adam K. , Kent Stephen J. , Khoury David S. , Davenport Miles P.
Published: February 2024

The ongoing evolution of the SARS-CoV-2 virus has led to a move to update vaccine antigens in 2022 and 2023. These updated antigens were chosen and approved based on in vitro neutralisation titres against recent SARS-CoV-2 variants. However, unavoidable delays in viral manufacture and distribution meant that the updated booster vaccine was no longer well matched to the circulating SARS-CoV-2 variant by the time of its deployment. Understanding whether the updating of booster vaccine antigens improves immune responses to subsequent SARS-CoV-2 circulating variants is a major priority in justifying future vaccine updates. Here we analyse all available data on the immunogenicity of variant containing SARS-CoV-2 vaccines and their ability to neutralise later circulating SARS-CoV-2 variants. We find that updated booster antigens give a 1.4-fold [95%CI 1.07-1.82] greater increase in neutralising antibody levels when compared with a historical vaccine immunogen. We then use this to predict the relative protection that can be expected from an updated vaccine even when the circulating variant has evolved away from the updated vaccine immunogen. These findings help inform the roll out of future booster vaccination programs.
Couple-Centered HIV Prevention and Care: Endorsement, Practice and Uncertainty among US Healthcare Providers in Western-Central Upstate New York
Authors: Leblanc Natalie M. , Muheriwa-Matemba Sadandaula R. , Vil Noelle St. , Alcena-Stiner Danielle , Bond Keosha T. , Glazier Alexander , Rosario-McCabe Luis , Lambert Faith , Smith Martez
Published: February 2024

Given the influence of provider perspective and practice in the uptake of HIV/STI prevention and care strategies, this qualitative description design study sought to illuminate perspectives of couples HIV testing and counseling (CHTC), and describe couple/partner-based practices among health providers in New York State. We utilized a purposive sampling strategy to recruit health providers (N = 27). Semi-structured in-depth interviews were conducted from Sept. 2019 to Feb. 2021. Four themes emerged: perspectives on engaging partners and couples-centered sexual health promotion; providers’ experiences with patients and partners in HIV prevention and care; provider endorsement of CHTC; and perceived CHTC implementation determinants. CHTC endorsement was prominently due to the perception of CHTC as a facilitator to enhance patient-provider engagement in HIV/STI treatment and care, especially in the communication and dissemination of information among partners. Providers reported that health literacy needs regarding HIV/STI testing and diagnosis, but primarily STIs treatment regimens warranted a joint approach. CHTC endorsement entailed the strategy’s perceived ability to enhance sexual health literacy among patients and patient’s partners. Lastly, CHTC endorsements entailed provider beliefs that it ensured knowledge equity and joint literacy in the communication of health information among health consumers. Determinants of CHTC implementation were factors that providers perceived to have a bearing on the facilitation or posed as barriers to jointly engaging partners in HIV/STI prevention and care and was subsequently a source of provider uncertainty. These determinants ranged from provider-level factors to organizational capacity issues that could impact CHTC implementation. Recommendations for CHTC are discussed.
STREAMLINED PROTEOME-WIDE IDENTIFICATION OF DRUG TARGETS INDICATES ORGAN-SPECIFIC ENGAGEMENT
Authors: Batth Tanveer Singh , Locard-Paulet Marie , Doncheva Nadezhda T. , Mendez Blanca Lopez , Jensen Lars Juhl , Olsen Jesper Velgaard
Published: February 2024

Proteins are the primary targets of almost all small molecule drugs. However, even the most selectively designed drugs can potentially target several unknown proteins. Identification of potential drug targets can facilitate design of new drugs and repurposing of existing ones. Current state-of-the-art proteomics methodologies enable screening of thousands of proteins against a limited number of drug molecules. Here we report the development of a label-free quantitative proteomics approach that enables proteome-wide screening of small organic molecules in a scalable, reproducible, and rapid manner by streamlining the proteome integral solubility alteration (PISA) assay. We used rat organsex-vivoto determine organ specific targets of medical drugs and enzyme inhibitors to identify novel drug targets for common drugs such as Ibuprofen. Finally, global drug profiling revealed overarching trends of how small molecules affect the proteome through either direct or indirect protein interactions.
Systematic benchmarking of deep-learning methods for tertiary RNA structure prediction
Authors: Bahai Akash , Kwoh Chee Keong , Mu Yuguang , Li Yinghui
Published: February 2024

The 3D structure of RNA critically influences its functionality, and understanding this structure is vital for deciphering RNA biology. Experimental methods for determining RNA structures are labour-intensive, expensive, and time-consuming. Computational approaches have emerged as valuable tools, leveraging physics-based-principles and machine learning to predict RNA structures rapidly. Despite advancements, the accuracy of computational methods remains modest, especially when compared to protein structure prediction. Deep learning methods, while successful in protein structure prediction, have shown some promise for RNA structure prediction as well but face unique challenges. This study systematically benchmarks state-of-the-art deep learning methods for RNA structure prediction across diverse datasets. Our aim is to identify factors influencing performance variation, such as RNA family diversity, sequence length, RNA type, multiple sequence alignment (MSA) quality, and deep learning model architecture. We show that generally ML-based methods perform much better than non-ML methods on most RNA targets, although the performance difference isn’t substantial when working with unseen novel or synthetic RNAs. The quality of the MSA and secondary structure prediction both play an important role and most methods aren’t able to predict non-Watson-Crick pairs in the RNAs. Overall, DeepFoldRNA has the best prediction followed by DRFold as the second best method. Finally, we also suggest possible mitigations to improve the quality of the prediction for future method development.
Microglia-astrocyte crosstalk regulates synapse remodeling via Wnt signaling
Authors: Faust Travis E. , Lee Yi-Han , O’Connor Ciara D. , Boyle Margaret A. , Gunner Georgia , Badimon Ana , Ayata Pinar , Schaefer Anne , Schafer Dorothy P.
Published: February 2024

Astrocytes and microglia are emerging key regulators of activity-dependent synapse remodeling that engulf and remove synapses in response to changes in neural activity. Yet, the degree to which these cells communicate to coordinate this process remains an open question. Here, we use whisker removal in postnatal mice to induce activity-dependent synapse removal in the barrel cortex. We show that astrocytes do not engulf synapses in this paradigm. Instead, astrocytes reduce their contact with synapses prior to microglia-mediated synapse engulfment. We further show that reduced astrocyte-contact with synapses is dependent on microglial CX3CL1-CX3CR1 signaling and release of Wnts from microglia following whisker removal. These results demonstrate an activity-dependent mechanism by which microglia instruct astrocyte-synapse interactions, which then provides a permissive environment for microglia to remove synapses. We further show that this mechanism is critical to remodel synapses in a changing sensory environment and this signaling is upregulated in several disease contexts.
Development of an improved blood-stage malaria vaccine targeting the essential RH5-CyRPA-RIPR invasion complex
Authors: Williams Barnabas G. , King Lloyd D. W. , Pulido David , Quinkert Doris , Lias Amelia M. , Silk Sarah E. , Ragotte Robert J. , Davies Hannah , Barrett Jordan R. , McHugh Kirsty , Rigby Cassandra A. , Alanine Daniel G. W. , Barfod Lea , Shea Michael W. , Cowley Li An , Dabbs Rebecca A. , Pattinson David J. , Douglas Alexander D. , Lyth Oliver R. , Illingworth Joseph J. , Jin Jing , Carnrot Cecilia , Kotraiah Vinayaka , Christen Jayne M. , Noe Amy R. , MacGill Randall S. , King C. Richter , Birkett Ashley J. , Soisson Lorraine A. , Skinner Katherine , Miura Kazutoyo , Long Carole A. , Higgins Matthew K. , Draper Simon J.
Published: February 2024

In recent years, reticulocyte-binding protein homologue 5 (RH5) has emerged as a leading blood-stagePlasmodium falciparummalaria vaccine antigen. The most advanced blood-stage vaccine candidate in a Phase 2b clinical trial, RH5.1/Matrix-M™, is based on a full-length soluble protein-with-adjuvant formulation. RH5 interacts with cysteine-rich protective antigen (CyRPA) and RH5- interacting protein (RIPR) to form an essential heterotrimeric “RCR-complex”. Here, we investigated whether a vaccine candidate based on the ternary RCR-complex could substantially improve upon the leading clinical candidate RH5.1/Matrix-M™ in preclinical studies. Using a panel of monoclonal antibodies (mAbs) we confirm that parasite growth-inhibitory epitopes on each antigen are exposed on the surface of the RCR-complex and that mAb pairs binding to different antigens can function additively or synergistically to mediate parasite growth inhibition activity (GIA)in vitro. However, immunisation of rats with the RCR-complex consistently fails to outperform RH5.1 alone. We show this is due to immuno-dominance of RIPR coupled with the inferior potency of anti-full length RIPR polyclonal IgG antibodies as compared to the anti-RH5 and anti-CyRPA response. To address this, we identified the growth-inhibitory antibody epitopes of RIPR are clustered within C-terminal EGF-like domains of RIPR. A fusion of these EGF domains to CyRPA, called “R78C”, combined with RH5.1, provided a new vaccination strategy that improves upon the levels ofin vitroGIA seen with RH5.1 alone. Superiority of the combination antigen vaccine candidate was achieved by the induction of a quantitatively higher, but qualitatively similar, polyclonal antibody response that demonstrated additive GIA across the three antigen targets. These preclinical data justified the advancement of the RH5.1+R78C/Matrix-M™ combination vaccine to a Phase 1 clinical trial.
Microbial Electrosynthesis from CO2reaches Productivity of Syngas and Chain Elongation Fermentations
Authors: Cabau-Peinado Oriol , Winkelhorst Marijn , Stroek Rozanne , de Kat Angelino Roderick , Straathof Adrie J.J. , Masania Kunal , Daran Jean Marc , Jourdin Ludovic
Published: February 2024

Microbial electrosynthesis allows the electrochemical upgrading of CO2. However, higher productivities and energy efficiencies are needed to reach a viability that can make the technology transformative. Here we show how a biofilm-based microbial porous cathode in a directed flow-through electrochemical system can continuously reduce CO2to even-chain C2-C6 carboxylic acids during 248 days. We demonstrate a 3-fold higher biofilm concentration, volumetric current density, and productivity than the state of the art, up to a new record of -35 kA m-3cathodeand 69 kgCm-3cathodeday-1, at 60-97% and 30-35% faradaic and energy efficiencies, respectively. Most notably, the volumetric productivity resembles those achieved in lab-scale and industrial syngas (CO-H2-CO2) fermentation and chain elongation fermentation. This work highlights key design parameters for efficient electricity-driven microbial CO2reduction. There is need and room to improve the rates of electrode colonization and microbe-specific kinetics to scale-up the technology.

Graphical abstract
Systematic annotation ofHelitron-like elements in eukaryote genomes using HELIANO
Authors: Li Zhen , Gilbert Clément , Peng Haoran , Pollet Nicolas
Published: February 2024

Helitron-like elements (HLEs) are widespread eukaryotic DNA transposons employing a rolling-circle transposition mechanism. Despite their prevalence in fungi, animals, and plant genomes, identifyingHelitronsremains challenging. We introduce HELIANO, a software for annotating and classifying autonomous and non-autonomousHelitronandHelentronsequences from whole genomes. HELIANO outperforms existing tools in speed and accuracy, demonstrated through benchmarking and its application to complex genomes (Xenopus tropicalis, Xenopus laevis, Oryza sativa), revealing numerous newly identifiedHelitronsandHelentrons.

In a comprehensive analysis of 404 eukaryote genomes, we found HLEs widely distributed across phyla, with exceptions in specific taxa.Helentronswere identified in numerous land plant species, and 20 protein domains were discovered integrated within specific autonomous HLE families. A global phylogenetic analysis confirmed the classification into main cladesHelentronandHelitron, revealing nine subgroups, some enriched in particular taxa. The future use of HELIANO will contribute to the global analysis of TEs across genomes and enhance our understanding of this transposon superfamily.
Mapping Serotonergic Dynamics using Drug-Modulated Molecular Connectivity
Authors: Ionescu TM , Amend M , Hafiz R , Maurer A , Biswal BB , Wehrl HF , Herfert K
Published: February 2024

Understanding the complex workings of the brain is one of the most significant challenges in neuroscience, providing insights into normal brain function, neurological diseases, and the effects of potential therapeutics. A major challenge in this field lies in the limitations of traditional brain imaging techniques, which often capture only fragments of the complex puzzle of brain function. Our research employs a novel approach termed “molecular connectivity” (MC), which combines the strengths of various imaging methods to provide a comprehensive view of how specific molecules, such as the serotonin transporter, interact across different brain regions and influence brain function.

This innovative technique bridges the gap between functional magnetic resonance imaging (fMRI), known for its ability to monitor brain activity by tracking blood flow, and positron emission tomography (PET), which visualizes specific molecular changes. By integrating these methods, we can better understand how drugs influence brain function. Our study focuses on the application of dynamic [11C]DASB PET scans to map the distribution of serotonin transporters, key players in regulating mood and emotions and examines how these transporters are altered following exposure to methylenedioxymethamphetamine (MDMA), which is commonly known as ecstasy.

Through a detailed comparison of MCs with traditional measures of brain connectivity, we reveal significant patterns that closely align with physiological changes. Our results revealed clear changes in molecular connectivity after a single dose of MDMA, establishing a direct link between the effects of drugs on serotonin transporter occupancy and changes in the functional brain network.

This work offers a novel methodology for the in-depth study of brain function at the molecular level and opens new pathways for understanding how drugs modulate brain activity.
Unfolded Von Willebrand Factor Binds Protein S and Reduces Anticoagulant Activity
Authors: Sim Martha M.S. , Mollica Molly Y. , Alfar Hammodah R. , Hollifield Melissa , Chung Dominic W. , Fu Xiaoyun , Gandhapudi Siva , Coenen Daniëlle M. , Prakhya Kanakanagavalli Shravani , Mahmood Dlovan F. D , Banerjee Meenakshi , Peng Chi , Li Xian , Thornton Alice C. , Porterfield James Z. , Sturgill Jamie L. , Sievert Gail A. , Barton-Baxter Marietta , Zheng Ze , Campbell Kenneth S. , Woodward Jerold G. , López José A. , Whiteheart Sidney W. , Garvy Beth A. , Wood Jeremy P.
Published: February 2024

Protein S (PS), the critical plasma cofactor for the anticoagulants tissue factor (TF) pathway inhibitor (TFPI) and activated protein C (APC), circulates in two functionally distinct pools: free (anticoagulant) or bound to complement component 4b-binding protein (C4BP) (anti-inflammatory). Acquired free PS deficiency is detected in several viral infections, but its cause is unclear. Here, we identified a shear-dependent interaction between PS and von Willebrand Factor (VWF) by mass spectrometry. Consistently, plasma PS and VWF comigrated in both native and agarose gel electrophoresis. The PS/VWF interaction was blocked by TFPI but not APC, suggesting an interaction with the C-terminal sex hormone binding globulin (SHBG) region of PS. Microfluidic systems, mimicking arterial laminar flow or disrupted turbulent flow, demonstrated that PS stably binds VWF as VWF unfolds under turbulent flow. PS/VWF complexes also localized to platelet thrombi under laminar arterial flow. In thrombin generation-based assays, shearing plasma decreased PS activity, an effect not seen in the absence of VWF. Finally, free PS deficiency in COVID-19 patients, measured using an antibody that binds near the C4BP binding site in SHBG, correlated with changes in VWF, but not C4BP, and with thrombin generation. Our data suggest that PS binds to a shear-exposed site on VWF, thus sequestering free PS and decreasing its anticoagulant activity, which would account for the increased thrombin generation potential. As many viral infections present with free PS deficiency, elevated circulating VWF, and increased vascular shear, we propose that the PS/VWF interaction reported here is a likely contributor to virus-associated thrombotic risk.

Key Points

Von Willebrand Factor (VWF) binds Protein S (PS) in a shear-dependent manner, reducing the free PS pool and its anticoagulant activity.

The PS/VWF complex forms under turbulent flow conditions, is stable in whole blood, and localizes to growing platelet thrombi.
Evaluating Bacterial Viability in Faecal Microbiota Transplantation: A Comparative Analysis of In Vitro Cultivation and Membrane Integrity Methods
Authors: Cibulková Ivana , Řehořová Veronika , Wilhelm Marek , Soukupová Hana , Hajer Jan , Duška František , Daňková Helena , Cahová Monika
Published: February 2024

Background

Faecal microbiota transplantation (FMT) is a developing therapy for disorders related to gut dysbiosis. Despite its growing application, standardized protocols for FMT filtrate preparation and quality assessment remain undeveloped. The viability of bacteria in the filtrate is crucial for FMT’s efficacy and for validating protocol execution. We compared two methods—in vitro cultivation and membrane integrity assessment—for their accuracy, reproducibility, and clinical applicability in measuring bacterial viability in frozen FMT stool filtrate.

Methods

Bacterial viability in stool filtrate was evaluated using (i) membrane integrity through fluorescent DNA staining with SYTO9 and propidium iodide, followed by flow cytometry; and (ii) culturable bacteria counts (colony-forming units, CFU) under aerobic or anaerobic conditions.

Results

We refined the bacterial DNA staining protocol integrated with flow cytometry for stool samples. Both the membrane integrity-based and cultivation-based methods exhibited significant variability in bacterial viability across different FMT filtrates, without correlation. The cultivation-based method showed a mean coefficient of variance of 17%, ranging from 5.3% to 52.9%. Conversely, the membrane integrity approach yielded highly reproducible results, with a median coefficient of variance for viable cells of 0.9%, ranging from 8.5% to 0.04%.

Conclusion

Bacterial viability assessment using cultivation-dependent methods produces inconsistent outcomes. In contrast, the membrane integrity method offers robust and precise data, making it a viable option for routine faecal material evaluation in FMT.
Diversity ofVibrio choleraeO1 through the human gastrointestinal tract during cholera
Authors: Lypaczewski Patrick , Chac Denise , Dunmire Chelsea N. , Tandoc Kristine M. , Chowdhury Fahima , Khan Ashraful I. , Bhuiyan Taufiqur , Harris Jason B. , LaRocque Regina C. , Calderwood Stephen B. , Ryan Edward T. , Qadri Firdausi , Shapiro B. Jesse , Weil Ana A.
Published: February 2024

Vibrio choleraeO1 causes the diarrheal disease cholera, and the small intestine is the site of active infection. During cholera, cholera toxin is secreted fromV. choleraeand induces a massive fluid influx into the small intestine, which causes vomiting and diarrhea. Typically,V. choleraegenomes are sequenced from bacteria passed in stool, but rarely from vomit, a fluid that may more closely represents the site of active infection. We hypothesized that theV. choleraeO1 population bottlenecks along the gastrointestinal tract would result in reduced genetic variation in stool compared to vomit. To test this, we sequencedV. choleraegenomes from ten cholera patients with paired vomit and stool samples. Genetic diversity was low in both vomit and stool, consistent with a single infecting population rather than co-infection with divergentV. choleraeO1 lineages. The number of single nucleotide variants decreased between vomit and stool in four patients, increased in two, and remained unchanged in four. The number of genes encoded in theV. choleraegenome decreased between vomit and stool in eight patients and increased in two. Pangenome analysis of assembled short-read sequencing demonstrated that the toxin-coregulated pilus operon more frequently contained deletions in genomes from vomit compared to stool. However, these deletions were not detected by PCR or long-read sequencing, indicating that interpreting gene presence or absence patterns from short-read data alone may be incomplete. Overall, we found thatV. choleraeO1 isolated from stool is genetically similar toV. choleraerecovered from the upper intestinal tract.

IMPORTANCE

Vibrio choleraeO1, the bacterium that causes cholera, is ingested in contaminated food or water and then colonizes the upper small intestine and is excreted in stool. ShedV. choleraegenomes are usually studied, butV. choleraeisolated from vomit may be more representative of whereV. choleraecolonizes in the upper intestinal epithelium.V. choleraemay experience bottlenecks, or large reductions in bacterial population sizes or genetic diversity, as it passes through the gut. Passage through the gut may select for distinctV. choleraemutants that are adapted for survival and gut colonization. We did not find strong evidence for such adaptive mutations, and instead observed that passage through the gut results in modest reductions inV. choleraegenetic diversity, and only in some patients. These results fill a gap in our understanding of theV. choleraelife cycle, transmission, and evolution.
A tension-induced morphological transition shapes the avian extra-embryonic territory
Authors: Michaut Arthur , Chamolly Alexander , Villedieu Aurélien , Corson Francis , Gros Jérôme
Published: February 2024

The segregation of the extra-embryonic lineage is one of the earliest events and a key step in amniote development. Whereas the regulation of extra-embryonic cell fate specification has been extensively studied, little is known about the morphogenetic events underlying the formation of this lineage. Here, taking advantage of the amenability of avian embryos to live and quantitative imaging, we investigate the cell- and tissue-scale dynamics of epiboly, the process during which the epiblast expands to engulf the entire yolk. We show that tension arising from the outward migration of the epiblast border on the vitelline membrane stretches extra-embryonic cells, which reversibly transition from a columnar to squamous morphology. The propagation of this tension is strongly attenuated in the embryonic territory, which concomitantly undergoes fluid-like motion, culminating in the formation of the primitive streak. We formulate a simple viscoelastic model in which the tissue responds elastically to isotropic stress but flows in response to shear stress, and show that it recapitulates the flows and deformation of both embryonic and extra-embryonic tissues. Together, our results clarify the mechanical basis of early avian embryogenesis and provide a framework unifying the divergent mechanical behaviors observed in the contiguous embryonic and extra-embryonic territories that make up the epiblast.

Highlights

The extra-embryonic region expands radially during epiboly

Cell area increase accounts for the rapid extra-embryonic expansion

Epiboly-induced tension reversibly stretches extra-embryonic cells

A simple viscoelastic model recovers the morphogenesis of the entire epiblast
Label free, capillary-scale blood flow mappingin vivoreveals that low intensity focused ultrasound evokes persistent dilation in cortical microvasculature
Authors: Shen YuBing , Jethe Jyoti V. , Reid Ashlan P. , Hehir Jacob , Amaral Marcello Magri , Ren Chao , Hao Senyue , Zhou Chao , Fisher Jonathan A. N.
Published: February 2024

Non-invasive, low intensity focused ultrasound (FUS) is an emerging neuromodulation technique that offers the potential for precision, personalized therapy. An increasing body of research has identified mechanosensitive ion channels that can be modulated by FUS and support acute electrical activity in neurons. However, neuromodulatory effects that persist from hours to days have also been reported. The brain’s ability to provide targeted blood flow to electrically active regions involve a multitude of non-neuronal cell types and signaling pathways in the cerebral vasculature; an open question is whether persistent effects can be attributed, at least partly, to vascular mechanisms. Using a novelin vivooptical approach, we found that microvascular responses, unlike larger vessels which prior investigations have explored, exhibit persistent dilation following sonication without the use of microbubbles. This finding and approach offers a heretofore unseen aspect of the effects of FUSin vivoand indicate that concurrent changes in neurovascular function may partially underly persistent neuromodulatory effects.
Genome-wide transcriptional silencing and mRNA stabilization allow the coordinated expression of the meiotic program in mice
Authors: Bellutti Laura , Pen Edith Chan Sock , Guerquin Marie-Justine , Messiaen Sébastien , Llano Elena , Cluzet Victoria , Dereli Ihsan , Rolland Antoine , Martini Emmanuelle , Tóth Attila , Pendás Alberto M. , Chalmel Frederic , Livera Gabriel
Published: February 2024

The transcriptional dynamic of mammalian cells when these transit from the ubiquitous mitotic to a meiotic specific program is key to understand this switch central to sexual reproduction. By quantifying active RNA polymerase II and nascent transcripts using single cell dataset and ethynyl-uridine pool-down with sorted cells from synchronised testes, we detailed the transcriptional activity of murine male germ cells. When spermatogonia differentiate, transcription slows down, reaching minimal activity at meiotic entry and resumes during pachytene stage. This event, we termed EMLT (for early meiotic low transcription), is distinct from the silencing of sex chromosomes as it is independent of Setdb1 though it is accompanied by the same chromatin mark, H3K9me3. EMLT is delayed in Stra8KO but occurs in mutants altering meiotic chromosome structure or DSB formation or repair. By comparing transcript abundance and neotranscription we unveil a massive event of mRNA stabilization that parallels EMLT. Altogether our data indicate that meiosis is initiated with a nearly silent genome and we propose that the stabilization of transcripts at that time facilitates the meiotic entry by synchronising the expression of several meiotic sub-programs.
BMP9 and BMP10 coordinate liver cellular crosstalk to maintain liver health
Authors: Zhao Dianyuan , Huang Ziwei , Li Xiaoyu , Wang Huan , Hou Qingwei , Wang Yuyao , Yan Fang , Yang Wenting , Liu Di , Yi Shaoqiong , Han Chunguang , Hao Yanan , Tang Li
Published: February 2024

The liver is the largest solid organ in the body and is primarily composed of HCs, ECs, KCs, and HSCs, which spatially interact and cooperate with each other to maintain liver homeostasis. However, the complexity and molecular mechanisms underlying the crosstalk between these different cell types remain to be revealed. Here, we generated mice with conditional deletion ofBmp9/10in different liver cell types and demonstrated that HSCs were the major source of BMP9 and BMP10 in the liver. Using transgenic ALK1 (receptor for BMP9/10) reporter mice, we found that ALK1 is expressed on KCs and ECs other than HCs and HSCs, and BMP9/10 secreted by HSCs promotes the differentiation of KCs and ECs and maintain their identity.Pdgfbexpression was significantly upregulated in KCs and ECs after BMP9 and BMP10 deletion, ultimately leading to HSCs activation and liver fibrosis. ECs express several angiocrine factors, such as BMP2, BMP6, Wnt2 and Rspo3, to regulate hepatocyte iron metabolism and metabolic zonation. We found that these angiocrine factors were significantly decreased in ECs fromBmp9/10HSC-KOmice, which further resulted in liver iron overload and disruption of HC zonation. In summary, we demonstrated that HSCs play a central role in mediating liver cell‒cell crosstalk via the production of BMP9/10, highlighting the important role of intercellular interaction in organ development and homeostasis.
Glial expression ofDrosophila UBE3Acauses spontaneous seizures modulated by 5-HT signaling
Authors: Sleep Megan , Landaverde Saul , Lacoste Andrew , Tan Selene , Schuback Reid , Reiter Lawrence T. , Iyengar Atulya
Published: February 2024

Misexpression of the E3 ubiquitin ligaseUBE3Ais thought to contribute to a range of neurological disorders. In the context of Dup15q syndrome, excess genomic copies ofUBE3Ais thought to contribute to the autism, muscle tone and spontaneous seizures characteristic of the disorder. In aDrosophilamodel of Dup 15q syndrome, it was recently shown glial-driven expression of theUBE3Aorthologdube3aled to a “bang-sensitive” phenotype, where mechanical shock triggers convulsions, suggesting glialdube3aexpression contributes to hyperexcitability in flies. Here we directly compare the consequences of glial- and neuronal-drivendube3aexpression on motor coordination and neuronal excitability in Drosophila. We utilized IowaFLI tracker and developed a hidden Markov Model to classify seizure-related immobilization. Both glial and neuronal drivendube3aexpression led to clear motor phenotypes. However, only glial-drivendube3aexpression displayed spontaneous immobilization events, that were exacerbated at high-temperature (38 °C). Using a tethered fly preparation we monitored flight muscle activity, we found glial-drivendube3aflies display spontaneous spike discharges which were bilaterally synchronized indicative of seizure activity. Neither control flies, nor neuronal-dube3aoverexpressing flies display such firing patterns. Prior drug screen indicated bang-sensitivity in glial-drivendube3aexpressing flies could be suppressed by certain 5-HT modulators. Consistent with this report, we found glial-drivendube3aflies fed the serotonin reuptake inhibitor vortioxetine and the 5HT2Aantagonist ketanserin displayed reduced immobilization and spike bursting. Together these findings highlight the potential for glial pathophysiology to drive Dup15q syndrome-related seizure activity.
Very low levels of physical activity among a broad group of patients hospitalized following hip fracture: A prospective cohort study (the HIP-ME-UP cohort study)
Authors: Hansen Maria Swennergren , Kristensen Morten Tange , Zilmer Camilla Kampp , Berger Anja L , Kirk Jeanette Wassar , Skibdal Kira Marie , Kallemose Thomas , Bandholm Thomas , Pedersen Mette Merete
Published: February 2024

Objectives: The evidence supports early and intensive mobilization and physical activity for hospitalized patients following a hip fracture. Since bedrest and inactivity during acute care are potentially fatal, we need updated knowledge of levels of physical activity in a diverse clinical population. Therefore, the objective was to determine levels of physical activity among a broad representation of patients hospitalized following hip fracture, and secondly to explore the association with 30-day post-discharge readmission, and mortality. Design: Prospective cohort study Setting: Data were collected at two university hospitals in the Capital Region of Denmark from March to June 2023. Participants: Patients hospitalized following hip fracture. Main outcome measures: 24-hr upright time (time standing and walking) was measured from inclusion (post-operative day (POD) 1-3) to discharge using a thigh-worn accelerometer. Readmission and mortality were verified by electronic patient records. Results: 101 patients (62 women) with a mean (SD) age of 79.9 (8.4) years were included. The median (IQR) 24-hr upright time on POD2-6 ranged from 15 (6.9:31.0) to a maximum of 34 (16:67) mins. Patients with cognitive impairment had less upright time than patients without. Post-surgery length of stay was a median of 7 (5:8) days. 25% of the patients were readmitted or had emergency ward referrals and 3% died within 30 days of discharge (no clear association with upright time). Conclusions: Physical activity seems extremely low among a broad representation of patients within the first week following a hip fracture but was not found to influence readmissions. Considering the strong evidence supporting physical activity during acute hospitalization, the low activity level in these patients calls for action. Clinicaltrials.gov-identifier:NCT05756517
Pre-vaccination carriage prevalence ofStreptococcus pneumoniaeserotypes among internally displaced people in Somaliland
Authors: van Zandvoort Kevin , Ibrahim Hassan Abdirahman , Bobe Mohamed , Pell Casey L. , Ahmed Mohammed Saed , Ortika Belinda D. , Ibrahim Saed , Abdi Mohamed Ismail , Karim Mustapha A , Eggo Rosalind M , Yusuf Sulieman , Hinds Jason , Soleman Saed Mohamood , Cummings Rachael , McGowan Catherine , Mulholland Kim , Hergeeye Mohamed Abdi , Satzke Catherine , Checchi Francesco , Flasche Stefan
Published: February 2024

Populations affected by humanitarian crises likely experience high burdens of pneumococcal disease. Streptococcus pneumoniae carriage estimates are essential to understand pneumococcal transmission dynamics and the potential impact of pneumococcal conjugate vaccines (PCV). Over 100 million people are forcibly displaced worldwide, yet here we present only the second pneumococcal carriage estimates for a displaced population.

In October 2019, we conducted a cross-sectional survey among internally displaced people (IDP) living in Digaale, a permanent IDP camp in Somaliland where PCV has not been implemented. We collected nasopharyngeal swab samples from 453 residents which were assessed for presence of pneumococci and serotyped using DNA microarray.

We found that pneumococcal carriage prevalence was 36% (95%CI 31 – 40) in all ages, and 70% (95%CI 64 – 76) in children under 5. The three most common serotypes were vaccine serotypes 6B, 19F, and 23. We estimated that the serotypes included in the 10-valent PNEUMOSIL vaccine were carried by 41% (95%CI 33 – 49) of all pneumococcal carriers and extrapolated that they caused 52% (95%CI 35 – 72) of invasive pneumococcal disease. We found some evidence that pneumococcal carriage was associated with recent respiratory symptoms, the total number of physical contacts made, and with malnutrition in children under 5. Through linking with a nested contact survey we projected that pneumococcal exposure of children under 2 was predominantly due to contact with children aged 2-5 (39%; 95%CI 32 – 48) and 6-14 (25%; 95%CI 18 – 33).

These findings suggest considerable potential for direct and indirect protection against pneumococcal disease in Digaale through PCV use in children and potentially adolescents.
Pion scattering, light resonances, and chiral symmetry restoration at nonzero chiral imbalance and temperature
Authors: Gómez Nicola Angel , Roa-Bravo Patricia , Vioque-Rodríguez Andrea
Published: February 2024

We calculate the pion scattering amplitude at nonzero temperature and nonzero μ5, the chemical potential associated to chiral imbalance in a locally P-breaking scenario. The amplitude is calculated up to next-to-leading order in Chiral Perturbation Theory and is unitarized with the Inverse Amplitude Method to generate the poles of the f0(500) and ρ(770) resonances. Within the saturation approach, the thermal f0(500) pole allows to determine Tc(μ5), the transition temperature for chiral symmetry restoration. Our results confirm the growing behavior of Tc(μ5) found in previous works and, through a fit to lattice results, we improve the uncertainty range of the low-energy constants associated to μ5 corrections in the chiral Lagrangian. The results for the ρ(770) pole are compatible with previous works regarding the dilepton yield in heavy-ion collisions.

Published by the American Physical Society
2024