bluuebunny/crossref_metadata_2025 huggingface dataset:
GENA-LM: A Family of Open-Source Foundational DNA Language Models for Long Sequences
Authors: Fishman Veniamin , Kuratov Yuri , Shmelev Aleksei , Petrov Maxim , Penzar Dmitry , Shepelin Denis , Chekanov Nikolay , Kardymon Olga , Burtsev Mikhail
Published: June 2023

Recent advancements in genomics, propelled by artificial intelligence, have unlocked unprecedented capabilities in interpreting genomic sequences, mitigating the need for exhaustive experimental analysis of complex, intertwined molecular processes inherent in DNA function. A significant challenge, however, resides in accurately decoding genomic sequences, which inherently involves comprehending rich contextual information dispersed across thousands of nucleotides. To address this need, we introduce GENA-LM, a suite of transformer-based foundational DNA language models capable of handling input lengths up to 36,000 base pairs. Notably, integration of the newly-developed Recurrent Memory mechanism allows these models to process even larger DNA segments. We provide pre-trained versions of GENA-LM, demonstrating their capability for fine-tuning and addressing a spectrum of complex biological tasks with modest computational demands. While language models have already achieved significant breakthroughs in protein biology, GENA-LM showcases a similarly promising potential for reshaping the landscape of genomics and multi-omics data analysis. All models are publicly available on GitHub https://github.com/AIRI-Institute/GENA LM and HuggingFace https://huggingface.co/AIRI-Institute.
The influence of social lifestyles on host-microbe symbioses in the bees
Authors: Mee Lauren , Barribeau Seth M
Published: June 2023

1

Microbiomes are increasingly recognised as critical for the health of an organism. In eusocial insect societies, frequent social interactions allow for high fidelity transmission of microbes across generations, leading to closer host-microbe coevolution. The microbial communities of bees with different social lifestyles are less well studied, and few comparisons have been made between taxa that vary in social structure. To address this gap, we leveraged a cloud-computing resource and publicly available transcriptomic data to conduct a survey of microbial diversity in bee samples from a variety of social lifestyles and taxa. We consistently recover the core microbes of well-studied corbiculates, supporting this method’s ability to accurately characterise microbial communities. We find that the bacterial communities of bees are influenced by host location, phylogeny, and social lifestyle, although no clear effect was found for fungal or viral microbial communities. Bee genera with more complex societies tend to harbour more diverse microbes, withWolbachiadetected more commonly in solitary tribes. We present the first description of the microbiota of Euglossine bees and find that they do not share the “corbiculate” core microbiome. Notably, we find that bacteria with known anti-pathogenic properties are present across social bee genera, suggesting that symbioses that enhance host immunity are important with higher sociality. Our approach provides an inexpensive means of exploring microbiomes of a given taxa and identifying avenues for further research. These findings contribute to our understanding of the relationships between bees and their associated microbial communities, highlighting the importance of considering microbiome dynamics in investigations of bee health.
Tetraspanins fromOpisthorchis viverrinistimulate cholangiocyte migration and inflammatory cytokine production
Authors: Ruangsuwast Apisit , Smout Michael J. , Brindley Paul J. , Loukas Alex , Laha Thewarach , Chaiyadet Sujittra
Published: June 2023

The liver flukeOpsithorchis viverrinisecretes extracellular vesicles (EVs) bearing CD63-like tetraspanins on their surface. Fluke EVs are actively internalized by host cholangiocytes in the bile ducts, where they drive pathology and promote neoplasia through induction of cellular proliferation and secretion of inflammatory cytokines. We investigated the effects of tetraspanins of the CD63 superfamily by co-culturing recombinant forms of the large extracellular loop (LEL) ofO. viverrinitetraspanin-2 (rLEL-Ov-TSP-2) and tetraspanin-3 (rLEL-Ov-TSP-3) with non-cancerous human bile duct (H69) and cholangiocarcinoma (CCA, M213) cell lines. The results showed that cell lines co-cultured with excretory/secretory products from adultO. viverrini(Ov-ES) underwent significantly increased cell proliferation at 48 hours but not 24 hours compared to untreated control cells (P<0.05), whereas rLEL-Ov-TSP-3 co-culture resulted in significantly increased cell proliferation at both 24 hr (P<0.05) and 48 hr (P<0.01) time points. In like fashion, H69 cholangiocytes co-cultured with bothOv-ES and rLEL-Ov-TSP-3 underwent significantly elevatedIl-6andIl-8gene expression for at least one of the time points assessed. Finally, both rLEL-Ov-TSP-and rLEL-Ov-TSP-3 significantly enhanced migration of both M213 and H69 cell lines. These findings indicated thatO. viverriniCD63 family tetraspanins can promote a cancerous microenvironment by enhancing innate immune responses and migration of biliary epithelial cells.
BERTrand - peptide:TCR binding prediction using Bidirectional Encoder Representations from Transformers augmented with random TCR pairing
Authors: Myronov Alexander , Mazzocco Giovanni , Król Paulina , Plewczynski Dariusz
Published: June 2023

Motivation

The advent of T cell receptor (TCR) sequencing experiments allowed for a significant increase in the amount of peptide:TCR binding data available and a number of machine learning models appeared in recent years. High-quality prediction models for a fixed epitope sequence are feasible, provided enough known binding TCR sequences are available. However, their performance drops significantly for previously unseen peptides.

Results

We prepare the dataset of known peptide:TCR binders and augment it with negative decoys created using healthy donors’ T-cell repertoires. We employ deep learning methods commonly applied in Natural Language Processing (NLP) to train part a peptide:TCR binding model with a degree of cross-peptide generalization (0.66 AUROC). We demonstrate that BERTrand outperforms the published methods when evaluated on peptide sequences not used during model training.

Availability

The datasets and the code for model training are available athttps://github.com/SFGLab/bertrand

Contact

alexander.myronov@gmail.com,dariusz.plewczynski@pw.edu.pl

Supplementary information

Supplementary data are available atBioinformaticsonline.
ModularImageAnalysis (MIA): Assembly of modularised image and object analysis workflows in ImageJ
Authors: Cross Stephen J. , Fisher Jordan D. J. R. , Jepson Mark A.
Published: June 2023

ModularImageAnalysis (MIA) is an ImageJ plugin providing a code-free graphical environment in which complex automated analysis workflows can be constructed and distributed. The near 200 included modules cover all stages of a typical analysis workflow, from image loading through image processing, object detection, extraction of measurements, measurement-based filtering, visualisation, and data exporting. MIA provides out-of-the-box compatibility with many advanced image processing plugins for ImageJ including Bio-Formats, DeepImageJ, MorphoLibJ and TrackMate, allowing these tools and their outputs to be directly incorporated into analysis workflows. By default, modules support spatially calibrated 5D images, meaning measurements can be acquired in both pixel and calibrated units. A hierarchical object relationship model allows for both parent-child (one-to-many) and partner (many-to-many) relationships to be established. These relationships underpin MIA’s ability to track objects through time, represent complex spatial relationships (e.g. skeletons) and measure object distributions (e.g. count puncta per cell). MIA features dual graphical interfaces: the “editing view” offers access to the full list of modules and parameters in the workflow, while the simplified “processing view” can be configured to display only a focused subset of controls. All workflows are batch-enabled by default, with image files within a specified folder being processed automatically and exported to a single spreadsheet. Beyond the included modules, functionality can be extended both internally, through integration with the ImageJ scripting interface, and externally, by developing third-party Java modules that extend the core MIA framework. Here we describe the design and functionality of MIA in the context of a series of real-world example analyses.
A hybrid RNA FISH immunofluorescence protocol onDrosophilapolytene chromosomes
Authors: Gilbonio Hannah E. , Puckett Gwyn L. , Nguyen Erica , Rieder Leila E.
Published: June 2023

Objectives

Investigating protein-DNA interactions is imperative to understanding fundamental concepts such as cell growth, differentiation, and cell development in many systems. Sequencing techniques such as ChIP-seq can yield genome-wide DNA binding profiles of transcription factors; however this assay can be expensive, time-consuming, may not be informative for repetitive regions of the genome, and depend heavily upon antibody suitability. Combining DNA fluorescence in situ hybridization (FISH) with immunofluorescence (IF) is a quicker and inexpensive approach which has historically been used to investigate protein-DNA interactions in individual nuclei. However, these assays are sometimes incompatible due to the required denaturation step in DNA FISH that can alter protein epitopes, hindering primary antibody binding. Additionally, combining DNA FISH with IF may be challenging for less experienced trainees. Our goal was to develop an alternative technique to investigate protein-DNA interactions by combining RNA FISH with IF.

Results

We developed a hybrid RNA FISH and IF protocol for use onDrosophila melanogasterpolytene chromosome spreads in order to visualize colocalization of proteins and DNA loci. We demonstrate that this assay is sensitive enough to determine if our protein of interest, Multi-sex combs (Mxc), localizes to single-copy target transgenes carrying histone genes. Overall, this study provides an alternative, accessible method for investigating protein-DNA interactions at the single gene level inDrosophila melanogasterpolytene chromosomes.
Combined direct/indirect detection allows identification of DNA termini in diverse sequencing datasets and supports a multiple-initiation-site model for HIV plus-strand synthesis
Authors: Wang William , Artiles Karen L. , Machida Shinichi , Benkirane Monsef , Jain Nimit , Fire Andrew Z.
Published: June 2023

Replication of genetic material involves the creation of characteristic termini. Determining these termini is important to refine our understanding of the mechanisms involved in maintaining the genomes of cellular organisms and viruses. Here we describe a computational approach combining direct and indirect readouts to detect termini from next-generation short-read sequencing. While a direct inference of termini can come from mapping the most prominent start positions of captured DNA fragments, this approach is insufficient in cases where the DNA termini are not captured, whether for biological or technical reasons. Thus, a complementary (indirect) approach to terminus detection can be applied, taking advantage of the imbalance in coverage between forward and reverse sequence reads near termini. A resulting metric (“strand bias”) can be used to detect termini even where termini are naturally blocked from capture or ends are not captured during library preparation (e.g., in tagmentation-based protocols). Applying this analysis to datasets where known DNA termini are present, such as from linear double-stranded viral genomes, yielded distinct strand bias signals corresponding to these termini. To evaluate the potential to analyze a more complex situation, we applied the analysis to examine DNA termini present early after HIV infection in a cell culture model. We observed both the known termini expected based on standard models of HIV reverse transcription (the U5-right-end and U3-left-end termini) as well as a signal corresponding to a previously described additional initiation site for plus-strand synthesis (cPPT [central polypurine tract]). Interestingly, we also detected putative terminus signals at additional sites. The strongest of these are a set that share several characteristics with the previously characterized plus-strand initiation sites (the cPPT and 3’ PPT [polypurine tract] sites): (i) an observed spike in directly captured cDNA ends, an indirect terminus signal evident in localized strand bias, (iii) a preference for location on the plus-strand, (iv) an upstream purine-rich motif, and (v) a decrease in terminus signal at late time points after infection. These characteristics are consistent in duplicate samples in two different genotypes (wild type and integrase-lacking HIV). The observation of distinct internal termini associated with multiple purine-rich regions raises a possibility that multiple internal initiations of plus-strand synthesis might contribute to HIV replication.
Late-life Rapamycin Treatment Enhances Cardiomyocyte Relaxation Kinetics and Reduces Myocardial Stiffness
Authors: Chakraborty Akash D. , Kooiker Kristi , Kobak Kamil A. , Cheng Yuanhua , Lee Chi Fung , Razumova Maria , Granzier H Henk , Regnier Michael , Rabinovitch Peter S. , Moussavi-Harami Farid , Chiao Ying Ann
Published: June 2023

Diastolic dysfunction is a key feature of the aging heart. We have shown that late-life treatment with mTOR inhibitor, rapamycin, reverses age-related diastolic dysfunction in mice but the molecular mechanisms of the reversal remain unclear. To dissect the mechanisms by which rapamycin improves diastolic function in old mice, we examined the effects of rapamycin treatment at the levels of single cardiomyocyte, myofibril and multicellular cardiac muscle. Compared to young cardiomyocytes, isolated cardiomyocytes from old control mice exhibited prolonged time to 90% relaxation (RT90) and time to 90% Ca2+transient decay (DT90), indicating slower relaxation kinetics and calcium reuptake with age. Late-life rapamycin treatment for 10 weeks completely normalized RT90and partially normalized DT90, suggesting improved Ca2+handling contributes partially to the rapamycin-induced improved cardiomyocyte relaxation. In addition, rapamycin treatment in old mice enhanced the kinetics of sarcomere shortening and Ca2+transient increase in old control cardiomyocytes. Myofibrils from old rapamycin-treated mice displayed increased rate of the fast, exponential decay phase of relaxation compared to old controls. The improved myofibrillar kinetics were accompanied by an increase in MyBP-C phosphorylation at S282 following rapamycin treatment. We also showed that late-life rapamycin treatment normalized the age-related increase in passive stiffness of demembranated cardiac trabeculae through a mechanism independent of titin isoform shift. In summary, our results showed that rapamycin treatment normalizes the age-related impairments in cardiomyocyte relaxation, which works conjointly with reduced myocardial stiffness to reverse age-related diastolic dysfunction.
Cytoplasmic Male Sterility and Abortive Seed Traits Generated through Mitochondrial Genome Editing Coupled with Allotopic Expression ofatp1in Tobacco
Authors: Dewey Ralph E. , Selote Devarshi , Griffin H. Carol , Dickey Allison N. , Jantz Derek , Smith J. Jeff , Matthiadis Anna , Strable Josh , Kestell Caitlin , Smith William A.
Published: June 2023

Allotopic expression is the term given for the deliberate relocation of gene function from an organellar genome to the nuclear genome. We hypothesized that the allotopic expression of an essential mitochondrial gene using a promoter that expressed efficiently in all cell types except those responsible for male reproduction would yield a cytoplasmic male sterility (CMS) phenotype once the endogenous mitochondrial gene was inactivated via genome editing. To test this, we repurposed the mitochondrially encodedatp1gene of tobacco to function in the nucleus under the transcriptional control of a CaMV 35S promoter (construct 35S:nATP1), a promoter that has been shown to be minimally expressed in early stages of anther development. The endogenousatp1gene was eliminated (Δatp1) from 35S:nATP1 tobacco plants using custom-designed meganucleases directed to the mitochondria. Vegetative growth of most 35S:nATP1/Δatp1plants appeared normal, but upon flowering produced malformed anthers that failed to shed pollen. When 35S:nATP1/Δatp1plants were cross-pollinated, ovary/capsule development appeared normal, but the vast majority of the resultant seeds were small, largely hollow and failed to germinate, a phenotype akin to the seedless trait known as stenospermocarpy. Characterization of the mitochondrial genomes from three independent Δatp1events suggested that spontaneous recombination over regions of microhomology and substoichiometric shifting were the mechanisms responsible foratp1elimination and genome rearrangement in response to exposure to theatp1-targeting meganucleases. Should the results reported here in tobacco prove to be translatable to other crop species, then multiple applications of allotopic expression of an essential mitochondrial gene followed by its elimination through genome editing can be envisaged. Depending on the promoter(s) used to drive the allotopic gene, this technology may have potential application in the areas of: (1) CMS trait development for use in hybrid seed production; (2) seedless fruit production; and (3) transgene containment.
High-Resolution Characterization of Human Brain Cortex with High-Fidelity Spatial Transcriptomic Slides (HiFi-Slides)
Authors: Xu Tianyang , Zhu Ekko , Zhang Chi , Calandrelli Riccardo , Lin Pei , Zhong Sheng
Published: June 2023

Spatial transcriptomic tools and platforms help researchers to inspect tissues and cells with fine details of how they differentiate in expressions and how they orient themselves. With the higher resolution we get and higher throughput of expression targets, spatial analysis can truly become the core player for cell clustering, migration study, and, eventually, the novel model for pathological study. We present the demonstration of HiFi-slide, a whole transcriptomic sequencing technique that recycles used sequenced-by-synthesis flow cell surfaces to a high-resolution spatial mapping tool that can be directly applied to tissue cell gradient analysis, gene expression analysis, cell proximity analysis, and other cellular-level spatial studies.
Mechanism of DNA unwinding by hexameric MCM8-9 in complex with HROB
Authors: Acharya Ananya , Bret Hélène , Huang Jen-Wei , Mütze Martin , Göse Martin , Kissling Vera , Seidel Ralf , Ciccia Alberto , Guérois Raphaël , Cejka Petr
Published: June 2023

The human MCM8-9 helicase functions in concert with HROB in the context of homologous recombination, but its precise function is unknown. To gain insights into how HROB regulates MCM8-9, we first used molecular modeling and biochemistry to define their interaction interface. We show that HROB makes important contacts with both MCM8 and MCM9 subunits, which directly promotes its DNA-dependent ATPase and helicase activities. MCM8-9-HROB preferentially binds and unwinds branched DNA structures, and single-molecule experiments reveal a low DNA unwinding processivity. MCM8-9 unwinds DNA as a hexameric complex that assembles from dimers on DNA in the presence of ATP, which is prerequisite for its helicase function. The hexamer formation thus involves two repeating protein-protein interfaces forming between the alternating MCM8 and MCM9 subunits. One of these interfaces is rather stable and forms an obligate heterodimer, while the other interface is labile and mediates the assembly of the hexamer on DNA, independently of HROB. The ATPase site composed of the subunits forming the labile interface disproportionally contributes to DNA unwinding. HROB does not affect the MCM8-9 ring formation, but promotes DNA unwinding downstream by possibly coordinating ATP hydrolysis with structural transitions accompanying translocation of MCM8-9 on DNA.
Understanding the Influence of Context on Real-World Walking Energetics
Authors: Baroudi Loubna , Barton Kira , Cain Stephen M. , Shorter K. Alex
Published: June 2023

Speeds that minimize energetic cost during steady-state walking have been observed during lab-based investigations of walking biomechanics and energetics. However, in real-world scenarios, humans walk in various contexts that elicit different walking strategies, which may not always prioritize minimizing energetic cost. To investigate whether individuals tend to select energetically optimal speeds in real-world situations and how contextual factors influence gait, we conducted a study combining data from lab and real-world experiments. Walking kinematics and context were measured during daily life over a week (N = 17) using wearable sensors and a mobile phone. To determine context, we utilized self-reported activity logs, GPS data, and follow-up exit interviews. Additionally, we estimated energetic cost using respirometry over a range of gait speeds in the lab. Cost of transport during these trials was used to identify an energetically optimal walking speed range for each participant. The proportion of real world steady-state stride speeds within this range was identified for all data and for each context. We found that participants walked significantly faster than what was energetically optimal, and the proportion of steady-state strides within the energetically optimal speed range was dependent on the context. On average, 45.6% of all steady-state stride speeds were energetically optimal across all contexts for all participants. These results suggest that while energetic cost is a factor considered by humans when selecting gait speed in daily life, it is not the sole determining factor. Context contributes to the observed variability in movement parameters both within and between individuals.
Impact ofBmal1rescue and time-restricted feeding on liver and muscle proteomes during the active phase in mice
Authors: Smith Jacob G. , Molendijk Jeffrey , Blazev Ronnie , Chen Wan Hsi , Zhang Qing , Litwin Christopher , Greco Carolina M. , Sassone-Corsi Paolo , Muñoz-Cánoves Pura , Parker Benjamin L. , Koronowski Kevin B.
Published: June 2023

Objective

Molecular clocks and daily feeding cycles support metabolism in peripheral tissues. Although the roles of local clocks and feeding is well defined at the transcriptional level, their impact on governing protein abundances in peripheral tissues is unclear. Here, we determine the relative contributions of the local molecular clock and daily feeding cycles on liver and muscle proteomes during feeding.

Methods

LC-MS/MS was performed on liver and skeletal muscle harvested four hours into the dark phase from wild-type (WT),Bmal1knockout (KO), and liver- and muscle-Bmal1-rescued (LMRE) mice housed under 12-h light/12-h dark cycles with eitherad libitumfeeding or nighttime-restricted feeding. Additional molecular and metabolic analyses were performed on liver and cultured hepatocytes.

Results

Feeding-fasting cycles had only minimal effects on liver and none on muscle. In contrast,Bmal1KO altered the abundance of 674 proteins in liver, and 80 in muscle. Rescue of liver and muscleBmal1restored 50% of proteins in liver and 25% in muscle. These included proteins involved in carbohydrate metabolism in muscle and in fatty acid oxidation in liver. For liver, proteins involved inde novolipogenesis were largely dependent onBmal1function in other tissues (i.e., the wider clock system). Proteins regulated by BMAL1 were enriched for secreted proteins; we determined that the maintenance of FGF1 abundance requires liver BMAL1, and that autocrine signaling through FGF1 is necessary and sufficient for mitochondrial respiration in hepatocytes.

Conclusions

BMAL1 in liver and muscle is a more potent regulator of dark phase proteomes than daily feeding cycles, highlighting the need to assess protein levels in addition to mRNA when investigating clock mechanisms. The proteome is more extensively regulated by BMAL1 in liver than in muscle, and numerous metabolic pathways in peripheral tissues are reliant on the function of the clock system as a whole.
A nucleoid-associated protein is involved in the emergence of antibiotic resistance by promoting the frequent exchange of the replicative DNA polymerase inM. smegmatis
Authors: Ng Wei L. , Rego E. Hesper
Published: June 2023

Antibiotic resistance inM. tuberculosisexclusively originates from chromosomal mutations, either during normal DNA replication or under stress, when the expression of error-prone DNA polymerases increases to repair damaged DNA. To bypass DNA lesions and catalyze error-prone DNA synthesis, translesion polymerases must be able to access the DNA, temporarily replacing the high-fidelity replicative polymerase. The mechanisms that govern polymerase exchange are not well understood, especially in mycobacteria. Here, using a suite of quantitative fluorescence imaging techniques, we discover that, as in other bacterial species, inM. smegmatis,the replicative polymerase, DnaE1, exchanges at a timescale much faster than that of DNA replication. Interestingly, this fast exchange rate depends on an actinobacteria-specific nucleoid-associated protein (NAP), Lsr2. In cells missinglsr2, DnaE1 exchanges less frequently, and the chromosome is replicated more faithfully. Additionally, in conditions that damage DNA, cells lackinglsr2load the complex needed to bypass DNA lesions less effectively and, consistently, replicate with higher fidelity but exhibit growth defects. Together, our results show that Lsr2 promotes dynamic flexibility of the mycobacterial replisome, which is critical for robust cell growth and lesion repair in conditions that damage DNA.

Importance

Unlike many other pathogens,M. tuberculosishas limited ability for horizontal gene transfer, a major mechanism for developing antibiotic resistance. Thus, the mechanisms that facilitate chromosomal mutagenesis are of particular importance in mycobacteria. Here, we show that Lsr2, a nucleoid-associated protein, has a novel role in DNA replication and mutagenesis in the model mycobacteriumM. smegmatis. We find that Lsr2 promotes the fast exchange rate of the replicative DNA polymerase, DnaE1, at the replication fork and is important for the effective loading of the DnaE2-ImuA’-ImuB translesion complex. Withoutlsr2,M. smegmatisreplicates its chromosome more faithfully and acquires resistance to rifampin at a lower rate, but at the cost of impaired survival to DNA damaging agents. Together, our work establishes Lsr2 as a potential factor in the emergence of mycobacterial antibiotic resistance.
Dominance Analysis: A formalism to uncover dominant energetic contributions to biomolecular condensate formation in multicomponent systems
Authors: Qian Daoyuan , Ausserwoger Hannes , Sneideris Tomas , Farag Mina , Pappu Rohit V. , Knowles Tuomas P. J.
Published: June 2023

Phase separation in aqueous solutions of macromolecules is thought to underlie the generation of biomolecular condensates in cells. Condensates are membraneless bodies, representing dense, macromolecule-rich phases that coexist with the dilute, macromolecule-deficient phase. In cells, condensates comprise hundreds of different macromolecular and small molecule solutes. Do all components contribute equally or very differently to the driving forces for phase separation? Currently, we lack a coherent formalism to answer this question, a gap we remedy in this work through the introduction of a formalism we term energy dominance analysis. This approach rests on model-free analysis of shapes of the dilute arms of phase boundaries, slopes of tie lines, and changes to dilute phase concentrations in response to perturbations of concentrations of different solutes. We present the formalism that underlies dominance analysis, and establish its accuracy and flexibility by deploying it to analyse phase spaces probedin silico, in vitro, andin cellulo.
refMLST: Reference-based Multilocus Sequence Typing Enables Universal Bacterial Typing
Authors: Khdhiri Mondher , Thomas Ella , de Smet Chanel , Chandar Priyanka , Madasu Vivek , Chandrakumar Induja , Davidson Jean M , Anderson Paul , Chorlton Samuel D
Published: June 2023

Commonly used approaches for genomic investigation of bacterial outbreaks, including SNP and gene-by-gene approaches, are limited by the requirement for curated allele schemes. As a result, they only work on a select subset of known organisms, and fail on novel or less studied pathogens. We introduce refMLST, a gene-by-gene approach using the reference genome of a bacterium to form a scalable, reproducible and robust method to perform outbreak investigation. When applied to 1263Salmonella entericagenomes, refMLST enabled consistent clustering, improved resolution and faster processing in comparison to chewieSnake. refMLST is applicable to any bacterial species with a public genome, does not require a curated scheme, and automatically accounts for genetic recombination.

Availability and Implementation

refMLST is freely available for academic use athttps://bugseq.com/academic.
Polarized Desmosome and Hemidesmosome Shedding via Exosomes is an Early Indicator of Outer Blood-Retina Barrier Dysfunction
Authors: Hernandez Belinda J. , Skiba Nikolai P. , Plößl Karolina , Strain Madison , Grigsby Daniel , Kelly Una , Cady Martha A. , Manocha Vikram , Maminishkis Arvydas , Watkins TeddiJo , Miller Sheldon S. , Ashley-Koch Allison , Stamer W. Daniel , Weber Bernhard H.F. , Rickman Catherine Bowes , Klingeborn Mikael
Published: June 2023

The retinal pigmented epithelium (RPE) constitutes the outer blood-retinal barrier, enables photoreceptor function of the eye, and is constantly exposed to oxidative stress. As such, dysfunction of the RPE underlies pathology leading to development of age-related macular degeneration (AMD), the leading cause of vision loss among the elderly in industrialized nations. A major responsibility of the RPE is to process photoreceptor outer segments, which relies on the proper functioning of its endocytic pathways and endosomal trafficking. Exosomes and other extracellular vesicles from RPE are an essential part of these pathways and may be early indicators of cellular stress. To test the role of exosomes that may underlie the early stages of AMD, we used a polarized primary RPE cell culture model under chronic subtoxic oxidative stress. Unbiased proteomic analyses of highly purified basolateral exosomes from oxidatively stressed RPE cultures revealed changes in proteins involved in epithelial barrier integrity. There were also significant changes in proteins accumulating in the basal-side sub-RPE extracellular matrix during oxidative stress, that could be prevented with an inhibitor of exosome release. Thus, chronic subtoxic oxidative stress in primary RPE cultures induces changes in exosome content, including basal-side specific desmosome and hemidesmosome shedding via exosomes. These findings provide novel biomarkers of early cellular dysfunction and opportunity for therapeutic intervention in age-related retinal diseases, (e.g., AMD) and broadly from blood-CNS barriers in other neurodegenerative diseases.
Characterisation ofPlasmodium vivaxlactate dehydrogenase dynamics inP. vivaxinfections
Authors: Cao Pengxing , Kho Steven , Grigg Matthew J. , Barber Bridget E. , Piera Kim A. , William Timothy , Poespoprodjo Jeanne R. , Jang Ihn Kyung , Simpson Julie A. , McCaw James M. , Anstey Nicholas M. , McCarthy James S. , Britton Sumudu
Published: June 2023

Plasmodium vivaxlactate dehydrogenase (PvLDH) is an essential enzyme in the glycolytic pathway ofPlasmodium vivax. It can also be used as a diagnostic biomarker. Quantitation of plasma PvLDH has been used as a measure ofP. vivaxbiomass in clinical studies of uncomplicated and severe vivax malaria. With the increasing importance of PvLDH in studyingP. vivaxdiagnosis and infection, improved characterisation of the dynamics of this biomarker is important. In this study, we developed mathematical models that capture parasite and matrix PvLDH dynamics inex vivoculture and the human host. We estimated the biological parameters usingex vivoandin vivolongitudinal data of parasitemia and PvLDH concentration collected fromP. vivax-infected humans using Bayesian hierarchical inference. We found that theex vivoandin vivoestimates of PvLDH in a parasitized red blood cell differed significantly across the asexual life cycle, within vivoestimates at least ten-fold higher thanex vivoestimates (for example, the median estimate of intraerythrocytic PvLDH mass at the end of the life cycle was 9.4×10−3ngin vivovs. 5.1×10−4ngex vivo). We also estimated theex vivoPvLDH half-life to be 65.3 h (95% credible interval: 60.8—70.7 h), which is approximately three times longer than the median estimate of thein vivoPvLDH half-life, 21.9 h (16.7—29.9 h). Our findings provide an important foundation to further improve quantitative understanding ofP. vivaxbiology and facilitate the development of PvLDH-based diagnostic tools.
Cryo-EM structures of a synthetic antibody against 22 kDa claudin-4 reveal its complex withClostridium perfringensenterotoxin
Authors: Erramilli Satchal K. , Dominik Pawel K. , Ogbu Chinemerem P. , Kossiakoff Anthony A. , Vecchio Alex J.
Published: June 2023

Claudins are a family of ∼25 kDa membrane proteins that integrate into tight junctions to form molecular barriers at the paracellular spaces between endothelial and epithelial cells. Humans have 27 subtypes, which homo- and hetero-oligomerize to impart distinct properties and physiological functions to tissues and organs. As the structural and functional backbone of tight junctions, claudins are attractive targets for therapeutics capable of modulating tissue permeability to deliver drugs or treat disease. However, structures of claudins are limited due to their small sizes and physicochemical properties—these traits also make therapy development a challenge. We have developed a synthetic antibody fragment (sFab) that binds human claudin-4 and used it to resolve structures of its complex withClostridium perfringensenterotoxin (CpE) using cryogenic electron microscopy (cryo-EM). The resolution of the structures reveals the architectures of 22 kDa claudin-4, the 14 kDa C-terminal domain of CpE, and the mechanism by which this sFab binds claudins. Further, we elucidate the biochemical and biophysical bases of sFab binding and demonstrate that this molecule exhibits subtype-selectivity by assaying homologous claudins. Our results provide a framework for developing sFabs against hard-to-target claudins and establishes the utility of sFabs as fiducial markers for determining cryo-EM structures of this small membrane protein family at resolutions that surpass X-ray crystallography. Taken together, this work highlights the ability of sFabs to elucidate claudin structure and function and posits their potential as therapeutics for modulating tight junctions by targeting specific claudin subtypes.
Microglial P2Y6calcium signaling promotes phagocytosis and shapes neuroimmune responses in epileptogenesis
Authors: Umpierre Anthony D. , Li Bohan , Ayasoufi Katayoun , Zhao Shunyi , Xie Manling , Thyen Grace , Hur Benjamin , Zheng Jiaying , Liang Yue , Wu Zhaofa , Yu Xinzhu , Sung Jaeyun , Johnson Aaron J. , Li Yulong , Wu Long-Jun
Published: June 2023

Microglial calcium signaling is rare in a baseline state but shows strong engagement during early epilepsy development. The mechanism and purpose behind microglial calcium signaling is not known. By developing anin vivoUDP fluorescent sensor, GRABUDP1.0, we discovered that UDP release is a conserved response to seizures and excitotoxicity across brain regions. UDP signals to the microglial P2Y6receptor for broad increases in calcium signaling during epileptogenesis. UDP-P2Y6signaling is necessary for lysosome upregulation across limbic brain regions and enhances production of pro-inflammatory cytokines—TNFα and IL-1β. Failures in lysosome upregulation, observed in P2Y6KO mice, can also be phenocopied by attenuating microglial calcium signaling in Calcium Extruder (“CalEx”) mice. In the hippocampus, only microglia with P2Y6expression can perform full neuronal engulfment, which substantially reduces CA3 neuron survival and impairs cognition. Our results demonstrate that calcium activity, driven by UDP-P2Y6signaling, is a signature of phagocytic and pro-inflammatory function in microglia during epileptogenesis.
Circulating Sex-specific Markers of Plaque Instability in Women and Men with Severe Carotid Atherosclerosis
Authors: Gasbarrino Karina , Zheng Huaien , Daskalopoulou Stella S.
Published: June 2023

Background

Differences in plaque composition and instability exist among men and women. Circulating markers that reflect sex-specific features in the plaque should be explored for better prediction of high-risk plaques in women and in men. This study aims to 1) investigate differences in the lipid, immune, and adipokine circulating profiles of men and women with stable versus unstable plaques, and 2) identify circulating markers that can better classify men and women according to plaque instability.

Methods

Pre-operative blood samples as well as plaque specimens were collected from men and women undergoing a carotid endarterectomy (n=460). Blood samples were used for adipokine, lipid, and immune profiling. Plaque stability was determined by gold-standard histological classifications.

Results

Men had more unstable plaques than women (P<0.001), exhibiting greater plaque hemorrhage, a larger lipid core, and more inflammation (P<0.001), as well as less favourable circulating profiles. Significant antagonistic interactions were observed between sex and white blood cell (WBC) counts, sex and basophil to WBC ratio, and sex and platelet counts on impacting plaque instability. Several circulating immune parameters served as independent sex-specific markers of plaque instability; low total white blood cell (WBC) counts, high monocyte to WBC ratio, and low basophil to WBC ratio were associated with greater plaque instability in men, while a higher basophil to WBC ratio was observed in women with unstable plaques.

Conclusions

Our findings demonstrated sex-specific differences between older men and postmenopausal women with severe carotid atherosclerosis, with women displaying more stable plaque phenotypes, and favourable circulating profiles compared to men. We identified several potential circulating markers that relate to sex-specific plaque phenotypes for better prediction of high-risk plaques in women and in men. Following future validation, these markers could be implemented into clinical practice to monitor when the plaque becomes unstable and better select men and women for intervention.
Rictor induces AKT signaling to regulate lymphatic valve formation
Authors: Banerjee Richa , Knauer Luz A. , Iyer Drishya , Barlow Sara E. , Scallan Joshua P. , Yang Ying
Published: June 2023

Lymphatic valves are specialized structures of the collecting lymphatic vessels and are crucial for preventing retrograde lymph flow. Mutations in valve-forming genes have been clinically implicated in the pathology of congenital lymphedema. Lymphatic valves form when oscillatory shear stress (OSS) from lymph flow signals through the PI3K/AKT pathway to promote the transcription of valve-forming genes that trigger the growth and maintenance of lymphatic valves throughout life. Conventionally, in other tissue types, AKT activation requires dual kinase activity and the mammalian target of rapamycin complex 2 (mTORC2) commands this process by phosphorylating AKT at Ser473. Here we showed that embryonic and postnatal lymphatic deletion ofRictor, a critical component of mTORC2, led to a significant decrease in lymphatic valves and prevented the maturation of collecting lymphatic vessels.RICTORknockdown in human lymphatic endothelial cells (hdLECs) not only significantly reduced the level of activated AKT and the expression of valve-forming genes under no-flow conditions, but also abolished the upregulation of AKT activity and valve-forming genes in response to flow. We further showed that the AKT target, FOXO1, a repressor of lymphatic valve formation, had increased nuclear activity inRictorknockout mesenteric LECs,in vivo. Deletion ofFoxo1inRictorknockout mice restored the number of valves to control levels in both mesenteric and ear lymphatics. Our work revealed a novel role of RICTOR signaling in the mechanotransduction signaling pathway, wherein it activates AKT and prevents the nuclear accumulation of the valve repressor, FOXO1, which ultimately allows the formation and maintenance of a normal lymphatic valve.
Non-differentiable activity in the brain
Authors: Tsubo Yasuhiro , Shinomoto Shigeru
Published: June 2023

Spike rasters of multiple neurons show vertical stripes, indicating that neurons exhibit synchronous activity in the brain. We seek to determine whether these coherent dynamics are caused by smooth brainwave activity or by something else. By analyzing biological data, we find that their cross-correlograms exhibit not only slow undulation but also a cusp at the origin, in addition to possible signs of monosynaptic connectivity. Here we show that undulation emerges if neurons are subject to smooth brainwave oscillations while a cusp results from non-differentiable fluctuations. While modern analysis methods have achieved good connectivity estimation by adapting the models to slow undulation, they still make false inferences due to the cusp. We devise a new analysis method that may solve both problems. We also demonstrate that oscillations and non-differentiable fluctuations may emerge in simulations of large-scale neural networks.
Quantitative Analysis of Macrophage Uptake and Retention of Fluorescent Organosilica Nanoparticles
Authors: Chou Hung-Chang , Chiu Shih-Jiuan , Hu Teh-Min
Published: June 2023

This study investigates the uptake and retention of stable fluorescent organosilica nanoparticles by macrophages, which play a vital role in scavenging environmental nanoparticles and nanomedicine within the body. We used rhodamine 6G-loaded fluorescent organosilica nanoparticles (SiNP-R6G) synthesized from a thiol-functionalized organosilane precursor. Our primary objective was to establish a quantitative relationship between fluorescent measurements and nanoparticle tracking analysis, enabling the precise “counting” of nanoparticles taken up by macrophages under kinetic measurement conditions. Our kinetic study demonstrated a concentration-dependent, saturable internalization of nanoparticles in a model macrophage (RAW 264.7 cells), with a maximum uptake rate (Vmax) of 7.9 × 104nanoparticles per hour per cell. The estimated number concentration of nanoparticles for half-maximum uptake was approximately 0.8 trillion nanoparticles per milliliter, and a significant portion (∼80%) of internalized SiNP-R6G remained entrapped within the cells for 48 hours, indicating the sustained particle retention capacity of macrophages. These findings highlight the successful development of a methodology to accurately “count” the cellular uptake of nanoparticles in macrophages, providing valuable insights into the kinetics and retention capabilities of macrophages for nanoparticles.
From Hot Water to Dry Dirt: Microbes Use Cytochrome ‘Nanowires’ of Similar Conductivity but Different Structure
Authors: Guberman-Pfeffer Matthew J.
Published: June 2023

Micron-scale electron transfer through polymeric cytochrome ‘nanowires’ powers prokaryotic life from hydrothermal vents to terrestrial soils in ways not fully understood. How much structural diversity optimizes electrical conductivity for survival in these different habitats is challenging to assess experimentally. Herein, physiologically relevant redox conduction is computationally assessed in cytochrome filaments fromGeobacter sulfurreducens(OmcE, OmcS, and OmcZ),Pyrobaculum calidifontis(A3MW92), andArchaeoglobus veneficus(F2KMU8). A newly implemented Python program, BioDC, is used and validated against redox currents predicted from considerably more expensive molecular dynamics and quantum mechanical/molecular mechanical calculations. BioDC uses the heme solvent accessibility, stacking geometry, and redox-linked change in electrostatic energy to estimate electron transfer energetics. Leveraging this efficiency, structurally diverse cytochrome ‘nanowires’ from different organisms are shown to have similar redox conductivities. A functionally robust heme chain ‘packaged’ in habitat-customized proteins is proposed to be a general evolutionary design principle for cytochrome ‘nanowires’ widely distributed among prokaryotes.

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The effectiveness of positive behaviour support in reducing challenging behaviour and increasing quality of life for individuals with intellectual disability
Authors: Dewey Katherine , Evans Sean , Horsley Sarah , Baker Ellis
Published: June 2023

Purpose

Intensive support teams (ISTs) are often poorly understood, despite reports of their effectiveness in managing behaviour that challenges for individuals with an intellectual disability. This paper aims to contribute to the understanding of ISTs through evaluating one IST’s process and their use of positive behaviour support (PBS) as an intervention.

Design/methodology/approach

Participants were obtained from the ISTs discharge database, and pre- and post-intervention data from these participants was used for analysis.

Findings

Three-paired sample t-tests found that there were significant differences between pre- and post-scores on the behaviour problem inventory, Health of the Nation Outcomes Scale for people with Learning Disabilities and periodic service reviews, which measure quality of targeted, individualised support. This indicates that challenging behaviour frequency and severity were lower post-intervention, quality of life improved post-intervention and staff teams implemented recommended strategies more consistently by the end of the intervention.

Originality/value

This service evaluation captured data over seven-year period, which helps to contribute to the understanding of the effectiveness of ISTs and the PBS framework.
Waiting for psychological therapy during the covid-19 pandemic: experiences of adults with an intellectual disability
Authors: Baxter Rebecca , Rawlings Gregg H. , Yates Luke , Beail Nigel
Published: June 2023

Purpose

Measures introduced to mitigate the spread of coronavirus-19 (COVID-19) may have contributed to an increase in waiting times for face-to-face psychological treatments. As adults with intellectual disabilities (ID) are more likely to encounter barriers when accessing remote therapies, it is important they receive appropriate support while waiting. To understand what care is needed, this service evaluation [aimed to] explored the experiences of service users with ID who have waited for treatment during the pandemic.

Design/methodology/approach

Seven individuals who had been waiting for psychological therapy during the COVID-19 pandemic, which included those waiting longer than the national health service target of 18 weeks, were interviewed. Data were analysed using framework analysis.

Findings

The following four key themes were identified: waiting has been “painful”, related to how service users continued to experience difficulties whilst they waited; tolerating the wait, highlighted that individuals understood the reasons for waiting; use of coping strategies where service users identified both internal and external strategies they had used to cope; and support and contact from the learning disability team, related to how individuals experienced the support they received from the service.

Originality/value

To the best of the authors’ knowledge, this service evaluation is the first to explore the experiences of service users with ID waiting for psychological therapy during the Covid-19 pandemic. Results guide suggestions on improving support whilst they are waiting to help prevent further decline.