bluuebunny/crossref_metadata_2025 huggingface dataset:
A step forward for Shiga toxin-producing Escherichia coli identification and characterization in raw milk using long-read metagenomics
Authors: Jaudou Sandra , Deneke Carlus , Tran Mai-Lan , Schuh Elisabeth , Goehler André , Vorimore Fabien , Malorny Burkhard , Fach Patrick , Grützke Josephine , Delannoy Sabine
Published: November 2022

Shiga toxin-producing

Escherichia coli

(STEC) are a cause of severe human illness and are frequently associated with haemolytic uraemic syndrome (HUS) in children. It remains difficult to identify virulence factors for STEC that absolutely predict the potential to cause human disease. In addition to the Shiga-toxin (stx genes), many additional factors have been reported, such as intimin (eae gene), which is clearly an aggravating factor for developing HUS. Current STEC detection methods classically rely on real-time PCR (qPCR) to detect the presence of the key virulence markers (stx and eae). Although qPCR gives an insight into the presence of these virulence markers, it is not appropriate for confirming their presence in the same strain. Therefore, isolation steps are necessary to confirm STEC viability and characterize STEC genomes. While STEC isolation is laborious and time-consuming, metagenomics has the potential to accelerate the STEC characterization process in an isolation-free manner. Recently, short-read sequencing metagenomics have been applied for this purpose, but assembly quality and contiguity suffer from the high proportion of mobile genetic elements occurring in STEC strains. To circumvent this problem, we used long-read sequencing metagenomics for identifying eae-positive STEC strains using raw cow's milk as a causative matrix for STEC food-borne outbreaks. By comparing enrichment conditions, optimizing library preparation for MinION sequencing and generating an easy-to-use STEC characterization pipeline, the direct identification of an eae-positive STEC strain was successful after enrichment of artificially contaminated raw cow's milk samples at a contamination level as low as 5 c.f.u. ml−1. Our newly developed method combines optimized enrichment conditions of STEC in raw milk in combination with a complete STEC analysis pipeline from long-read sequencing metagenomics data. This study shows the potential of the innovative methodology for characterizing STEC strains from complex matrices. Further developments will nonetheless be necessary for this method to be applied in STEC surveillance.
Finding Haplotypic Signatures in Proteins
Authors: Vašíček Jakub , Skiadopoulou Dafni , Kuznetsova Ksenia G. , Wen Bo , Johansson Stefan , Njølstad Pål R. , Bruckner Stefan , Käll Lukas , Vaudel Marc
Published: November 2022

The non-random distribution of alleles of common genomic variants produces haplotypes, which are fundamental in medical and population genetic studies. Consequently, protein-coding genes with different co-occurring sets of alleles can encode different amino acid sequences: protein haplotypes. These protein haplotypes are present in biological samples, and detectable by mass spectrometry, but are not accounted for in proteomic searches. Consequently, the impact of haplotypic variation on the results of proteomic searches, and the discoverability of peptides specific to haplotypes remain unknown. Here, we study how common genetic haplotypes influence the proteomic search space and investigate the possibility to match peptides containing multiple amino acid substitutions to a publicly available data set of mass spectra. We found that for 9.96 % of the discoverable amino acid substitutions encoded by common haplotypes, two or more substitutions may co-occur in the same peptide after tryptic digestion of the protein haplotypes. We identified 342 spectra that matched to such multi-variant peptides, and out of the 4,251 amino acid substitutions identified, 6.63 % were covered by multi-variant peptides. However, the evaluation of the reliability of these matches remains challenging, suggesting that refined error rate estimation procedures are needed for such complex proteomic searches. As these become available and the ability to analyze protein haplotypes increases, we anticipate that proteomics will provide new information on the consequences of common variation, across tissues and time.
HAPPY: A deep learning pipeline for mapping cell-to-tissue graphs across placenta histology whole slide images
Authors: Vanea Claudia , Džigurski Jelisaveta , Rukins Valentina , Dodi Omri , Siigur Siim , Salumäe Liis , Meir Karen , Parks W. Tony , Hochner-Celnikier Drorith , Fraser Abigail , Hochner Hagit , Laisk Triin , Ernst Linda M. , Lindgren Cecilia M. , Nellåker Christoffer
Published: November 2022

Accurate placenta pathology assessment is essential for managing maternal and newborn health, but the placenta’s heterogeneity and temporal variability pose challenges for histology analysis. To address this issue, we developed the ‘Histology Analysis Pipeline.PY’ (HAPPY), a deep learning hierarchical method for quantifying the variability of cells and micro-anatomical tissue structures across placenta histology whole slide images. HAPPY differs from patch-based features or segmentation approaches by following an interpretable biological hierarchy, representing cells and cellular communities within tissues at a single-cell resolution across whole slide images. We present a set of quantitative metrics from healthy term placentas as a baseline for future assessments of placenta health and we show how these metrics deviate in placentas with clinically significant placental infarction. HAPPY’s cell and tissue predictions closely replicate those from independent clinical experts and placental biology literature.
CLARUS: An Interactive Explainable AI Platform for Manual Counterfactuals in Graph Neural Networks
Authors: Beinecke Jacqueline , Saranti Anna , Angerschmid Alessa , Pfeifer Bastian , Klemt Vanessa , Holzinger Andreas , Hauschild Anne-Christin
Published: November 2022

Background

Lack of trust in artificial intelligence (AI) models in medicine is still the key blockage for the use of AI in clinical decision support systems (CDSS). Although AI models are already performing excellently in systems medicine, their black-box nature entails that patient-specific decisions are incomprehensible for the physician. This is especially true for very complex models such as graph neural networks (GNNs), a common state-of-the-art approach to model biological networks such as protein-protein-interaction graphs (PPIs) to predict clinical outcomes. The aim of explainable AI (XAI) algorithms is to “explain” to a human domain expert, which input features, such as genes, influenced a specific recommendation. However, in the clinical domain, it is essential that these explanations lead to some degree of causal understanding by a clinician in the context of a specific application.

Results

We developed the CLARUS platform, aiming to promote human understanding of GNN predictions by allowing the domain expert to validate and improve the decision-making process. CLARUS enables the visualisation of the patient-specific biological networks used to train and test the GNN model, where nodes and edges correspond to gene products and their interactions, for instance. XAI methods, such as GNNExplainer, compute relevance values for genes and interactions. The CLARUS graph visualisation highlights gene and interaction relevances by color intensity and line thickness, respectively. This enables domain experts to gain deeper insights into the biological network by identifying the most influential sub-graphs and molecular pathways crucial for the decision-making process. More importantly, the expert can interactively alter the patient-specific PPI network based on the acquired understanding and initiate re-prediction or retraining. This interactivity allows to ask manual counterfactual questions and analyse the resulting effects on the GNN prediction.

Conclusion

To the best of our knowledge, we present the first interactive XAI platform prototype, CLARUS, that allows not only the evaluation of specific human counterfactual questions based on user-defined alterations of patient PPI networks and a re-prediction of the clinical outcome but also a retraining of the entire GNN after changing the underlying graph structures. The platform is currently hosted by the GWDG onhttps://rshiny.gwdg.de/apps/clarus/.
Variation in near-surface soil temperature drives plant assemblage insurance potential
Authors: Simpson Elizabeth G. , Fraser Ian , Woolf Hillary , Pearse William D.
Published: November 2022

Studying how assemblages vary across environmental gradients provides a baseline for how assemblages may respond to climate change. Per the biological insurance hypothesis, assemblages with more variation in functional diversity will maintain ecosystem functions when species are lost. In complement, environmental heterogeneity supports landscape-scale ecosystem functionality (i.e. spatial insurance), when that variation includes environments with more abundant resources.

We use the relationship between vascular plant functional diversity and microenvironment to identify where assemblages are most likely to maintain functionality in a mountainous fieldsite in northeastern Utah, USA. We assessed how life history strategies and information about phylogenetic differences affect these diversity-environment relationships.

We found less functionally dispersed assemblages, that were shorter and more resource-conservative on hotter, more variable, south-facing slopes. In contrast, we found more functionally dispersed assemblages, that were taller and more resource-acquisitive on cooler, less variable, north-facing slopes. Herbaceous and woody perennials drove these trends. Additionally, including information about phylogenetic differences in a dispersion metric indicated that phylogeny accounts for traits we did not measure.

Synthesis. At our fieldsite, soil temperature acts as an environmental filter across aspect. If soil temperature increases and becomes more variable, the function of north- vs. south-facing assemblages may be at risk for contrasting reasons. On south-facing slopes, assemblages may not have the variance in functional diversity needed to respond to more intense, stressful conditions. Conversely, assemblages on north-facing slopes may not have the resource-conservative strategies needed to persist if temperatures become hotter and more variable. We suggest that studying dispersal traits, especially of perennial species, will provide additional insight into whether this landscape will maintain function as climate changes.
Bioresorbable Mesenchymal Stem Cell–Loaded Electrospun Polymeric Scaffold Inhibits Neointimal Hyperplasia Following Arteriovenous Fistula Formation in a Rat Model of Chronic Kidney Disease
Authors: Barcena Allan John R. , Perez Joy Vanessa D. , Bernardino Marvin R. , Damasco Jossana A. , Cortes Andrea , Del Mundo Huckie C. , San Valentin Erin Marie D. , Klusman Carleigh , Canlas Gino Martin , Heralde Francisco M. , Avritscher Rony , Fowlkes Natalie , Bouchard Richard R. , Cheng Jizhong , Huang Steven Y. , Melancon Marites P.
Published: November 2022

In the setting of chronic kidney disease (CKD), the periadventitial injection of mesenchymal stem cells (MSCs) has shown significant potential in improving arteriovenous fistula (AVF) maturation by inhibiting neointimal hyperplasia (NIH). However, the rapid clearance of MSCs remains a challenge. Hence, we fabricated an electrospun perivascular scaffold from polycaprolactone (PCL) to support MSC attachment and allow gradual MSC elution at the outflow vein, the AVF site most prone to NIH. We performed a 5/6thnephrectomy to induce CKD in Sprague-Dawley rats, followed by direct AVF formation and perivascular scaffold application. We then compared the following groups of CKD rats: no perivascular scaffold (i.e., control), PCL scaffold, and PCL+MSC scaffold. On ultrasonography, the PCL and PCL+MSC groups showed significantly reduced wall thickness and wall-to-lumen ratio and increased luminal diameter and flow rate. Of note, the PCL+MSC group showed greater improvement in luminal diameter and flow rate compared to PCL alone. Moreover,18F-fluorodeoxyglucose positron emission tomography showed that only PCL+MSC resulted in a significant reduction in uptake. On histology, the PCL and PCL+MSC groups showed significantly reduced neointima-to-lumen and neointima-to-media ratios and reduced neointimal CD45, α-SMA, and vimentin fluorescence staining compared to the control. Although the two scaffold treatments did not differ significantly in histology, in vivo imaging suggested that the addition of MSCs promoted greater luminal expansion and blood flow and reduced the inflammatory process underlying NIH. Our results demonstrate the utility of mechanical support loaded with MSCs at the outflow vein immediately after AVF formation to support maturation by minimizing NIH.
Proteome mapping of the human pancreatic islet microenvironment reveals endocrine-exocrine signaling sphere of influence
Authors: Gosline Sara JC , Velickovic Marija , Pino James , Day Le Z. , Attah Isaac K. , Swensen Adam C. , Danna Vincent , Rodland Karin D. , Chen Jing , Matthews Clayton E. , Campbell-Thompson Martha , Laskin Julia , Burnum-Johnson Kristin , Zhu Ying , Piehowski Paul D.
Published: November 2022

The need for a clinically accessible method with the ability to match protein activity within heterogeneous tissues is currently unmet by existing technologies. Our proteomics sample preparation platform, named microPOTS (Microdroplet Processing in One pot for Trace Samples), can be used to measure relative protein abundance in micron-scale samples alongside the spatial location of each measurement, thereby tying biologically interesting proteins and pathways to distinct regions. However, given the smaller sample number and amount of tissue measu red, standard mass spectrometric analysis pipelines have proven inadequate. Here we describe how existing computational approaches can be adapted to focus on the specific biological questions asked in spatial proteomics experiments. We apply this approach to present an unbiased characterization of the human islet microenvironment comprising the entire complex array of tissues involved while maintaining spatial information and the degree of the islet’s sphere of influence. We identify specific functional activity unique to the pancreatic islet cells and demonstrate how far their signature can be measured. Our results show that we can distinguish pancreatic islet cells from the neighboring exocrine tissue environment, recapitulate known biological functions of islet cells, and identify a spatial gradient in the expression of RNA processing proteins within the islet microenvironment.
k2v: A Containerized Workflow for Creating VCF Files from Kintelligence Targeted Sequencing Data
Authors: Turner Stephen D. , Peck Michelle A.
Published: November 2022

The ForenSeq Kintelligence kit developed by Verogen is a targeted Illumina sequencing assay that genotypes 10,230 single nucleotide polymorphisms designed for forensic genetic genealogy, forensic DNA phenotyping, and ancestry inference. We developedk2v, a containerized workflow for creating standard specification-compliant variant call format (VCF) files from the custom output data produced by the Kintelligence Universal Analysis Software. VCF files produced withk2venable the use of many pre-existing, widely used, community-developed tools for manipulating and analyzing genetic data in the standard VCF format. Here we describe thek2vimplementation, demonstrate its usage, and use the VCF produced byk2vto demonstrate downstream analyses that can easily be performed with pre-existing tools using VCF data as input: concordance analysis, ancestry inference, and relationship estimation.k2vis distributed as a Docker container available on Docker Hub. Documentation and source code fork2vis freely available under the GNU Public License (GPL-3.0) athttps://github.com/signaturescience/k2v.
excluderanges: exclusion sets for T2T-CHM13, GRCm39, and other genome assemblies
Authors: Ogata Jonathan D. , Mu Wancen , Davis Eric S. , Xue Bingjie , Harrell J. Chuck , Sheffield Nathan C. , Phanstiel Douglas H. , Love Michael I. , Dozmorov Mikhail G.
Published: November 2022

Exclusion regions are sections of reference genomes with abnormal pileups of short sequencing reads. Removing reads overlapping them improves biological signal, and these benefits are most pronounced in differential analysis settings. Several labs created exclusion region sets, available primarily through ENCODE and Github. However, the variety of exclusion sets creates uncertainty which sets to use. Furthermore, gap regions (e.g., centromeres, telomeres, short arms) create additional considerations in generating exclusion sets. We generated exclusion sets for the latest human T2T-CHM13 and mouse GRCm39 genomes and systematically assembled and annotated these and other sets in theexcluderangesR/Bioconductor data package, also accessible via the BEDbase.org API. The package provides unified access to 82 GenomicRanges objects covering six organisms, multiple genome assemblies and types of exclusion regions. For human hg38 genome assembly, we recommendhg38.Kundaje.GRCh38_unified_blacklistas the most well-curated and annotated, and sets generated by the Blacklist tool for other organisms.

Availability and implementation

https://bioconductor.org/packages/excluderanges/

Contact

Mikhail G. Dozmorov (mdozmorov@vcu.edu)

Supplementary information

Package website:https://dozmorovlab.github.io/excluderanges/
Differential requirement ofnanoshomologs in the germline suggests the evolutionary path toward an inheritance mechanism of primordial germ cell formation in the silkmothBombyx
Authors: Nakao Hajime , Takasu Yoko
Published: November 2022

The lepidopteran insectBombyx moripossesses unique embryogenesis characteristics among insects.nanos(nos) has conserved functions in metazoan primordial germ cell formation.Bombyxpossesses fournosgenes (M, N, O, P), a unique feature found in lepidopterans examined so far. Of these, maternalnosOmRNA exhibits a localization pattern: it may act as a primordial germ cell (PGC) determinant. A previous knock-out experiment ofnosOshowed that this localized mRNA is dispensable for PGC formation in laboratory environment and has limited involvement in PGC specification. This study examined whether other nos genes act redundantly withnosOin germline using RNAi and gene editing. Although individual embryonic RNAi exhibited no detectable phenotypic alterations, simultaneous RNAi ofnosO/nosPmarkedly reduced oocyte number and male fecundity. Additionally,nosPKO almost completely sterilized both sexes. BecausenosPis broadly expressed in the posterior of embryos in non-germline specific manner, these results could reflect an evolutionary step taken byBombyxtoward its unique inheritance mechanisms. This study also suggests thatnosgenes inBombyxdo not affect anterior-posterior axis specification. This could reflect its characteristic embryogenesis.
Re-evaluating management of established pests including the European wasp,Vespula germanicausing biocontrol agents
Authors: Cacho Oscar , Hester Susie , Tait Peter , Kwong Raelene , Lefoe Greg , Rutherford Paul , Kriticos Darren
Published: November 2022

Established pests such as the European wasp (Vespula germanica) are often overlooked as candidates for management programmes (eradication and/or containment) because the use of traditional surveillance and control techniques over very large areas becomes uneconomic. Use of biological control agents that persist in the environment is usually the only economically feasible option, however the processes around approvals for release of biocontrol agents can take significant amounts of time and resources, especially if screening and testing of potential agents is required.

This project investigates whether the European wasp could be a candidate for a renewed management programme in south-eastern Australia given the availability of a biocontrol agent following successful screening and testing of an agent,Sphecophaga vesparum vesparum, in the 1980s. Whether a biological control programme is worthwhile pursuing depends on the size of the benefits to industry, community and the environment from a reduction in European wasp abundance. This project explores the benefits and costs of European wasp management using a biocontrol agent, and importantly, includes valuation of the social and environmental impacts of the pest.
Structure and function ofPlasmodiumactin II in the parasite mosquito stages
Authors: Lopez Andrea J. , Andreadaki Maria , Vahokoski Juha , Deligianni Elena , Calder Lesley J. , Camerini Serena , Freitag Anika , Bergmann Ulrich , Rosenthal Peter B. , Sidén-Kiamos Inga , Kursula Inari
Published: November 2022

Actins are filament-forming, highly-conserved proteins in eukaryotes. They are involved in essential processes in the cytoplasm and have also nuclear functions. Malaria parasites (Plasmodiumspp.) have two actin isoforms that differ from each other and from canonical actins in structure and filament-forming properties. Actin I has an essential role in motility and is fairly well characterized. The structure and function of actin II are not as well understood, but mutational analyses have revealed two essential functions in male gametogenesis and in the zygote. Here, we present expression analysis, high-resolution filament structures, and biochemical characterization ofPlasmodiumactin II. We show that it is expressed in male gametocytes and zygotes and associated with the nucleus in both stages in filament-like structures. Unlike actin I, actin II readily forms long filamentsin vitro, and near-atomic structures in the presence or absence of jasplakinolide reveal very similar structures. Small but significant differences compared to other actins in the openness and twist, the active site, the D-loop, and the plug region contribute to filament stability. Mutational analyses suggest that long and stable filaments are necessary for male gametogenesis, while the second function in the zygote stage also requires finetuned regulation by methylation of histidine 73. Actin II polymerizesviathe classical nucleation-elongation mechanism and has a critical concentration of ~0.1 μM at the steady-state, like actin I and canonical actins. Similarly to actin I, dimers are a stable form of actin II at equilibrium.

Significance statement

Malaria is a parasitic infection caused byPlasmodiumspp., which belong to the phylum Apicomplexa. In 2020, approximately 627000 people died from nearly 241 million malaria cases registered worldwide. In contrast to other apicomplexan parasites,Plasmodiumspp. encode two actin isoforms. While actin I is part of the glideosome complex, essential for locomotion, actin II is present in the mosquito stages, where it has functions in gametogenesis and in the zygote. The exact function of actin II is still unclear, and information at the molecular level is limited. We show here that actin II is associated with the nucleus in gametocytes and zygotes and performs specific functions requiring both long filaments and fine-tuning by methylation of histidine 73. We determined the structures of the filamentous form of actin II at near-atomic resolution and characterized its polymerization propertiesin vitro. Our study provides a molecular basis for the differences between actins of the malaria parasite and humans, as well as between the two parasite actin isoforms. Furthermore, thein vivostudies provide insights into the function of actin II in the parasite.
Integrated Multi-omics Analyses of NFKB1 patients B cells points towards an up regulation of NF-κB network inhibitors
Authors: Camacho-Ordonez Nadezhda , Ramirez Neftali , Posadas-Cantera Sara , Caballero-Oyteza Andrés , Fliegauf Manfred , Zhao Fangwen , Guarini Maria , Gernedl Victoria , Pecoroaro Mateo , Warnatz Klaus , Bock Christoph , Ballestar Esteban , Geiger Roger , Proietti Michele , Grimbacher Bodo
Published: November 2022

The transcription factor NF-κB plays a pivotal role in the adaptive immune response. Pathogenic variants inNFKB1are the most common genetic etiology of common variable immunodeficiency (CVID). Patients frequently present with impaired terminal B cell differentiation, autoimmunity, and hyperinflammatory immune dysregulation. NF-κB signaling and target gene expression are expected to be dysregulated inNFKB1-mutated patients. Here, we performed a multi-omics characterization of B cells from a cohort of clinically affected and unaffectedNFKB1mutation carriers. Our analysis identified specific epigenetic dysregulation and gene expression differences on B cells fromNFKB1-mutated patients. We observed an aberrant expression of negative regulators of NF-κB signaling inNFKB1mutation carriers, which may be a key factor for the autoinflammatory phenotype of these patients. Moreover, our analysis points towards a dysregulation ofXBP1andBCL3, key players of B cell activation and proliferation at different stages of B cell differentiation. The reduced expression of negative regulators of the NF-κB network is likely to be one of several mechanisms responsible for the aberrant NF-κB signaling, which impairs the maintenance of a normal humoral immune response. In summary, our findings highlight epigenetic and gene expression changes in B cells associated withNFKB1mutations. Our data give insight into future therapeutic opportunities for patients withNFKB1(haplo)insufficiency.
Monkeypox virus quadrivalent mRNA vaccine induces antibody responses and cellular immunity and protects mice against Vaccinia virus
Authors: Sang Ye , Zhang Zhen , Liu Fan , Lu Haitao , Yu Changxiao , Sun Huisheng , Long Jinrong , Cao Yiming , Mai Jierui , Wang Xin , Fang Jiaxin , Wang Youchun , Huang Weijin , Yang Jing , Wang Shengqi
Published: November 2022

There is an urgent need for efficient and safe vaccines against the monkeypox virus (MPXV) in response to the rapidly spreading monkeypox epidemic. In the age of COVID-19, mRNA vaccines have been highly successful and emerged as platforms enabling rapid development and large-scale preparation. Here, we have developed two MPXV quadrivalent mRNA vaccines, named mRNA-A-LNP and mRNA-B-LNP, based on two IMVs (A29L and M1R) and two EEVs (A35R and B6R). By administering mRNA-A-LNP and mRNA-B-LNP intramuscularly twice, mice have induced MPXV-specific IgG antibodies and potent Vaccinia virus (VACV)-specific neutralizing antibodies. Additionally, it elicited durable MPXV-specific killer memory T-cell immunity as well as memory B-cell immunity in mice. Furthermore, the passive transfer of sera from mRNA-A-LNP and mRNA-B-LNP-immunized mice protected nude mice against the VACV challenge. In addition, two doses of mRNA-A-LNP and mRNA-B-LNP were also protective against the VACV challenge in mice. Overall, our results demonstrated that mRNA-A-LNP and mRNA-B-LNP appear to be safe and effective vaccine candidates against monkeypox epidemics, as well as against outbreaks caused by other orthopoxviruses, including the smallpox virus.
Identification of differential polypharmacology between the PARP inhibitor rucaparib and its major metabolite
Authors: Hu Huabin , Serra Carme , Llebaria Amadeu , Antolin Albert A.
Published: November 2022

The (poly)pharmacology of drug metabolites is seldom comprehensively characterized in drug discovery and development. However, some drug metabolites can reach high plasma concentrations and display relevant in vivo activity, which can be distinct from its parent drug. Here, we use computational and experimental methods to comprehensively characterise the kinase polypharmacology of M324, the major metabolite of the FDA-approved PARP inhibitor rucaparib. We experimentally demonstrate that M324 displays a distinct in vitro kinome profile from its parent drug, characterized by potent in vitro inhibition of GSK3A and PLK2 at clinically-relevant concentrations. These confirmed kinase activities of M324 could have potential implications for the efficacy and safety of rucaparib and therefore warrant further clinical investigation. The study reported here highlights the importance of thoroughly characterizing the activity of significant drug metabolites to better understanding drug responses in the clinic and maximally exploit the current drug arsenal in personalized and precision medicine.
NAT10, an RNA acetyl cytidine transferase restrains ferroptosis in cancer cells by maintaining SLC7A11 RNA stability
Authors: Dalhat Mahmood Hassan , Choudhry Hani , Khan Mohammad Imran
Published: November 2022

Recently, we reported that N-acetyltransferase 10 (NAT10) regulates fatty acid metabolism through ac4C-dependent RNA modification of key genes in cancer cells. During this work, we noticed ferroptosis as one of the most negatively enriched pathways among other pathways in NAT10 depleted cancer cells. In the current work, we explored the possibility of whether NAT10 acts as an epitrancriptomic regulator of ferroptosis pathway in cancer cells. Global ac4C levels and expression of NAT10 with other ferroptosis-related genes were assessed via dotblot and RT-qPCR respectively. Flow cytometry and biochemical analysis were used to assess oxidative stress and ferroptosis features. The ac4C mediated mRNA stability was conducted RIP-PCR and mRNA stability assay. Metabolites were profiled using LC-MS/MS. Our results showed significant downregulation in expression of essential genes related to ferroptosis namelySLC7A11, GCLC, MAP1LC3A, andSLC39A8in NAT10 depleted cancer cells. Further, we noticed a reduction in cystine uptake and reduced GSH levels along with elevated ROS, and lipid peroxidation levels in NAT10 depleted cells. Consistently, overproduction of oxPLs as well as increased mitochondrial depolarization and decreased activities of antioxidant enzymes support the notion of ferroptosis induction in NAT10 depleted cancer cells. Mechanistically, reduced ac4C level shortens the half-life ofGCLCandSLC7A11mRNA, resulting in low levels of intracellular cystine and reduced GSH, failing to detoxify ROS leading to increased cellular oxPLs which facilitates ferroptosis induction. Collectively, our findings suggest that NAT10 restrains ferroptosis by stabilizing the SLC7A11 mRNA transcripts to avoid oxidative stress that induces oxidation of phospholipids to initiate ferroptosis.
MuVEH and mitoMuVEH improve discovery of genetic variation from single cells
Authors: Ransom Monica R. , Engel Krysta L. , Stevens Brett M. , Jordan Craig T. , Gillen Austin E.
Published: November 2022

Understanding the genetic underpinnings and clonal structure of malignancies at single-cell resolution is critical to accurately predicting drug response and understanding mechanisms of drug resistance and disease evolution in heterogeneous populations of cells. Here, we introduce an accessible, multiplexable, targeted mutation enrichment approach and end-to-end analysis pipeline called MuVEH (Multiplexed Variant Enrichment by Hybridization) that increases the resolution of variant detection in scRNA-seq analysis. When applied specifically to the mitochondrial chromosome (“mitoMuVEH”), this technique can also be used to reconstruct and trace clonal relationships between individual cells. We applied both approaches to two pairs of primary bone marrow specimens from acute myelogenous leukemia (AML) patients collected at diagnosis and after relapse following Venetoclax+Azacitidine (Ven/Aza) therapy. Used together, MuVEH and mitoMuVEH reveal clonal evolution and changing mutational burden in response to treatment at single-cell resolution in these patients. Ultimately, these approaches have the potential to extract additional biological insights from precious patient samples and provide insight into the contributions clonality and genotype have during disease progression.
A singleVerticillium dahliaeeffector determines pathogenicity on tomato by targeting auxin response factors
Authors: Li Jinling , Faino Luigi , Fiorin Gabriel L. , Bashyal Sagar , Schaveling Arno , van den Berg Grardy C.M. , Seidl Michael F. , Thomma Bart P.H.J.
Published: November 2022

Verticillium dahliaeis a xylem-invading fungal pathogen that causes devastating vascular wilt diseases on hundreds of plant hosts, including tomato (Solanum lycopersicum). Although individualV. dahliaestrains are typically characterized by their broad host range, differential pathogenicity occurs on nearly all hosts. Currently, the molecular basis underlying such pathogenicity differences remains unknown. We used comparative genomics to identify a single effector gene that specifically occurs in tomato-pathogenicV. dahliaestrains and is expressed during tomato colonization. Functional analyses showed that this Tom1 effector governs pathogenicity on tomato, asTom1deletion prohibited tomato colonization, while introduction ofTom1into non-pathogenicV. dahliaeor into saprophytic sister speciesV. tricorpusandV. nubilumresulted in disease. Through proteomics-based approaches, auxin response factors (ARFs) were identified asin plantatargets of Tom1. Intriguingly, repression ofSlARF2aexpression by virus-induced gene silencing fully impairedV. dahliaecolonization of tomato, solidifying its role as susceptibility target.Collectively, our findings indicate that a single effector, Tom1, mediates pathogenicity ofV. dahliaeon tomato by targeting auxin response factors.
Pump it up: bioelectric stimulation controls tissue shape and size
Authors: Shim Gawoon , Breinyn Isaac B. , Martínez-Calvo Alejandro , Sameeksha  , Cohen Daniel J.
Published: November 2022

Epithelial tissues sheath many organs, separating ‘outside’ from ‘inside’ and exquisitely regulating ion and water transport electromechanically to maintain homeostatic balance and tissue hydrostatic pressure. While it is increasingly clear that the ionic microenvironment and external electric stimuli can affect epithelial function and behavior, the coupling between electrical perturbation and tissue form remain unclear. We investigated this by combining electrical stimulation with three-dimensional epithelial tissues with hollow ‘lumens’—both kidney cysts and complex intestinal stem cell organoids. Our core finding is that physiological strength electrical stimulation of order 1-3 V/cm (with both direct and alternating currents) can drive powerful and rapid inflation of hollow tissues through a process we call ‘electro-inflation’, inducing up to a threefold increase in tissue volume and striking asymmetries in tissue form. Electro-inflation is primarily driven by field-induced ion crowding on the outer surface of the hollow tissue that creates an ion gradient across the epithelial shell, which drives increased ionic flux mediated by ion channels/transporters and subsequent osmotic water flow into the lumen. This influx generates hydrostatic pressure, and inflation results from a competition between this pressure and cell cytoskeletal tension. We validated these interpretations with computational models connecting ion crowding around tissues to tissue mechanics. Electrically stimulated cysts and organoids also exhibited pronounced asymmetry, where the epithelial shell thickened on the cathode-facing side and thinned on the anode-facing side of the tissue. We discovered that this process is the result of 3D electrotaxis–directed migration of cells in an electric field–causing a redistribution of cells around the shell. The ability of electrical cues to dramatically regulate tissue size and shape highlight the key role of the electrical micro-environment and the potential offered by manipulating these signals.
A Novel Approach to Topological Network Analysis for the Identification of Metrics and Signatures in Non-Small Cell Lung Cancer
Authors: Wu Isabella , Wang Xin
Published: November 2022

Non-small cell lung cancer (NSCLC), the primary histological form of lung cancer, accounts for about 25% - the highest - of all cancer deaths. As NSCLC is often undetected until symptoms appear in the late stages, it is imperative to discover more effective tumor-associated biomarkers for early diagnosis. Topological data analysis is one of the most powerful methodologies applicable to biological networks. However, current studies fail to consider the biological significance of their quantitative methods and utilize popular scoring metrics without verification, leading to low performance. To extract meaningful insights from genomic data, it is essential to understand the relationship between geometric correlations and biological function mechanisms. Through bioinformatics and network analyses, we propose a novel composite selection index, the C-Index, that best captures significant pathways and interactions in gene networks to identify biomarkers with the highest efficiency and accuracy. Furthermore, we establish a 4-gene biomarker signature that serves as a promising therapeutic target for NSCLC and personalized medicine. We designed a Cascading machine learning model to validate both the C-Index and the biomarkers discovered. The methodology proposed for finding top metrics can be applied to effectively select biomarkers and early diagnose many diseases, revolutionizing the approach to topological network research for all cancers.
Not all pathways are the same – unique adaptations to submerged environments emerge from comparative seagrass genomics
Authors: Bayer Philipp E , Fraser Matthew W. , Martin Belinda C. , Petereit Jakob , Severn-Ellis Anita A. , Sinclair Elizabeth A. , Batley Jacqueline , Kendrick Gary A. , Edwards David
Published: November 2022

Seagrasses are an ecologically important group of plants that have returned to the sea from terrestrial ancestors on at least three occasions (Cymodoceaceae, Posidoniaceae, Zosteraceae). Seagrass-specific genomic adaptations to marine life are known from the Zosteraceae. However, independent lineages may have devised different solutions to life underwater. Here, we present two new genome assemblies from endemic Australian seagrasses,Amphibolis antarctica(Cymodoceaceae) andPosidonia australis(Posidoniaceae). We found large differences in genome size betweenAmphibolisandPosidoniadriven by repeat expansion inPosidonia. We show that parts of ethylene pathways known to be lost in Zosteraceae are partially retained in older seagrass lineages (Cymodoceaeceae and Posidoniaceae). We describe adaptations within salinity, disease resistance, cell wall, and photosynthesis-related pathways not shared with other seagrasses. These findings provide insight into the impact of recolonising marine environments on formerly terrestrial plant genomes, with some adaptations previously thought to be universal to marine living not having occurred inA. antarcticaandP. australis.
The synergistic anti-Warburg efficacy of temozolomide, metformin and epigallocatechin gallate in glioblastoma
Authors: Kuduvalli Shreyas S. , Precilla Daisy S , Biswas Indrani , Anitha T. S
Published: November 2022

Background

An important hallmark of glioblastoma aggressiveness is its altered metabolism of glucose. This metabolic shift wherein the tumor cells employ aerobic glycolysis regardless of oxygen availability via reprogramming of mitochondrial oxidative phosphorylation is known as the Warburg effect. Previous literatures have linked this metabolic reprograming to tumor progression glioblastoma cell proliferation making it a key target for targeted drug therapy.

Objective

To evaluate the anti-Warburg efficacies of the triple-drug combination of temozolomide, metformin and epigallocatechin gallate in preclinical glioblastoma models.

Methodology

Based on this lacuna, the current study aimed to explore the therapeutic efficacy of the triple-drug combination of temozolomide, metformin and epigallocatechin gallate in attenuating Warburg effect and glucose uptake in glioblastoma bothin vitroandin vivo.

Results

Our results showed that the triple-drug combination had significantly reduced glucose uptake and reversed the Warburg effect in glioblastoma cells and in the xenograft-induced glioma rat model.

Conclusion

Thus, the triple-drug combination would serve as an effective therapeutic regime to hamper glioblastoma progression via altering glucose metabolism and improve the overall prognosis in patient setting.
Functional promiscuity of small multidrug resistance transporters fromStaphylococcus aureus,Pseudomonas aeruginosa, andFrancisella tularensis
Authors: Spreacker Peyton J. , Porter Colin J. , Wegrzynowicz Andrea , Beeninga Will F. , Demas Sydnye , Powers Emma N. , Henzler-Wildman Katherine A.
Published: November 2022

Small multidrug resistance (SMR) transporters efflux toxic substrates from bacterial cells and were recently divided into two subfamilies: specific toxic metabolite transporters and promiscuous drug exporters. These drug exporters are thought to function similarly to EmrE, the model system for this subfamily of SMR transporters. Studies of EmrE homologs indicate that they are able to confer resistance to EmrE substrates inE. coliand in their native organisms. Recent work from our lab showed that functional EmrE can confer resistance or susceptibilityin vivodepending on the drug substrate. Here, we test whether this functional promiscuity of EmrE extends to SMR transporters from three additional human or animal pathogens: SAsmr fromStaphylococcus aureus, PAsmr fromPseudomonas aeruginosa, and FTsmr fromFrancisella tularensis. We find that these SMR homologs can confer either resistance or susceptibility to different toxic substrates inE. coli. This demonstrates that the ability of a single transporter to lead to opposite biological outcomes when transporting different substrates is a general property of the promiscuous multidrug transporters in the SMR family. It also suggests the potential for novel antibiotic development targeting these transporters with small molecules that trigger susceptibility. Such a strategy does not require that the target be the primary mode for antibiotic resistance because the goal is not simple inhibition of activity, but rather activation of an alternative transport function that is detrimental to bacteria.
Holistic monitoring of freshwater and terrestrial vertebrates by camera trapping and environmental DNA
Authors: Rubæk Holm Anne Marie , Knudsen Steen Wilhelm , Månsson Malene , Pedersen Ditte Elmgreen , Nordfoss Pauli Holm , Johansson Daniel Klingberg , Gramsbergen Marthe , Havmøller Rasmus Worsøe , Sigsgaard Eva Egelyng , Thomsen Philip Francis , Olsen Morten Tange , Møller Peter Rask
Published: November 2022

The anthropogenic impact on the world’s ecosystems is severe and the need for non-invasive, cost-effective tools for monitoring and understanding those impacts are therefore urgent. Here we combine two such methods in a comprehensive multi-year study; camera trapping (CT) and analysis of environmental DNA (eDNA), in river marginal zones of a temperate, wetland Nature Park in Denmark. CT was performed from 2015 to 2019 for a total of 8,778 camera trap days and yielded 24,376 animal observations. The CT observations covered 87 taxa, of which 78 were identified to species level, and 73 were wild native species. For eDNA metabarcoding, a total of 114 freshwater samples were collected from eight sites in all four seasons from 2017 to 2018. The eDNA results yielded a total detection of 80 taxa, of which 74 were identified to species level, and 65 were wild native species. While the number of taxa detected with the two methods were comparable, the species overlap was only 20 %. In combination, CT and eDNA monitoring thus yielded a total of 115 wild species (20 fishes, four amphibians, one snake, 23 mammals and 67 birds), representing half of the species found via conventional surveys over the last ca. 20 years (83% of fishes, 68 % of mammals, 67 % of amphibians, 41 % of birds and 20 % of reptiles). Our study demonstrates that a holistic approach combining two non-invasive methods, CT and eDNA metabarcoding, has great potential as a cost-effective biomonitoring tool for vertebrates.
Prophage regulation ofShewanella fidelis3313 motility and biofilm formation: implications for gut colonization dynamics inCiona robusta
Authors: Natarajan Ojas , Gibboney Susanne L. , Young Morgan N. , Lim Shen Jean , Nguyen Felicia , Pluta Natalia , Atkinson Celine G.F. , Liberti Assunta , Kees Eric D. , Leigh Brittany A. , Breitbart Mya , Gralnick Jeffrey A. , Dishaw Larry J.
Published: November 2022

Lysogens, bacteria that contain viruses (prophages) integrated into their genomes, are abundant in the gut of animals. Prophages often influence bacterial traits; however, the influence of prophages on the gut microbiota-host immune axis in animals remains poorly understood.Here, we investigate the influence of the prophage SfPat onShewanella fidelis3313, a persistent member of the gut microbiome of the model marine tunicate,Ciona robusta. Establishment of a SfPat deletion mutant (ΔSfPat) reveals the influence of this prophage on bacterial physiologyin vitroand during colonization of theCionagut.In vitro, deletion of SfPat reducesS. fidelis3313 motility and swimming while increasing biofilm formation. To understand thein vivoimpact of these prophage-induced changes in bacterial traits, we exposed metamorphic stage 4Cionajuveniles to wildtype (WT) and ΔSfPat strains. During colonization, ΔSfPat localizes to distinct areas of the gut compared to the WT strain. We tested the differential expression of various regulators of cyclic-di-GMP, a secondary signaling molecule known to mediate biofilm formation and motility. The pdeB gene, which encodes a bacterial phosphodiesterase known to influence biofilm formation and motility by degrading cyclic-di-GMP, is upregulated in the WT strain but not in ΔSfPat when examinedin vivo. Expression of theCionagut immune effector, VCBP-C, is enhanced during colonization by ΔSfPat compared to the WT strain; however, VCBP-C binding to the WT strain does not promote the excision of SfPat in an SOS-dependent pathway. Our findings suggest that SfPat influences host perception of this important colonizing commensal and highlights the significance of investigating interkingdom dynamics between prophages, bacteria, and their animal hosts to better understand the gut microbiota-host immune axis.
miRNome profiling of lung cancer metastases revealed a key role for miRNA-PD-L1 axis in the modulation of chemotherapy response
Authors: Cuttano Roberto , Colangelo Tommaso , Guarize Juliana , Dama Elisa , Cocomazzi Maria Pia , Mazzarelli Francesco , Melocchi Valentina , Palumbo Orazio , Marino Elena , Belloni Elena , Montani Francesca , Vecchi Manuela , Barberis Massimo , Graziano Paolo , Pasquier Andrea , Sanz-Ortega Julian , Montuenga Luis M. , Carbonelli Cristiano , Spaggiari Lorenzo , Bianchi Fabrizio
Published: November 2022

Locally-advanced non–small-cell lung cancer (NSCLC) is frequent at diagnosis and requires multimodal treatment approaches. Neoadjuvant chemotherapy (NACT) followed by surgery is the treatment of choice for operable locally-advanced NSCLC (Stage IIIA). However, the majority of patients are NACT-resistant and shows persistent lymph nodal metastases (LNmets) and an adverse outcome. Therefore, the identification of mechanisms and biomarkers of NACT resistance is paramount for ameliorating prognosis of patients with Stage IIIA NSCLC. Here, we investigated the miRNome and transcriptome of chemo naïve LNmets collected from patients with Stage IIIA NSCLC (N=64). We found that a microRNA signature accurately predicts NACT response. Mechanistically, we discovered a miR-455-5p/PD-L1 regulatory axis which drives chemotherapy resistance, hallmarks metastases with active IFN-γresponse pathway (an inducer of PD-L1 expression), and impacts T cells viability and relative abundances in tumor-microenviroment (TME). Our data provides new biomarkers to predict NACT response and adds molecular insights relevant for improving the management of patients with locally-advanced NSCLC.