bluuebunny/crossref_metadata_2025_split huggingface dataset:
An adapted method for Cas9-mediated editing reveals the species-specific role of β-glucoside utilization driving competition betweenKlebsiellaspecies
Authors: d. H. Almási Éva , Knischewski Nele , Osbelt Lisa , Muthukumarasamy Uthayakumar , Mouali Youssef El , Vialetto Elena , Beisel Chase L. , Strowig Till 
Published: September 2023

AbstractCas9-based gene editing tools have revolutionized genetics, enabling the fast and precise manipulation of diverse bacterial species. However, widely applicable genetic tools for non-model gut bacteria are unavailable. Here, we present a two-plasmid Cas9-based system designed for gene deletion and knock-in complementation in three members of theKlebsiella oxytocaspecies complex (KoSC), which we applied to study the genetic factors underlying the role of these bacteria in competition againstKlebsiella pneumoniae. The system allowed efficient and precise editing via enhanced lambda Red expression and functionally-validated complementation with the use of universal ‘bookmark’ targets inK. oxytoca, Klebsiella michiganensis, andKlebsiella grimontii. We revealed that the carbohydrate permease CasA is critical inex vivoassays forK. pneumoniaeinhibition byK. oxytocabut is neither sufficient nor required forK. michiganensisandK. grimontii. Thus, the adaptation of state-of-the-art genetic tools to KoSC allows the identification of species-specific functions in microbial competition.* ImportanceCas9-based gene editing tools have revolutionized bacterial genetics, yet, their application to non-model gut bacteria is frequently hampered by various limitations. We utilized a two-plasmid Cas9-based system designed for gene deletion inKlebsiella pneumoniaeand demonstrate after optimization its utility for gene editing in three members of theKlebsiella oxytocaspecies complex (KoSC) namelyK. oxytoca, K. michiganensisandK. grimontii. We then adapted a recently developed protocol for functional complementation based on universal ‘bookmark’ targets applicable to all tested species. In summary, species specific adaptation of state-of-the-art genetic tools allow efficient gene deletion and complementation in types strains as well as natural isolates of KoSC members to study microbial interactions.
Int&in: a machine learning-based web server for split site identification in inteins
Authors: Schmitz Mirko , Ballestin Jara Ballestin , Liang Junsheng , Tomas Franziska , Freist Leon , Voigt Karsten , Ventura Barbara Di , Öztürk Mehmet Ali 
Published: September 2023

AbstractInteins are proteins that excise themselves out of host proteins and ligate the flanking polypeptides in an auto-catalytic process called protein splicing. They are gaining momentum in synthetic biology for their ability to post-translationally modify proteins of interest. In nature, inteins are either contiguous or split, in which case the two intein fragments must first form a complex for the splicing to occur. So far, heuristic methods have been employed whenever a new split site in an intein had to be identified. To make the process of split site identification in inteins faster, easier and less costly, we developed Int&in, a web server that uses a gaussian Naïve Bayes machine learning model to predict active and inactive split sites with high accuracy. The model was trained on a data set generated by us and validated using a large diverse data set from the literature, resulting in an accuracy of 0.76. Int&in will facilitate the engineering of novel split inteins for applications in biotechnology and synthetic biology.
The interaction between NLRP1 and oxidized TRX1 involves a transient disulfide bond
Authors: Geeson Michael B. , Hsiao Jeffrey C. , Tsamouri Lydia P. , Ball Daniel P. , Bachovchin Daniel A. 
Published: September 2023

SUMMARYNLRP1 is an innate immune receptor that detects pathogen-associated signals, assembles into a multiprotein structure called an inflammasome, and triggers a proinflammatory form of cell death called pyroptosis. We previously discovered that the oxidized, but not the reduced, form of thioredoxin-1 directly binds to NLRP1 and represses inflammasome formation. However, the molecular basis for NLRP1’s selective association with only the oxidized form of TRX1 has not yet been established. Here, we leveraged Alphafold-Multimer, site-directed mutagenesis, thiol-trapping experiments, and mass spectrometry to reveal that a specific cysteine residue (C427 in humans) on NLRP1 forms a transient disulfide bond with oxidized TRX1. Overall, this work demonstrates how NLRP1 monitors the cellular redox state, further illuminating an unexpected connection between the intracellular redox potential and the innate immune system.
The genetic architecture of the pepper metabolome provides insights into the regulation of capsianoside biosynthesis
Authors: Nauen Julia , Tripodi Pasquale , Wendenburg Regina , Tringovska Ivanka , Nakar Amol N. , Stoeva Veneta , Pasev Gancho , Klemmer Annabella , Todorova Velichka , Bulut Mustafa , Tikunov Yury , Bovy Arnaud , Gechev Tsanko , Kostova Dimitrina , Fernie Alisdair R. , Alseekh Saleh 
Published: September 2023

AbstractCapsicum(pepper) is among the most economically important species worldwide, the fruit accumulates specialized metabolites with essential roles in plant environmental interaction and potential health benefits. However, the underlying genetic basis of their biosynthesis remains largely unknown. In this study, we developed and assessed both wild genetic variance and a bespoke mapping population to determine the genetic architecture of the pepper metabolome. The genetic analysis provided over 30 metabolic quantitative trait loci (mQTL) for over 1100 metabolites. We identified 92 candidate genes involved in various mQTL. Among the identified loci, we described and validated by transient overexpression a domestication gene cluster of eleven UDP-glycosyltransferases involved in monomeric capsianoside biosynthesis. We additionally constructed the biosynthetic reactions and annotated the genes involved in capsianoside biosynthesis in pepper. Given that differential glycosylation of acyclic diterpenoid glycosides contributes to plant resistance and acts as anticancer agents in humans, our data provide new insight, and resources for better understanding the biosynthesis of beneficial natural compounds to improve human health.
Heat ‘n Beat: A universal high-throughput end-to-end proteomics sample processing platform in under an hour
Authors: Xavier Dylan , Lucas Natasha , Williams Steven G , Koh Jennifer M. S. , Ashman Keith , Loudon Clare , Reddel Roger , Hains Peter G. , Robinson Phillip J. 
Published: September 2023

AbstractProteomic analysis by mass spectrometry (MS) of small (≤2 mg) solid tissue samples from diverse formats requires high throughput and comprehensive proteome coverage. We developed a near universal, rapid and robust protocol for sample preparation, suitable for high-throughput projects that encompass most cell or tissue types. This end-to-end workflow extends from original sample to loading the mass spectrometer and is centred on a one tube homogenisation and digestion method called Heat ‘n Beat (HnB). It is applicable to most tissues, regardless of how they were fixed or embedded. Sample preparation was divided to separate challenges. The initial sample washing, and final peptide clean-up steps were adapted to three tissue sources: fresh frozen (FF), optimal cutting temperature (OCT) compound embedded (FF-OCT), and formalin-fixed paraffin-embedded (FFPE). Thirdly, for core processing, tissue disruption and lysis were decreased to a 7 min heat and homogenisation treatment, and reduction, alkylation and proteolysis were optimised into a single step. The refinements produced near doubled peptide yield, delivered consistently high digestion efficiency of 85-90%, and required only 38 minutes for core processing in a single tube, with total processing time being 53-63 minutes. The robustness of HnB was demonstrated on six organ types, a cell line and a cancer biopsy. Its suitability for high throughput applications was demonstrated on a set of 1,171 FF-OCT human cancer biopsies, which were processed for end-to-end completion in 92 hours, producing highly consistent peptide yield and quality for over 3,513 MS runs.Graphical Abstract
Decision support system to evaluate VENTilation in the Acute Respiratory Distress Syndrome
Authors: Patel Brijesh , Mumby Sharon , Johnson Nicholas , Falaschetti Emanuela , Handslip Rhodri , Patel Sunil , Lee Teresa , Andersen Martin S , Adcock Ian M , McAuley Danny , Takata Masao , Staudinger Thomas , Karbing Dan S. , Jabaudon Matthieu , Schellongowski Peter , Rees Stephen E. 
Published: September 2023

AbstractRationaleThe acute respiratory distress syndrome (ARDS) shows significant heterogeneity in responsiveness to changes in mechanical ventilation and lacks personalisation.ObjectivesInvestigate the clinical efficacy of a physiologic-based ventilatory decision support system (DSS) on ARDS patients.MethodsAn international, multi-centre, randomized, open-label study enrolling patients with ARDS during the COVID-19 pandemic. The primary outcome was to detect a reduction in average driving pressure between groups. Secondary outcomes included several clinically relevant measures of respiratory physiology, ventilator free days; time from control mode to support mode; number of changes in ventilator settings per day; percentage of time in control and support mode ventilation; ventilation related and device related adverse events; and number of times the advice is followed.Measurements and Main Results. 95 patients were randomized to this study. The DSS showed was no effect in the average driving pressure between arms. Patients in the intervention arm had statistically improved oxygenation index when in support mode ventilation (−1.41, 95% CI: −2.76, −0.08; p=0.0370). Ventilatory ratio was also significantly improved in the intervention arm for patients in control mode ventilation (−0.63, 95% CI: −1.08, −0.17, p= 0.0068). The application of the DSS resulted in a significantly increased number of ventilator changes for pressure settings and respiratory frequency.ConclusionsThe application of a physiological model-based decision support system for advice on mechanical ventilation in patients with COVID-19 and non-COVID-19 ARDS showed that application of about 60% of advice improved physiological state, despite no significant difference in driving pressure as a primary outcome measure.
Genome sequencing as a generic diagnostic strategy for rare disease
Authors: Schobers Gaby , Derks Ronny , den Ouden Amber , Swinkels Hilde , van Reeuwijk Jeroen , Bosgoed Ermanno , Lugtenberg Dorien , Sun Su Ming , Corominas Galbany Jordi , Weiss Marjan , Blok Marinus J. , Olde Keizer Richelle A.C.M. , Hofste Tom , Hellebrekers Debby , de Leeuw Nicole , Stegmann Alexander , Kamsteeg Erik-Jan , Paulussen Aimee D.C. , Ligtenberg Marjolijn J.L. , Bradley Xiangqun Zheng , Peden John , Gutierrez Alejandra , Pullen Adam , Payne Tom , Gilissen Christian , van den Wijngaard Arthur , Brunner Han G. , Nelen Marcel , Yntema Helger G. , Vissers Lisenka E.L.M. 
Published: September 2023

AbstractBackgroundTo diagnose the full spectrum of hereditary and congenital diseases, genetic laboratories use many different workflows, ranging from karyotyping to exome sequencing. A single generic high-throughput workflow would greatly increase efficiency. We assessed whether genome sequencing (GS) can replace these existing workflows aimed at germline genetic diagnosis for rare disease.MethodsWe performed GS (NovaSeq™6000; 37x mean coverage) on 1,000 cases with 1,271 known clinically relevant variants, identified across different workflows, representative of our tertiary diagnostic centers. Variants were categorized into small variants (single nucleotide variants and indels <50 bp), large variants (copy number variants and short tandem repeats) and other variants (structural variants and aneuploidies). Variant calling format files were queried per variant, from which workflow-specific true positive rates (TPRs) for detection were determined. A TPR of ≥98% was considered the lower threshold for transition to GS. A GS-first scenario was generated for our laboratory, using diagnostic efficacy and predicted false negative as primary outcome measures. As input, we modeled the diagnostic path for all 24,570 individuals referred in 2022, combining the clinical referral, the transition of the underlying workflow(s) to GS, and the variant type(s) to be detected.ResultsOverall, 95% (1,206/1,271) of variants were detected. Detection rates differed per variant category: small variants in 96% (826/860), large variants in 93% (341/366), and other variants in 87% (39/45). TPRs varied between workflows (79-100%), with 7/10 being replaceable by GS. Models for our laboratory indicate that a GS-first strategy would be feasible for 84.9% of clinical referrals (750/883), translating to 71% of all individuals (17,444/24,570) receiving GS as their primary test. An estimated false negative rate of 0.3% could be expected.ConclusionGS can capture clinically relevant germline variants in a ‘GS-first strategy’ for the majority of clinical indications in a genetics diagnostic lab.
Multiscale chromatin dynamics and high entropy in plant iPSC ancestors
Authors: Rutowicz Kinga , Lüthi Joel , de Groot Reinoud , Holtackers René , Yakimovich Yauhen , Pazmiño Diana M. , Gandrillon Olivier , Pelkmans Lucas , Baroux Célia 
Published: September 2023

AbstractPlant protoplasts constitute the starting material to induce pluripotent cell massesin vitrocompetent for tissue regeneration. Dedifferentiation is associated with large-scale chromatin reorganisation and massive transcriptome reprogramming, characterized by stochastic gene expression. How this cellular variability reflects on chromatin organisation in individual cells and what are the factors influencing chromatin transitions during culturing is largely unknown. High-throughput imaging and a custom, supervised image analysis protocol extracting over 100 chromatin features unravelled a rapid, multiscale dynamics of chromatin patterns which trajectory strongly depends on nutrients availability. Decreased abundance in H1 (linker histones) is hallmark of chromatin transitions. We measured a high heterogeneity of chromatin patterns indicating an intrinsic entropy as hallmark of the initial cultures. We further measured an entropy decline over time, and an antagonistic influence by external and intrinsic factors, such as phytohormones and epigenetic modifiers, respectively. Collectively, our study benchmarks an approach to understand the variability and evolution of chromatin patterns underlying plant cell reprogrammingin vitro.
Control of iron homeostasis by a regulatory protein-protein interaction inBacillus subtilis: The FurA (YlaN) acts as an antirepressor to the ferric uptake regulator Fur
Authors: Demann Lorenz , Bremenkamp Rica , Stahl Kolja , Hormes Björn , Warneke Robert , Rappsilber Juri , Stülke Jörg 
Published: September 2023

ABSTRACTIron is essential for most organisms. However, two problems are associated with the use of iron for aerobically growing organisms: (i) its accumulation leads to the formation of toxic reactive oxygen species and (ii) it is present mainly as the highly insoluble ferric iron which makes the access to iron difficult. As a consequence, a tight regulation of iron homeostasis is required. This regulation is achieved in many bacteria by the ferric uptake repressor Fur. The way how the activity of Fur is controlled, has so far remained elusive. Here, we have identified the Fur antirepressor FurA (previously YlaN) in the model bacteriumBacillus subtilisand describe its function to release Fur from the DNA under conditions of iron limitation. The FurA protein physically interacts with Fur, and this interaction prevents Fur from binding to its target sites due to a complete re-orientation of the protein. Bothin vivoandin vitroexperiments using a reporter fusion and Fur-DNA binding assays, respectively, demonstrate that the Fur-FurA interaction prevents Fur from binding DNA and thus from repressing the genes required for iron uptake. Accordingly, the lack of FurA results in the inability of the cell to express the genes for iron uptake under iron-limiting conditions. This explains why thefurAgene was identified as being essential under standard growth conditions inB. subtilis. Phylogenetic analysis suggests that the control of Fur activity by the antirepressor FurA is confined to, but very widespread in bacteria of the class Bacilli.IMPORTANCEIron is essential for most bacteria since it is required for many redox reactions. Under aerobic conditions, iron is both essential and toxic due to radical formation. Thus, iron homeostasis must be faithfully controlled. The transcription factor Fur is responsible for this regulation in many bacteria; however, the control of Fur activity has remained open. Here we describe the FurA protein, a so far unknown protein which acts as an antirepressor to Fur inBacillus subtilis. This mechanism seems to be widespread inB. subtilisand several important pathogens and might be a promising target for drug development.
Male pathology regardless of behaviour drives transmission in an avian host-pathogen system
Authors: Sauer Erin L. , Connelly Chloe , Perrine Weston , Love Ashely C. , DuRant Sarah E. 
Published: September 2023

AbstractHost sex is an important source of heterogeneity in the severity of epidemics. Pinpointing the mechanisms causing this heterogeneity can be difficult because differences in behaviour among sexes (e.g. greater territorial aggression in males) can bias exposure risk, obfuscating the role of immune function, which can lead to differences in pathology, in driving differential susceptibility between sexes. Thus, sex-biased transmission driven by differences in immune function independent of behaviour is poorly understood, especially in non-mammalian systems. Here we examine the previously unexplored potential for male-biased pathology to affect transmission using an avian host-pathogen system. We employ a sex-dependent multistate transmission model parameterized with isolated, individual-based experimental exposures of domestic canaries and experimental transmission data of house finches. The experiment revealed that male birds have shorter incubation periods, longer recovery periods, higher pathogen burdens, and greater disease pathology, than females. Our model revealed that male-biased pathology led to epidemic size rapidly increasing with the proportion of male birds, with a nearly 16-fold increase in total epidemic size from an all-female to an all-male simulation. Our results demonstrate that female-biased resistance, independent of male behaviour, can drive sex-dependent transmission in wildlife, indicating that sex-based differences in immune function, not just differences in exposure risk, can shape epidemic dynamics.
Bacterial efflux pumps excrete SYTO™dyes from bacteria and lead to false-negative staining results
Authors: Minero G. A. , Larsen P. B. , Hoppe M. E. , Meyer R. L. 
Published: September 2023

Multidrug efflux pumps excrete a range of small molecules from bacterial cells. In this study, we show that bacterial efflux pumps have affinity for a range of SYTO™ dyes that are commonly used to label bacteria. Efflux pump activity will there lead to false negative results from bacterial staining and SYTO™ dyes should be used with caution on live samples.Abstract Figure
Structural basis of lipid head group entry to the Kennedy pathway by FLVCR1
Authors: Son Yeeun , Kenny Timothy C. , Khan Artem , Birsoy Kıvanç , Hite Richard K. 
Published: September 2023

AbstractPhosphatidylcholine and phosphatidylethanolamine, the two most abundant phospholipids in mammalian cells, are synthesizedde novoby the Kennedy pathway from choline and ethanolamine, respectively1–6. Despite the importance of these lipids, the mechanisms that enable the cellular uptake of choline and ethanolamine remain unknown. Here, we show that FLVCR1, whose mutation leads to the neurodegenerative syndrome PCARP7–9, transports extracellular choline and ethanolamine into cells for phosphorylation by downstream kinases to initiate the Kennedy pathway. Structures of FLVCR1 in the presence of choline and ethanolamine reveal that both metabolites bind to a common binding site comprised of aromatic and polar residues. Despite binding to a common site, the larger quaternary amine of choline interacts differently with FLVCR1 than does the primary amine of ethanolamine. Structure-guided mutagenesis identified residues that are critical for the transport of ethanolamine, while being dispensable for choline transport, enabling functional separation of the entry points into the two branches of the Kennedy pathway. Altogether, these studies reveal how FLCVR1 is a high-affinity metabolite transporter that serves as the common origin for phospholipid biosynthesis by two branches of the Kennedy pathway.
Disruption of Epithelium Integrity by Inflammation-Associated Fibroblasts through Prostaglandin Signaling
Authors: Dong Yi , Johnson Blake A. , Ruan Linhao , Zeineldin Maged , Liu Albert Z. , Raychaudhuri Sumana , Chiu Ian , Zhu Jin , Smith Barbara , Zhao Nan , Searson Peter , Watanabe Shigeki , Donowitz Mark , Larman Tatianna C. , Li Rong 
Published: September 2023

AbstractInflammation-associated fibroblasts (IAFs) are associated with the progression and drug resistance of chronic inflammatory diseases such as inflammatory bowel disease (IBD), but their direct impact on epithelial function and architecture is unknown. In this study, we developed anin vitromodel whereby human colon fibroblasts are induced to become IAFs by specific cytokines and recapitulate key features of IAFsin vivo. When co-cultured with patient-derived colon organoids (colonoids), IAFs induced rapid colonoid swelling and barrier disruption due to swelling and rupture of individual epithelial cells. Epithelial cells co-cultured with IAFs also exhibit increased DNA damage, mitotic errors, and proliferation arrest. These IAF-induced epithelial defects are mediated through a paracrine pathway involving prostaglandin E2 (PGE2) and the PGE2 receptor EP4, leading to PKA-dependent activation of the CFTR chloride channel. Importantly, EP4-specific chemical inhibitors effectively prevented colonoid swelling and restored normal proliferation and genome stability of IAF-exposed epithelial cells. These findings reveal a mechanism by which IAFs could promote and perpetuate IBD and suggest a potential treatment to mitigate inflammation-associated epithelial injury.TeaserInflammation-associated fibroblasts compromise colon epithelial barrier integrity and genome stability via PGE2-EP4 signaling.