bluuebunny/crossref_metadata_2025 huggingface dataset:
Apoptotic proteins inLeishmania donovani:In silicoscreening, modelling, and validation by knock-out and gene expression analysis
Authors: Kumar Ketan , Crobu Lucien , Sterkers Yvon , Prajapati Vijay Kumar
Published: April 2024

Visceral leishmaniasis (VL), a life-threatening vector-borne illness that disproportionately affects children and elderly immunocompromised people, is a primary tropical neglected disease. No apoptotic partner proteins inL. donovanihave been reported yet, which might contribute to the knowledge of parasite cell death and the establishment of alternative therapeutics. We used the Orthologues algorithm to search for the mammalian Bcl-2 family proteins orthologs, one anti-apoptotic and two pro-apoptotic, inL. donovani. We also included a pro-death aquaporin (AQP) protein due to its characteristic BH3 domain, which is known to interact with pro-apoptotic proteins in mammals. Molecular docking and molecular dynamics simulation studies were conducted to assess the protein-protein interaction between the identified apoptotic proteins and mimic mammalian intrinsic apoptotic pathways. The results showed that the pro-apoptotic protein interacted with the hydrophobic pocket of the anti-apoptotic ortholog, forming a stable complex, which may represent a critical event in the apoptotic pathways of leishmaniasis. To further establish an apoptotic pathway inL. donovani, we used several CRISPR-Cas9 approaches to target the identified proteins. The pure knocked population mutants, and episomal over-expressing mutant cells were exposed to apoptotic stimuli. TUNEL assay and quantitative expression profiling suggested that these proteins are needed during the parasite’s apoptosis and could play a role in the parasite’s survival.

Author Summary

Visceral leishmaniasis, a fatal systemic infection affecting internal organs, is one of three types of leishmaniasis in mammals alongside cutaneous and mucocutaneous leishmaniasis. It predominantly occurs in tropical and subtropical climatic zones,Leishmania donovanipredominant in the Indian subcontinent andLeishmania infantumin the Mediterranean basin, the Middle East, Central Asia, South America, and Central America. This disease primarily affects children, immunocompromised adults, and the elderly.L donovani,transmitted by the infected sandflies complete its life cycle in humans, serving as reservoir. During its life cycle, at a particular stage, the parasite undergoes apoptotic-like events, yet underlying proteins or key factors remain unidentified. Using computational methods, we screened theL. donovanigenome for potential candidate genes of the Bcl-2 family apoptotic proteins. We biologically/experimentally validated ourin-silicofindings using molecular editing tools like CRISPR-Cas9, advancing our understanding of the parasite’s apoptotic pathway. Targeting this pathway could lead to more effective therapeutics against visceral leishmaniasis.
Cyr61 delivery promotes angiogenesis during bone fracture repair
Authors: Lang Annemarie , Eastburn Emily A. , Younesi Mousa , Nijsure Madhura , Siciliano Carly , Pranatharthi Haran Annapurna , Panebianco Christopher J. , Seidl Elizabeth , Tang Rui , Alsberg Eben , Willett Nick J. , Gottardi Riccardo , Huh Dongeun , Boerckel Joel D.
Published: April 2024

Compromised vascular supply and insufficient neovascularization impede bone repair, increasing risk of non-union. Cyr61, Cysteine-rich angiogenic inducer of 61kD (also known as CCN1), is a matricellular growth factor that is regulated by mechanical cues during fracture repair. Here, we map the distribution of endogenous Cyr61 during bone repair and evaluate the effects of recombinant Cyr61 delivery on vascularized bone regeneration. In vitro, Cyr61 treatment did not alter chondrogenesis or osteogenic gene expression, but significantly enhanced angiogenesis. In a mouse femoral fracture model, Cyr61 delivery did not alter cartilage or bone formation, but accelerated neovascularization during fracture repair. Early initiation of ambulatory mechanical loading disrupted Cyr61-induced neovascularization. Together, these data indicate that Cyr61 delivery can enhance angiogenesis during bone repair, particularly for fractures with stable fixation, and may have therapeutic potential for fractures with limited blood vessel supply.
Early hippocampal high-amplitude rhythmic spikes predict post-traumatic epilepsy in mice
Authors: Shannon Tyler , Levine Noah , Dirickson Rina , Shen Yiyun , Cotter Christopher , Yi Yoon-Jae , Rajjoub Noora , de Villena Fernando Pardo-Manuel , Kokiko-Cochran Olga , Gu Bin
Published: April 2024

Oscillations, a highly conserved brain function across mammalian species, are pivotal in brain physiology and pathology. Traumatic brain injury (TBI) often leads to subacute and chronic brain oscillatory alterations associated with complications like post-traumatic epilepsy (PTE) in patients and animal models. Our recent work longitudinally recorded local field potential from the contralateral hippocampus of 12 strains of recombinant inbred Collaborative Cross (CC) mice alongside classical laboratory inbred C57BL/6J mice after lateral fluid percussion injury. In this study, we profiled the acute (<12 hr post-injury) and subacute (12-48 hr post-injury) hippocampal oscillatory responses to TBI and evaluated their predictive value for PTE. We found dynamic high-amplitude rhythmic spikes with elevated power density and reduced entropy that prevailed during the acute phase in CC031 mice who later developed PTE. This characteristic early brain oscillatory alteration is absent in CC031 sham controls or other CC and reference C57BL/6J strains that did not develop PTE after TBI. Our work provides quantitative measures linking early brain oscillation to PTE at a population level in mice under controlled experimental conditions. These findings will offer insights into circuit mechanisms and potential targets for neuromodulatory intervention.
Brilacidin, a host defense peptide mimetic, potentiates ibrexafungerp antifungal activity against the human pathogenic fungusAspergillus fumigatus
Authors: dos Reis Thaila Fernanda , Diehl Camila , Pinzan Camila Figueiredo , de Castro Patrícia Alves , Goldman Gustavo H.
Published: April 2024

Aspergillus fumigatusis the primary etiological agent of aspergillosis. Here, we show that the host defense peptide mimetic, brilacidin (BRI) can potentiate ibrexafungerp (IBX) against clinical isolates ofA. fumigatus. CAS-resistant strains with mutations infks1that encodes the 1,3-β-D-glucan synthase are not IBX-resistant and BRI+IBX can inhibit their growth. The combination of BRI+IBX plays a fungicidal role, increases the fungal cell permeability and decreases the fungal survival in the presence of A549 epithelial cells.
Sinorhizobium melilotiBR-bodies promote fitness during host colonization
Authors: Mallikaarachchi Kaveendya S. , Huang Jason L. , Madras Shanmukha , Cuellar Rodrigo A. , Huang Zhenzhong , Gega Alisa , Rathnayaka-Mudiyanselage Imalka W. , Al-Husini Nadra , Saldaña-Rivera Natalie , Ma Loi H. , Ng Eric , Chen Joseph C. , Schrader Jared M.
Published: April 2024

Biomolecular condensates, such as the nucleoli or P-bodies, are non-membrane-bound assemblies of proteins and nucleic acids that facilitate specific cellular processes. Like eukaryotic P-bodies, the recently discovered bacterial ribonucleoprotein bodies (BR-bodies) organize the mRNA decay machinery, yet the similarities in molecular and cellular functions across species have been poorly explored. Here, we examine the functions of BR-bodies in the nitrogen-fixing endosymbiontSinorhizobium meliloti, which colonizes the roots of compatible legume plants. Assembly of BR-bodies into visible foci inS. meliloticells requires the C-terminal intrinsically disordered region (IDR) of RNase E, and foci fusion is readily observedin vivo, suggesting they are liquid-like condensates that form via mRNA sequestration. Using Rif-seq to measure mRNA lifetimes, we found a global slowdown in mRNA decay in a mutant deficient in BR-bodies, indicating that compartmentalization of the degradation machinery promotes efficient mRNA turnover. While BR-bodies are constitutively present during exponential growth, the abundance of BR-bodies increases upon cell stress, whereby they promote stress resistance. Finally, usingMedicago truncatulaas host, we show that BR-bodies enhance competitiveness during colonization and appear to be required for effective symbiosis, as mutants without BR-bodies failed to stimulate plant growth. These results suggest that BR-bodies provide a fitness advantage for bacteria during infection, perhaps by enabling better resistance against the host immune response.

Significance

While eukaryotes often organize their biochemical pathways in membrane-bound organelles, bacteria generally lack such subcellular structures. Instead, membraneless compartments called biomolecular condensates have recently been found in bacteria to enhance biochemical activities. Bacterial ribonucleoprotein bodies (BR-bodies), as one of the most widespread biomolecular condensates identified to date, assemble the mRNA decay machinery via the intrinsically disordered regions (IDRs) of proteins. However, the implications of such assemblies are unclear. Using a plant-associated symbiont, we show that the IDR of its mRNA degradation protein is necessary for condensate formation. Absence of BR-bodies results in slower mRNA decay and ineffective symbiosis, suggesting that BR-bodies play critical roles in regulating biochemical pathways and promoting fitness during host colonization.
Elasmobranchs Exhibit Species-Specific Epidermal Microbiomes Guided by Denticle Topography
Authors: Goodman Asha Z. , Papudeshi Bhavya , Mora Maria , Kerr Emma N. , Torres Melissa , Moffatt Jennifer Nero , Lima Laís F.O. , Niesman Ingrid R. , Moreno Isabel Y. , Doane Michael P. , Dinsdale Elizabeth A.
Published: April 2024

Elasmobranch epidermal microbiomes are species-specific, yet microbial assembly and retainment drivers are mainly unknown. The contribution of host-derived factors in recruiting an associated microbiome is essential for understanding host-microbe interactions. Here, we focus on the physical aspect of the host skin in structuring microbial communities. Each species of elasmobranch exhibits unique denticle morphology, and we investigate whether microbial communities and functional pathways are correlated with the morphological features or follow the phylogeny of the three species. We extracted and sequenced the DNA from the epidermal microbial communities of three captive shark species: Horn (Heterodontus francisci), Leopard (Triakis semifasciata), and Swell shark (Cephaloscyllium ventriosum) and use electron microscopy to measure the dermal denticle features of each species. Our results outline species-specific microbial communities, as microbiome compositions vary at the phyla level;C. ventriosumhosted a higher relative abundance of Pseudomonadota and Bacillota, whileH. francisciwere associated with a higher prevalence of Euryarchaeota and Aquificae, and Bacteroidota and Crenarchaeota were ubiquitous withT. semifasciata. Functional pathways performed by each species’ respective microbiome were species-specific metabolic. Microbial genes associated with aminosugars and electron-accepting reactions were correlated with the distance between dermal denticles, whereas desiccation stress genes were only present when the dermal denticle overlapped. Microbial genes associated with Pyrimidines, chemotaxis and virulence followed the phylogeny of the sharks. Several microbial genera display associations that resemble host evolutionary lineage, while others had linear relationships with interdenticle distance. Therefore, denticle morphology was a selective influence for some microbes and functions in the microbiome contributing to the phylosymbiosis.

Importance

Microbial communities form species-specific relationships with vertebrate hosts, but the drivers of these relationships remain an outstanding question. We explore the relationship between a physical feature of the host and the microbial community. A distinguishing feature of the subclass Elasmobranchii (sharks, rays, and skates), is the presence of dermal denticles on the skin. These structures protrude through the epidermis providing increased swimming efficiency for the host and an artificial model skin affect microbial recruitment and establishment of cultured microbes but has not been tested on natural microbiomes. Here, we show some naturally occurring microbial genera and functional attributes were correlated with dermal denticle features, suggesting they are one, but not only contributing factor in microbiome structure on benthic sharks.
Requirements for efficient endosomal escape by designed mini-proteins
Authors: Giudice Jonathan , Brauer Daniel D. , Zoltek Madeline , Vázquez Maldonado Angel L. , Kelly Mark , Schepartz Alanna
Published: April 2024

ZF5.3 is a compact, rationally designed mini-protein that escapes efficiently from the endosomes of multiple cell types. Despite its small size (27 amino acids), ZF5.3 can be isolated intact from the cytosol of treated cells and guides multiple classes of proteins into the cytosol and/or nucleus. In the best cases, delivery efficiencies reach or exceed 50% to establish nuclear or cytosolic concentrations of 500 nM or higher. But other than the requirement for unfoldable cargo and an intact HOPS complex, there is little known about how ZF5.3 traverses the limiting endocytic membrane. Here we delineate the attributes of ZF5.3 that enable efficient endosomal escape. We confirm that ZF5.3 is stable at pH values between 5.5 and 7.5, with no evidence of unfolding even at temperatures as high as 95 °C. The high-resolution NMR structure of ZF5.3 at pH 5.5, also reported here, shows a canonical p zinc-finger fold with the penta-arg motif integrated seamlessly into the C-terminal ⍺-helix. At lower pH, ZF5.3 unfolds cooperatively as judged by both circular dichroism and high-resolution NMR. Unfolding occurs upon protonation of a single Zn(II)-binding His side chain whose pKacorresponds almost exactly to that of the late endosomal lumen. pH-induced unfolding is essential for endosomal escape, as a ZF5.3 analog that remains folded at pH 4.5 fails to efficiently reach the cytosol, despite high overall uptake. Finally, using reconstituted liposomes, we identify a high-affinity interaction of ZF5.3 with a specific lipid–BMP–that is selectively enriched in the inner leaflet of late endosomal membranes. This interaction is 10-fold stronger at low pH than neutral pH, providing a molecular picture for why escape occurs preferentially and in a HOPS-dependent manner from late endosomal compartments. The requirements for programmed endosomal escape identified here should aid and inform the design of proteins, peptidomimetics, and other macromolecules that reach cytosolic or nuclear targets intact and at therapeutically relevant concentrations.
Astrocyte modulation of synaptic plasticity mediated by activity-dependent Sonic hedgehog signaling
Authors: Le Anh Duc , Fu Marissa , Kumar Riya , Zacharias Gloria , Garcia A. Denise R.
Published: April 2024

The influence of neural activity on astrocytes and their reciprocal interactions with neurons has emerged as an important modulator of synapse function. Astrocytes exhibit activity-dependent changes in gene expression, yet the molecular mechanisms by which they accomplish this have remained largely unknown. The molecular signaling pathway, Sonic hedgehog (Shh), mediates neuron-astrocyte communication and regulates the organization of cortical synapses. Here, we demonstrate that neural activity stimulates Shh signaling in cortical astrocytes and upregulates expression of Hevin and SPARC, astrocyte derived molecules that modify synapses. Whisker stimulation and chemogenetic activation both increase Shh activity in deep layers of the somatosensory cortex, where neuron-astrocyte Shh signaling is predominantly found. Experience-dependent Hevin and SPARC require intact Shh signaling and selective loss of pathway activity in astrocytes occludes experience-dependent structural plasticity. Taken together, these data identify Shh signaling as an activity-dependent, neuronal derived cue that stimulates astrocyte interactions with synapses and promotes synaptic plasticity.
Structural basis for CCR6 modulation by allosteric antagonists
Authors: Wasilko David Jonathan , Gerstenberger Brian S. , Farley Kathleen A. , Li Wei , Alley Jennifer , Schnute Mark E. , Unwalla Ray J. , Victorino Jorge , Crouse Kimberly K. , Ding Ru , Sahasrabudhe Parag V. , Vincent Fabien , Frisbie Richard K. , Dermenci Alpay , Flick Andrew , Choi Chulho , Chinigo Gary , Mousseau James J. , Trujillo John I. , Nuhant Philippe , Mondal Prolay , Lombardo Vincent , Lamb Daniel , Hogan Barbara J. , Minhas Gurdeep Singh , Segala Elena , Oswald Christine , Windsor Ian W. , Han Seungil , Rappas Mathieu , Cooke Robert M. , Calabrese Matthew F. , Berstein Gabriel , Thorarensen Atli , Wu Huixian
Published: April 2024

The CC chemokine receptor 6 (CCR6) is a potential target for chronic inflammatory diseases such as psoriasis and inflammatory bowel disease. Previously, we reported an active CCR6 structure in complex with its cognate chemokine CCL20, revealing the molecular basis of CCR6 activation mediated by CCL20. Here, we present two inactive CCR6 structures determined by cryo-EM in ternary complexes with different allosteric antagonists, CCR6/SQA1/OXM1 and CCR6/SQA1/OXM2. OXM1 and OXM2 are oxomorpholine (OXM) analogues which are highly selective for CCR6 and disrupt the molecular network critical for receptor activation by binding to an extracellular allosteric pocket within the transmembrane domain. A U-shaped conformation stabilized by intramolecular interactions was revealed by structural and NMR studies of active OXM analogues. SQA1 is a squaramide (SQA) derivative with close-in analogues that were previously reported to be antagonists of CCR6 and other chemokine receptors. Our structures reveal an intracellular pocket occupied by SQA1 that overlaps with the G protein binding site. In addition, SQA1 stabilizes a closed conformation of the intracellular pocket, a hallmark of the inactive state of GPCRs. Minimal communication was found between the two allosteric pockets. Overall, our work provides new evidence of the versatility of GPCR antagonism by small molecules, complementing previous knowledge on CCR6 activation, and sheds light on drug discovery approaches to target CCR6 for autoimmune disorders.
Antigenic cartography using hamster sera identifies SARS-CoV-2 JN.1 evasion seen in human XBB.1.5 booster sera
Authors: Wang Wei , Bhushan Gitanjali L. , Paz Stephanie , Stauft Charles B. , Selvaraj Prabhu , Goguet Emilie , Bishop-Lilly Kimberly A. , Subramanian Rahul , Vassell Russell , Lusvarghi Sabrina , Cong Yu , Agan Brian , Richard Stephanie A. , Epsi Nusrat J. , Fries Anthony , Fung Christian K. , Conte Matthew A. , Holbrook Michael R. , Wang Tony T. , Burgess Timothy H. , Mitre Edward , Pollett Simon D. , Katzelnick Leah C. , Weiss Carol D.
Published: April 2024

Antigenic assessments of SARS-CoV-2 variants inform decisions to update COVID-19 vaccines. Primary infection sera are often used for assessments, but such sera are rare due to population immunity from SARS-CoV-2 infections and COVID-19 vaccinations. Here, we show that neutralization titers and breadth of matched human and hamster pre-Omicron variant primary infection sera correlate well and generate similar antigenic maps. The hamster antigenic map shows modest antigenic drift among XBB sub-lineage variants, with JN.1 and BA.4/BA.5 variants within the XBB cluster, but with five to six-fold antigenic differences between these variants and XBB.1.5. Compared to sera following only ancestral or bivalent COVID-19 vaccinations, or with post-vaccination infections, XBB.1.5 booster sera had the broadest neutralization against XBB sub-lineage variants, although a five-fold titer difference was still observed between JN.1 and XBB.1.5 variants. These findings suggest that antibody coverage of antigenically divergent JN.1 could be improved with a matched vaccine antigen.
Landscape-scale endophytic community analyses in replicated grapevine stands reveal that dieback disease is not caused by specific fungal communities
Authors: Monod Vinciane , Hofstetter Valérie , Viret Olivier , Zufferey Vivian , Gindro Katia , Croll Daniel
Published: April 2024

Tree diebacks are complex and multi-factorial diseases with suspected biotic and abiotic components. Microbiome effects on plant health are challenging to assess due to the complexity of fungal and bacterial communities. Grapevine wood dieback is the main threat to sustainable production worldwide and no causality with microbial species has been established. Here, we aimed to test the hypothesis that grapevine esca disease progression has reproducible drivers in the fungal species community. For this, we analyzed a set of 21 vineyards planted simultaneously with a single susceptible cultivar to provide replication at the landscape scale. We sampled a total of 496 plants across vineyards in two different years to perform deep amplicon sequencing analyses of the fungal communities inhabiting grapevine trunks. The communities were highly diverse with a total of 4,129 amplified sequence variants assigned to 697 distinct species. Individual plants varied in fungal community composition depending on the year of sampling, vineyard location, and disease status. However, we detect no specific fungal species driving symptom development across the vineyards contrary to long-standing expectations. Our study shows how landscape-scale replicated field surveys allow for powerful hypothesis-testing for complex dieback disease drivers and prioritize future research towards additional factors.
Test-retest repeatability of intravoxel incoherent motion (IVIM) measurements in the cervical cord
Authors: Lebret Anna , Lévy Simon , Freund Patrick , Callot Virginie , Seif Maryam
Published: April 2024

This work aimed at assessing the reliability of intravoxel incoherent motion (IVIM) parameters sensitive to perfusion changes in the cervical cord by determining the test-retest variability across subjects and different post-processing fitting algorithms. IVIM test-retest scans were acquired in the cervical cord (C1-C3) of 10 healthy subjects on a 3T MRI scanner, with a 15-minute break in-between. IVIM parameters, including microvascular volume fraction (F), pseudo-diffusion coefficient (D*), blood flow-related coefficient (F·D*), and diffusion coefficient (D), were derived using voxel-wise and region of interest (ROI)-wise fits. The reliability of each IVIM parameter was determined with coefficients of variation (CV), intraclass correlation coefficients (ICC), Bland-Altman analysis and linear regression. To assess the effects of the different fitting approaches, a two-way repeated-measures analysis of variance (ANOVA) was conducted on the CVs calculated across fitting algorithms. Mean CVs of IVIM parameters calculated across subjects using the voxel-wise fit was lower in the white matter (WM) and grey matter (GM) (2.6% to 15.6%; 2.2% to 16.4%, respectively) compared with those calculated using the ROI-wise fit approach (WM: 4.5%-32.2%; GM: 3.4%-53.4%). The voxel-wise fit in the WM yielded higher ICC values (good-to-excellent, 0.71 – 0.97) compared to the ROI-wise fit approach (0.49 – 0.90). IVIM parameters, derived using the voxel-wise fitting approach, demonstrated a high reliability in the cervical cord. Robust IVIM metrics, observed across scans and subjects, can facilitate studies targeting perfusion impairment and pave the way to future clinical trials assessing perfusion impairment as a potential quantitative biomarker.
Leveraging large-scale biobank EHRs to enhance pharmacogenetics of cardiometabolic disease medications
Authors: Sadler Marie C. , Apostolov Alexander , Cevallos Caterina , Ribeiro Diogo M. , Altman Russ B. , Kutalik Zoltán
Published: April 2024

Electronic health records (EHRs) coupled with large-scale biobanks offer great promises to unravel the genetic underpinnings of treatment efficacy. However, medication-induced biomarker trajectories stemming from such records remain poorly studied. Here, we extract clinical and medication prescription data from EHRs and conduct GWAS and rare variant burden tests in the UK Biobank (discovery) and the All of Us program (replication) on ten cardiometabolic drug response outcomes including lipid response to statins, HbA1c response to metformin and blood pressure response to antihypertensives (N = 740-26,669). Our findings at genome-wide significance level recover previously reported pharmacogenetic signals and also include novel associations for lipid response to statins (N = 26,669) nearLDLRandZNF800. Importantly, these associations are treatment-specific and not associated with biomarker progression in medication-naive individuals. Furthermore, we demonstrate that individuals with higher genetically determined low-density and total cholesterol baseline levels experience increased absolute, albeit lower relative biomarker reduction following statin treatment. In summary, we systematically investigated the common and rare pharmacogenetic contribution to cardiometabolic drug response phenotypes in over 50,000 UK Biobank and All of Us participants with EHR and identified clinically relevant genetic predictors for improved personalized treatment strategies.
BPP43_05035 is aBrachyspira pilosicolicell surface adhesin that weakens the integrity of the epithelial barrier during infection
Authors: Rajan Anandi , Gallego Pablo , Dolan Brendan , Patel Piyush , Dwibedi Chinmay , Luis Ana S. , Trillo-Muyo Sergio , Arike Liisa , van der Post Sjoerd , Simrén Magnus , Pelaseyed Thaher
Published: April 2024

The anaerobic spirocheteBrachyspiracauses intestinal spirochetosis, characterized by the intimate attachment of bacterial cells to the colonic mucosa, potentially leading to symptoms such as diarrhea, abdominal pain, and weight loss. Despite the clinical significance ofBrachyspirainfections, the mechanism behind the interaction betweenBrachyspiraand the colonic epithelium is not known. In this study, we characterized the molecular mechanism ofB. pilosicoli-epithelium interaction and its impact on the epithelial barrier during infection. Through a proteomics approach, we identified BPP43_05035 as a candidateB. pilosicoliadhesion protein that mediates bacterial attachment to cultured human colonic epithelial cells. The crystal structure of BPP43_05035 revealed a globular lipoprotein with a six-bladed beta-propeller domain. Blocking the native BPP43_05035 onB. pilosicoli, either with a specific antibody or via competitive inhibition, abrogated its binding to epithelial cells. Furthermore, the binding of BPP43_05035 to epithelial cells required surface-exposed hostN-glycans. Proximity labeling and interaction assays revealed that BPP43_05035 bound to tight junctions, thereby increasing the permeability of the epithelial monolayer. Extending our investigation to human patients, we identified a downregulation of tight junction and brush border genes inB. pilosicoli-infected patients carrying detectible levels of epithelium-bound BPP43_05035. Collectively, our findings identify BPP43_05035 as aB. pilosicoliadhesin that weakens the colonic epithelial barrier during infection.
Transformers do not outperform Cellpose
Authors: Stringer Carsen , Pachitariu Marius
Published: April 2024

In a recent publication, Ma et al [1] claim that a transformer-based cellular segmentation method called Mediar [2] — which won a Neurips challenge — outperforms Cellpose [3] (0.897 vs 0.543 median F1 score). Here we show that this result was obtained by artificially impairing Cellpose in multiple ways. When we removed these impairments, Cellpose outperformed Mediar (0.861 vs 0.826 median F1 score on the updated test set). To further investigate the performance of transformers for cellular segmentation, we replaced the Cellpose backbone with a transformer. The transformer-Cellpose model also did not outperform the standard Cellpose (0.848 median F1 test score). Our results suggest that transformers do not advance the state-of-the-art in cellular segmentation.
Genome-wide profiling of the hierarchical control of autophagy dynamics using deep learning
Authors: Chica Nathalia , Andersen Aram N. , Orellana-Muñoz Sara , Garcia Ignacio , Nguéa P Aurélie , Ayuda-Durán Pilar , Håkensbakken Linda , Rødningen Eline , Putnam Christopher D. , Zucknick Manuela , Rusten Tor Erik , Enserink Jorrit M.
Published: April 2024

Recycling of nutrients through autophagy is a crucial mechanism for cells to sustain internal stability in a fluctuating environment. Dysregulation of the process has been associated with a range of human disorders, and the core components of autophagy have been comprehensively outlined. However, limited insight into its systems-wide dynamical control has hampered predictive modeling and effectivein vivomanipulation. Here, we mapped yeast genomic influences on autophagy dynamics in response to changes in nitrogen levels. Using time-resolved high-content imaging coupled with deep learning, we examined the kinetics of autophagy activation and inactivation in 5919 gene deletion mutants, and classified their profiles based on temporal responsiveness and activation potential, as well as their contribution to autophagosome formation and clearance. By integrating these profiles with functional and genetic network data, we unveiled a hierarchical and multi-layered control of autophagy dynamics and exposed novel regulatory features of the core components and well-established nutrient-sensing pathways. Furthermore, by leveraging multi-omics resources and explainable machine learning to model genetic perturbation effects, we identified the retrograde pathway as a central time-varying transcriptional modulator of autophagy execution. These findings offer valuable insights into the systems-wide tuning of autophagy, and advance our understanding of the dynamical control by providing genome-wide quantitative data under concurrent genetic and environmental interventions. We further anticipate that our study can serve as a blueprint for high-content, deep learning-driven exploration of complex dynamical processes in any organism.
Mechanism of enterovirus VP0 maturation cleavage based on the structure of a stabilised assembly intermediate
Authors: Kingston Natalie J , Snowden Joseph S , Grehan Keith , Hall Philippa K , Hietanen Eero V , Passchier Tim C , Polyak Stephen J , Filman David J , Hogle James M , Rowlands David J , Stonehouse Nicola J
Published: April 2024

Molecular details of genome packaging are little understood for the majority of viruses. In enteroviruses (EVs), cleavage of the structural protein VP0 into VP4 and VP2 is initiated by the incorporation of RNA into the assembling virion and is essential for infectivity. We have applied a combination of bioinformatic, molecular and structural approaches to generate the first high-resolution structure of an intermediate in the assembly pathway, termed a provirion, which contains RNA and intact VP0. We have demonstrated an essential role of VP0 E096 in VP0 cleavage independent of RNA encapsidation and generated a new model of capsid maturation, supported by bioinformatic analysis. This provides a molecular basis for RNA-dependence, where RNA induces conformational changes required for VP0 maturation, but that RNA packaging itself is not sufficient to induce maturation. These data have implications for understanding production of infectious virions and potential relevance for future vaccine and antiviral drug design.
The structure of the monobactam-producing thioesterase domain of SulM forms a unique complex with the upstream carrier protein domain
Authors: Patel Ketan D. , Oliver Ryan A. , Lichstrahl Michael S. , Li Rongfeng , Townsend Craig A. , Gulick Andrew M.
Published: April 2024

Nonribosomal peptide synthetases (NRPSs) are responsible for the production of important biologically active peptides. The large, multidomain NRPSs operate through an assembly line strategy in which the growing peptide is tethered to carrier domains that deliver the intermediates to neighboring catalytic domains. While most NRPS domains catalyze standard chemistry of amino acid activation, peptide bond formation and product release, some canonical NRPS catalytic domains promote unexpected chemistry. The paradigm monobactam antibiotic sulfazecin is produced through the activity of a terminal thioesterase domain that catalyzes an unusual β-lactam forming reaction in which the nitrogen of the C-terminalN-sulfo-2,3-diaminopropionate residue attacks its thioester tether to release the β-lactam product. We have determined the structure of the thioesterase domain as both a free-standing domain and a didomain complex with the upstreamholopeptidyl-carrier domain. The structure illustrates a constrained active site that orients the substrate properly for β-lactam formation. In this regard, the structure is similar to the β-lactone forming thioesterase domain responsible for the production of obafluorin. Analysis of the structure identifies features that are responsible for this four-membered ring closure and enable bioinformatic analysis to identify additional, uncharacterized β-lactam-forming biosynthetic gene clusters by genome mining.
The membrane-targeting-sequence motif is required for exhibition of recessive resurrection inEscherichia coliRNase E
Authors: Basak Papri , Ekka Manjula , Pandiyan Apuratha , Tandon Smriti , Gowrishankar Jayaraman
Published: April 2024

The essential homo-tetrameric endoribonuclease RNase E ofEscherichia coliparticipates in global RNA turnover as well as stable RNA maturation. The protomer’s N-terminal half (residues 1-529) bears the catalytic, allosteric and tetramerization domains, including the critical active site residues D303 and D346. The C-terminal half (CTH, residues 530-1061) is dispensable for viability. We have previously described a phenomenon of recessive resurrection in RNase E that requires the CTH, wherein the wild-type homo-tetramer apparently displays nearly identical activity in vivo as a hetero-tetramer comprised of three catalytically dead subunits (with D303A/D346A substitutions) and one wild-type subunit. Here we show that recessive resurrection is exhibited even in dimeric RNase E with the CTH, and that it is largely dependent on presence of the membrane-targeting-sequence motif (residues 565-582). A single F575E substitution also impaired recessive resurrection, whereas other CTH motifs (such as those for binding of RNA or of partner proteins) were dispensable. The phenomenon was independent of RNA 5’-monophosphate sensing by the enzyme. We propose that membrane-anchoring of RNase E renders it processive for endoribonucleolytic action, and that recessive resurrection and dominant negativity associated with mutant protomers are mutually exclusive manifestations of, respectively, processive and distributive catalytic mechanisms in a homo-oligomeric enzyme.
Nuclear release of eIF1 globally increases stringency of start-codon selection to preserve mitotic arrest physiology
Authors: Ly Jimmy , Xiang Kehui , Su Kuan-Chung , Sissoko Gunter B. , Bartel David P. , Cheeseman Iain M.
Published: April 2024

Regulated start-codon selection has the potential to reshape the proteome through the differential production of uORFs, canonical proteins, and alternative translational isoforms. However, conditions under which start-codon selection is altered remain poorly defined. Here, using transcriptome-wide translation initiation site profiling, we reveal a global increase in the stringency of start-codon selection during mammalian mitosis. Low-efficiency initiation sites are preferentially repressed in mitosis, resulting in pervasive changes in the translation of thousands of start sites and their corresponding protein products. This increased stringency of start-codon selection during mitosis results from increased interactions between the key regulator of start-codon selection, eIF1, and the 40S ribosome. We find that increased eIF1-40S ribosome interactions during mitosis are mediated by the release of a nuclear pool of eIF1 upon nuclear envelope breakdown. Selectively depleting the nuclear pool of eIF1 eliminates the changes to translational stringency during mitosis, resulting in altered mitotic proteome composition. In addition, preventing mitotic translational rewiring results in substantially increased cell death and decreased mitotic slippage following treatment with anti-mitotic chemotherapeutics. Thus, cells globally control translation initiation stringency with critical roles during the mammalian cell cycle to preserve mitotic cell physiology.
Structural plasticity of 2A proteins in the Parechovirus family
Authors: Zhu Ling , Pichon Marion , Pietras Zuzanna , Wang Xiangxi , Ren Jingshan , Fry Elizabeth E. , Stuart David. I. , Perrakis Anastassis , von Castelmur Eleonore
Published: April 2024

Parechoviruses, includingParechovirus Athat infects humans as well asParechovirus B(formerly Ljungan virus) andParechovirus C(formerly Sebokele virus) that infect rodents, belong to a group of picornaviruses whose 2A proteins, instead of being proteases, contain a conserved H-box and NC-motif and are homologous to a small cellular lipid-modifying enzyme (PLAAT3) that acts as a host factor, enabling the picornavirus life cycle. Despite the common evolutionary origin, 2AH/NCproteins and PLAAT3 have no conserved function, as the active site of the viral proteins cannot support catalysis. Here, we set out to find if allParechovirusspecies share the structural rearrangement that destroys the active site configuration of the cellular enzyme. This has revealed a remarkable structural plasticity of these 2AH/NCproteins that arises not only from sequence differences between species, but also from differences in the length of the recombinantly expressed proteins, resulting in large structural rearrangements. These include rerouting of a large internal loop and repositioning of the C-terminal helix with respect to the central β-sheet, and these in turn influence the oligomeric state of the protein. We discuss how this structural plasticity could correlate with the function of these proteins in the viral life cycle and how this could recapitulate the possible evolution of this protein from host factor to viral 2AH/NCprotein, with new independent functions in RNA replication.
The association between HDL-C and stroke in people over 40 years of age: A NHANES cross-sectional study
Authors: Patel Kevin
Published: April 2024

1.

Background

Previously, studies have indicated that high-density cholesterol (HDL-C) levels are associated with lower risk of cardiovascular disease. However, this has been challenged in recent years.

Method

This cross-sectional study was conducted on data from the National Health and Nutrition Examination Survey 2011-2020. There were total of 15232 out of 45462 individuals who were age >40 years old, not missing HDL-C levels and not missing stroke data. The independent variable was HDL-Cholesterol. The outcome variable was stroke. We used multivariate logistic regression models to assess the relationship between stroke and HDL-C in three models consisting of the following explanatory variables: 1. HDL-C alone (unadjusted model), 2. HDL-C and non-modifiable risk factors (age, sex and race), 3. HDL-C, non-modifiable, and modifiable risk factors [hypertension, diabetes and smoking status] risk factors. We also conducted analyses stratified by sex and race.

Results

HDL-C categories 40-59 mg/dl (unadjusted model- 0.759, CI: 0.639, 0.901; non-modifiable risk factors adjusted- 0.668, CI: 0.558, 0.798) and 60-79 mg/dl (unadjusted model- 0.733, CI: 0.598, 0.898; non-modifiable risk factors adjusted- 0.565, CI: 0.455, 0.701) had significantly lower risk of stroke relative to HDL-C category <40 mg/dl. But when adjusted for modifiable risk factors, risk of stroke HDL-C categories 40-59 mg/dl and 60-79 mg/dl categories were not significantly different from the HDL-C <40 mg/dl category. The risk of stroke HDL-C categories 80-89 mg/dl and >90 mg/dl categories were not significantly different from the HDL-C <40 mg/dl category in all models. In stratified analyses, HDL-C was significantly inversely associated with the risk of stroke in male gender and in non-Hispanic White race.

Conclusion

Our study reveals various inverse relationship between HDL-C and risk of stroke. These findings suggest that relevant levels of HDL-C may be beneficial in preventing stroke in the absence of modifiable risk-factors.
Soft-lithographically defined template for arbitrarily patterned acoustic bioassembly
Authors: Chen Sihan , Wang Jibo , Jiang Shanqing , Fan Yuhang , Zhao Yang , Zhao Wen , Wan Zian , Zhou Qin , Chen Yun , Chen Pu
Published: April 2024

Acoustic bioassembly is recently regarded as a highly efficient biofabrication tool to generate functional tissue mimics. Despite their capacity of directly patterning live cells with close intercellular proximity, most acoustic bioassembly techniques are currently limited to generate some specific simple types of periodic and symmetric patterns, which represents an urgent challenge to emulate geometrically complex cytoarchitecture in human tissue. To address this challenge, we herein demonstrate a soft-lithographically defined acoustic bioassembly (SLAB) technique that enables to assemble live cells into geometrically defined arbitrary multicellular structures. Particularly, we employed a widely accessible soft lithography technique to fabricate a PDMS construct that works as an amplitude modulation template to define the pressure distribution of near-field acoustic waves. We found that zero pressure areas of the near-field acoustic waves at the PDMS surface distribute above the air-filling regions of the PDMS construct when both the PDMS top layer and air layer are approximately one-tenth of the acoustic wavelength. Using this technique, bioparticles can be assembled into symmetrical or asymmetrical patterns. Specifically, we have demonstrated the SLAB of endothelial spheroids and hepatic cells into liver tissue mimics (LTMs). The functional analysis further indicates that the formed LTMs displayed liver-specific functions, including albumin secretion, urea synthesis, glucose metabolism, and lipid storage. We expect this SLAB technique will be broadly used to construct complex functional tissues for tissue engineering and regenerative medicine.
Differential Causal Effects of Type 1 and Type 2 Diabetes on Osteomyelitis Risk: Insights from Mendelian Randomization Analysis
Authors: Li YanHui , Zhou Chuanyang , Yang Liming , Tan Lei
Published: April 2024

Background

Osteomyelitis (OM) poses a significant clinical challenge, especially among individuals with diabetes mellitus (DM). While both type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM) have been linked to an elevated risk of OM, the precise causal relationships remain uncertain.

Methods

We conducted Mendelian randomization (MR) analyses using summary statistics from genome-wide association studies (GWAS) to explore the causal effects of T1DM, T2DM, their complications, and glycemic traits on OM risk. The study utilized the inverse variance weighted (IVW) method, along with weighted median and MR-Egger for causal estimation, and performed various sensitivity analyses to ensure robustness. Multivariable MR (MVMR) analysis assessed direct effects, while two-step mediation MR analyses investigated the mediating role of DM between rheumatoid arthritis (RA) and OM.

Results

The MR analysis unveiled distinct causal effects of T1DM and T2DM on OM risk. Genetically determined T2DM, rather than its complications, significantly increased OM risk (primary dataset: IVW: OR = 1.13, 95% CI 1.056–1.209,p= 4E-04; validation dataset: IVW: OR = 1.317, 95% CI 1.14–1.522,p=2E-04; Meta-analysis: OR=1.206; 95% CI 1.037–1.402;p=0.014), with no observable heterogeneity or horizontal pleiotropy. MVMR analysis confirmed the robustness of the causal association between T2DM and OM, even after adjusting for potential confounders such as body mass index. Conversely, T1DM and its complications showed no significant causal link with OM in either the primary dataset (IVW:p= 0.071), the validation dataset (IVW:p= 0.276), or the meta-analysis (IVW:p= 0.242). Additionally, there was no robust evidence supporting the causal risk of glycemic traits on OM. Mediation MR analysis underscored T2DM as a pivotal contributor to the differential effects of RA on OM.

Conclusions

Mendelian randomization analysis provides compelling evidence of a significant causal relationship between genetically determined T2DM and increased OM risk, while T1DM exhibits distinct causal effects. Additionally, our findings highlight the role of T2DM in mediating the association between RA and OM. Further research is warranted to elucidate the underlying mechanisms and guide targeted interventions for OM prevention and management in diabetic populations.
State of affairs: the US platelet supply and the role of whole blood–derived platelets
Authors: Razatos Anna , King Aspen
Published: April 2024

Purpose

The US platelet supply is almost exclusively dependent on apheresis donors who are “aging out.” As a result, blood centers and hospitals have been experiencing spot shortages and have resorted to transfusing low-dose platelets. This paper explores using whole blood–derived platelets (WB-PLTs) to supplement the apheresis platelet (APH-PLT) supply.

Design/methodology/approach

This paper reviews the history leading to the current state of the US platelet supply and includes the impact of recent events such as the COVID-19 pandemic and the implementation of the US Food and Drug Administration (FDA)-mandated bacterial mitigation strategies.

Findings

WB-PLTs represent a viable source of platelets that can be used to supplement the APH-PLT supply. Whole blood automation represents a new methodology to more easily prepare WB-PLTs. Advances in donor testing and screening as well as pre-storage leukoreduction have improved the safety of WB-PLTs to the same level as APH-PLTs. Blood services in the US and abroad transfuse WB-PLTs interchangeably in all patient populations.

Originality/value

This paper highlights how the US blood industry is essentially “sole-sourced” in terms of APH-PLTs. In this post-COVID-19 period, when most industries are building redundancies in their supply chains, blood centers should consider WB-PLTs as an additional source of platelets to bolster the US platelet supply.