bluuebunny/crossref_metadata_2025 huggingface dataset:
Environment, plant genetics, and their interaction shape important aspects of sunflower rhizosphere microbial communities
Authors: Bueno de Mesquita Clifton P. , Walsh Corinne M. , Attia Ziv , Koehler Brady D. , Tarble Zachary J. , Van Tassel David L. , Kane Nolan C. , Hulke Brent S.
Published: August 2024

Associations with soil microorganisms are crucial for plants’ overall health and functioning. While much work has been done to understand drivers of rhizosphere microbiome structure and function, the relative importance of geography, climate, soil properties, and plant genetics remains unclear, as results have been mixed and comprehensive studies across many sites and genotypes are limited. Rhizosphere microbiomes are crucial for crop resistance to pathogens, stress tolerance, nutrient availability, and ultimately yield. Here we quantify the relative roles of plant genotype, environment, and their interaction in shaping soil rhizosphere communities, using 16S and ITS gene sequencing of rhizosphere soils from 10 common sunflower (Helianthus annuus) genotypes from 15 sites across the Great Plains of the United States. While site generally outweighed genotype overall in terms of effects on archaeal, bacterial and fungal richness, community composition, and taxa relative abundances, there was also a significant interaction such that genotype exerted a significant influence on archaeal, bacterial and fungal microbiomes in certain sites. Site effects were attributed to a combination of spatial distance and differences in climate and soil properties. Microbial taxa that were previously associated with resistance to the fungal necrotrophic pathogenSclerotiniawere present in most sites but differed significantly in relative abundance across sites. Our results have implications for plant breeding and agronomic microbiome manipulations for agricultural improvement across different geographic regions.

Importance

Despite the importance of plant breeding in agriculture, we still have a limited understanding of how plant genetic variation shapes soil microbiome composition across broad geographic regions. Using 15 sites across the Great Plains of North America, we show that cultivated sunflower rhizosphere archaeal, bacterial and fungal communities are driven primarily by site soil and climatic differences, but that genotype can interact with site to influence composition, especially in warmer and drier sites with lower overall microbial richness. We also show that all taxa that were previously found to be associated with resistance to the fungal pathogenSclerotinia sclerotiorumwere widespread but significantly affected by site, while a subset were also significantly affected by genotype. Our results contribute to a broader understanding of rhizosphere archaeal, bacterial and fungal community assembly, and provide foundational knowledge for plant breeding efforts and potential future microbiome manipulations in agriculture.
Reducing Offsite Modification using 2-mercaptoethanol for Proteome Analysis
Authors: Suto Arisa , Matsui Takashi , Kodera Yoshio
Published: August 2024

Alkylation of the thiol group in cysteine (Cys) residues using halide reagents is a significant step in proteomics. However, non-specific modifications to the N-terminus and other amino acids are known. Thus promiscuous offsite alkylation in the peptide further complicated the MS spectra and thus made the difficulty of identification and quantification of all peptides. 2-mercaptoethanol (2-ME) is not only a regent for the reduction of the disulfide bond but also is bound to the Cys residue. Furthermore, it is known that dimethyl sulfoxide (DMSO) enhances the disulfide bond formation. Thus, based on these facts, we developed a method for specifical modification of Cys residues using 2-ME and DMSO. The specific modification of Cys residue by 2-ME were promoted by the concentration-dependent manner of DMSO with quite less offsite modification reaction compared with recent procedures. This Cys-specific modification technique may not only improve the quantification of peptides containing cysteine but also enhance the quantification accuracy of all peptides.
Cryo-EM structure and evolutionary history of the conjugation surface exclusion protein TraT
Authors: Seddon Chloe , David Sophia , Wong Joshua LC , Ishimoto Naito , He Shan , Bradshaw Jonathan , Low Wen Wen , Frankel Gad , Beis Konstantinos
Published: August 2024

Conjugation plays a major role in dissemination of antimicrobial resistance genes. Following transfer of IncF-like plasmids, recipients become refractory to a second wave of conjugation with the same plasmid via entry (TraS) and surface (TraT) exclusion mechanisms. Here, we show that TraT from the pKpQIL and F plasmids (TraTpKpQILand TraTF) exhibits plasmid surface exclusion specificity. The cryo-EM structures of TraTpKpQILand TraTFrevealed that they oligomerise into decameric champagne bottle cork-like structures, which are anchored to the outer membrane via a diacylglycerol modified α-helical barrel domain. Unexpectedly, we identified chromosomal TraT homologues from multiple Gram-negative phyla which formed numerous deep-branching lineages in a phylogenetic tree of TraT sequences. Plasmid-associated TraT sequences largely cluster into two separate lineages that have more recently evolved, incorporating TraT from Enterobacterales IncF and Legionellaceae F-like plasmids. These findings suggest that different plasmid backbones have acquired and co-opted TraT on independent occasions.
Type I TARPs regulate Kv7.2 potassium channels and susceptibility to seizures
Authors: Rodrigues Marina V. , Inácio Ângela S. , Soldovieri Maria Virginia , Ribau Sara , Leal Telmo M. , Baurberg Theo , Mosca Ilaria , Ambrosino Paolo , Amado Luísa , Beltrão Nuno , Caldeira Gladys L. , Ribeiro Luís F. , Li Ka Wan , Groc Laurent , Ferreira Joana S. , Taglialatela Maurizio , Carvalho Ana Luisa
Published: August 2024

The M-current is a low-threshold potassium current that modulates neuronal excitability and suppresses repetitive firing. However, the mechanisms regulating M-channel function remain unclear. We identified type I Transmembrane AMPA receptor Regulatory Proteins (TARPs) as M-channel Kv7.2 subunit interactors in cortical neurons, with their interaction increasing upon neuronal depolarization. Co-expression of TARPs with Kv7.2 increased channel surface expression and Kv7.2-mediated currents, while disrupting TARP-γ2 expression in neurons perturbed dendritic Kv7.2 nano-clusters and decreased M-currents. Knock-in mice with an intellectual disability-associated TARP-γ2 variant showed reduced hippocampal M-currents and increased seizure susceptibility, indicating that disrupting TARP-γ2 regulation of Kv7.2-M-channels is epileptogenic. These findings show that TARP-γ2, a synaptic protein crucial for excitatory transmission, also controls intrinsic excitability via M-channels. This discovery provides a link between synaptic transmission and neuronal excitability, with implications for disease, as the interplay between synaptic and intrinsic plasticity is pivotal to how the brain adapts to varying input signals.

Highlights

Type I TARPs bind to Kv7.2-M-channels and enhance Kv7.2-mediated currents.

TARP-γ2 governs the neuronal nano-organization and function of Kv7.2 channels.

Intellectual disability-associated TARP-γ2 variant impairs M-currents and facilitates seizures.

Type I TARPs can serve as molecular integrators of synaptic and intrinsic excitability.
EVE is an open modular data analysis software for event-based localization microscopy
Authors: Weber Laura M. , Martens Koen J.A. , Cabriel Clément , Gates Joel J. , Albecq Manon , Vermeulen Fredrik , Hein Katarina , Izeddin Ignacio , Endesfelder Ulrike
Published: August 2024

Event-based sensors (EBS), or neuromorphic vision sensors, offer a novel approach to imaging by recording light intensity changes asynchronously, unlike conventional cameras that capture light over fixed exposure times. This capability results in high temporal resolution, reduced data redundancy, and a wide dynamic range. This makes EBS ideal for Single-Molecule Localization Microscopy (SMLM) as SMLM relies on the sequential imaging of sparse, blinking fluorescent emitters to achieve super-resolution. Recent studies have shown that EBS can effectively capture these emitters, achieving spatial resolution comparable to traditional cameras. However, existing analyses of event-based SMLM (eveSMLM) data have relied on converting event lists into image frames for conventional analysis, limiting the full potential of the technology.

To overcome this limitation, we developed EVE, a specialized software for analyzing eveSMLM data. EVE offers an integrated platform for detection, localization, and post-processing, with various algorithmic options tailored for the unique structure of eveSMLM data. EVE is user-friendly and features an open, modular infrastructure that supports ongoing development and optimization. EVE is the first dedicated tool for event-based SMLM, transforming the analysis process to fully utilize the spatiotemporal data generated by EBS. This allows researchers to explore the full potential of eveSMLM and encourages the development of new analytical methods and experimental improvements.
Genome-wide analysis of somatic non-coding mutation patterns and mitochondrial heteroplasmy in type B1 and B2 thymomas
Authors: Fujikura Kohei , Correa Isabel , Heck Susanne , King Juliet , McLean Emma , Bille Andrea , Nonaka Daisuke
Published: August 2024

Introduction

Type B1 and B2 thymomas are lymphocyte-rich malignant tumors with few somatic mutations in protein-coding regions of the nuclear genome; nonetheless, non-coding regions remain uncharacterized. Here, we developed a rigorous tumor isolation method from lymphocyte-rich thymoma tissues and identified somatic mutations in non-coding and mitochondrial DNA.

Methods

CD205+CD45-pure tumor cells were isolated from fresh-frozen tissues using DEPArray system. Deep whole-genome sequencing was performed, and recurrent somatic alterations in coding, non-coding, and mitochondria regions were systemically identified by computational framework. The mutations were classified according to gene function, cis-regulatory element, and mutational signature.

Results

The total number of somatic mutations was approximately 80 times higher in non-coding regions than in coding regions in type B1-2 thymomas (1,671.3 vs. 21.1 per case). Coding mutations were identified in epigenetic regulators, DNA repair genes, and some other genes. Nevertheless, 40% of cases exhibited fewer than four mutations in coding regions. A systematic non-coding analysis identified a total of 405.0 mutations per case on cis-regulatory elements, and detected six recurrent mutations: one interferon regulatory factor (IRF8), two E3 ubiquitin ligases (UBR2andRNF213), and three intergenic regions. Mitochondrial heteroplasmy was observed in 90% of cases, with a significant proportion of mutations located in D-loop region. The single-base substitution pattern was signature 12.

Conclusions

Numerous non-coding mutations and mitochondrial heteroplasmy were detected in type B1 and B2 thymomas. Given the paucity of coding mutations observed in this disease entity, disruption of the non-coding landscape and mitochondrial heteroplasmic shift may be the primary cause of thymoma.
Quantitative comparison of single-cell RNA sequencing versus single-molecule RNA imaging for quantifying transcriptional noise
Authors: Khetan Neha , Zuckerman Binyamin , Calia Giuliana P. , Chen Xinyue , Arceo Ximena Garcia , Weinberger Leor S.
Published: August 2024

Stochastic fluctuations (noise) in transcription generate substantial cell-to-cell variability. However, how best to quantify genome-wide noise, remains unclear. Here we utilize a small-molecule perturbation (IdU) to amplify noise and assess noise quantification from numerous scRNA-seq algorithms on human and mouse datasets, and then compare to noise quantification from single-molecule RNA FISH (smFISH) for a panel of representative genes. We find that various scRNA-seq analyses report amplified noise, without altered mean-expression levels, for ∼90% of genes and that smFISH analysis verifies noise amplification for the vast majority of genes tested. Collectively, the analyses suggest that most scRNA-seq algorithms are appropriate for quantifying noise including a simple normalization approach, although all of these systematically underestimate noise compared to smFISH. From a practical standpoint, this analysis argues that IdU is a globally penetrant noise-enhancer molecule—amplifying noise without altering mean-expression levels—which could enable investigations of the physiological impacts of transcriptional noise.

MOTIVATION

Cell-to-cell variability in isogenic populations is predominantly attributed to the stochastic fluctuations (i.e., noise) in transcription. However, the quantification of this noise, particularly on a genome-wide scale, remains an open question. To address this general question, here we utilize a small-molecule perturbation reported to amplify transcriptional noise. Previous single-cell RNA-sequencing (scRNA-seq) analysis indicated that the pyrimidine nucleobase 5′-iodo-2′-deoxyuridine (IdU) amplifies noise but technical drawbacks of scRNA-seq may have obscured the penetrance and degree of noise amplification. Consequently, here we assess numerous scRNA-seq algorithms, on two different scRNA-seq datasets for a human and mouse cell type, for their ability to quantify noise amplification and then compare the results to noise quantification from single-molecule RNA FISH (smFISH) imaging for a panel of representative genes. The specific questions addressed are whether IdU amplifies noise in a globally or partially penetrant manner, and what scRNA-seq algorithm is most appropriate for quantifying transcriptional noise.

Graphical Abstract
Circadian and permanent pools of extracellular matrix co-exist in tendon tissue, but have distinct rates of turnover and differential responses to ageing
Authors: Hoyle Anna , Chang Joan , Cutiongco Marie FA , O’Cualain Ronan , Warwood Stacey , Knight David , Meng Qing-Jun , Kadler Karl E , Swift Joe
Published: August 2024

Heavy carbon isotopes in the tendons of people who grew up in the age of nuclear bomb testing have shown that the extracellular matrix (ECM), assembled during development, stays with us for life. However, recent work suggests that type-I collagen in ECM-rich mouse tendon exists in two pools: a permanent matrix, and a more soluble, circadian-regulated matrix. Despite this, the underlying regulation of such distinct pools is not understood. Here, we demonstrate using stable isotope labelling coupled with mass spectrometry proteomics that circadian and permanent matrix pools have significantly different half-lives. Furthermore, the properties of the matrix pools are altered during development and ageing. Tail tendon tissue was harvested from mice fed on a heavy-lysine diet; protein was then extracted for analysis using a sequential two-step protocol. The first, soluble fraction (‘F1’) was found to contain intracellular proteins, and a range of core and associated extracellular matrix proteins, including a pool of type-I collagen shown to be circadian-regulated. The remaining fraction (‘F2’) contained primarily collagens, including type-I collagen which did not show rhythmicity. In adult mice, matrix proteins extracted in the F1 pool had significantly shorter half-lives than F2, including type-I collagen which had half-lives of 4 ± 2 days in F1, compared to 700 ± 100 days in F2. Circadian-regulated matrix proteins were found to have significantly faster turnover than non-circadian in adult mice, but this distinction was lost in older animals. This work identifies protein turnover as the underlying mechanism for the circadian/permanent model of tendon matrix, and suggests a loss of circadian regulation as a characteristic of ECM ageing.
Phosphate transporter(Pht) gene families in rye (Secale cerealeL.) – genome-wide identification and sequence diversity assessment
Authors: Chan-Rodriguez David , Koboyi Brian Wakimwayi , Werghi Sirine , Till Bradley J. , Maksymiuk Julia , Shoormij Fatemeh , Hilderlith Abuya , Hawliczek Anna , Królik Maksymilian , Bolibok-Brągoszewska Hanna
Published: August 2024

Background

Phosphorus is a macronutrient indispensable for plant growth and development. Plants utilize specialized transporters (PHT) to take up inorganic phosphorus and distribute it throughout the plant. The PHT transporters are divided into five families: PHT1 to PHT5. Each PHT family has a particular physiological and cellular function. Rye (Secale cereale L.) is a member ofTriticeae, and an important source of variation for wheat breeding. It is considered to have the highest tolerance of nutrient deficiency, amongTriticeae. To date, there is no report about genes involved in response to phosphorus deficiency in rye. The aim of this study was to: (i) identify and characterize putative members of different phosphate transporter families in rye, (i) assess their sequence diversity in a collection of diverse rye accessions via low-coverage resequencing (DArTreseq), and (iii) evaluate the expression of putative ryePhtgenes under phosphate-deficient conditions.

Results

We identified 29 and 35 putativePhttransporter genes in the rye Lo7 and Weining reference genomes, respectively, representing all knownPhtfamilies. Phylogenetic analysis revealed a close relationship of rye PHT with previously characterized PHT proteins from other species. Quantitative RT PCR carried out on leaf and root samples of Lo7 plants grown in Pi-deficient and control condition demonstrated thatScPht1;6, ScPht2andScPht3;1are Pi-deficiency responsive. Based on DArTreseq genotyping of 94 diverse rye accessions we identified 820 polymorphic sites within ryeScPht, including 12 variants with a putatively deleterious effect. SNP density varied markedly betweenScPhtgenes.

Conclusions

This report is the first step toward elucidating the mechanisms of rye’s response to Pi deficiency. Our findings point to multiple layers of adaptation to local environments, ranging from gene copy number variation to differences in level of polymorphism acrossPhtfamily members. DArTreseq genotyping permits for a quick and cost-effective assessment of polymorphism levels across genes/gene families and supports identification and prioritization of candidates for further studies. Collectively our findings provide the foundation for selecting most promising candidates for further functional characterization.
Optimising future rhino population management strategies using insights from genetic health assessments across India
Authors: Ghosh Tista , Kakati Parikshit , Sharma Amit , Mondol Samrat
Published: August 2024

Various species conservation paradigms are facing enormous challenges during the ongoing Anthropocene. While the widely-used reintroduction/translocation-based approaches have supported many endangered species population recoveries, they seldom use detailed genetic information during initial planning. The Indian greater one-horned rhino typifies such assisted migration-driven species recovery, but currently facing long-term survival concerns due to their mostly small, isolated populations reaching respective carrying capacities. We assessed nation-wide rhino genetic health, identified suitable source populations and provided future translocation scenarios for all extant and proposed rhino habitats. Analyses with 504 unique rhino genotypes across all seven Indian rhino-bearing parks revealed six genetically-isolated populations with overall moderately low genetic diversity. Our results showed that Kaziranga and Manas NPs (Assam) to have the best rhino genetic health, whereas Jaldapara and Gorumara NPs (West Bengal) undergoing strong genetic erosions. Forward genetic simulations suggested that annual supplementation efforts from only few Assam rhino populations (Kaziranga NP, Orang NP and Pobitora WLS) are best suited for genetic rescue of most of the extant populations. Overall, the genetic diversity and differentiation patterns mimics the complex evolutionary history and individual recovery histories. We suggest park-specific management solutions (ranging from protection measures, grassland restoration, livestock and conflict management, regular supplementation events etc.) to ensure the species’ long-term persistence and prevent the alarming loss of grassland habitats and its associate biodiversity. We insist on utilising such genetic health indices-driven population management solutions to identify targeted mitigative measures in other species.
Rapid GIT transit time in volant vertebrates, with implications for convergence in microbiome composition
Authors: Cornelius Ruhs Emily , McFerrin Katherine , Jones Devin N. , Cortes-Delgado Natalia , Ravelomanantsoa Ny Anjara Fifi , Yeoman Carl J. , Plowright Raina K. , Brook Cara E.
Published: August 2024

Flying birds and bats have simplified gastrointestinal tracts (GITs) and low intestinal mass to facilitate flight. Previous work showed reduced GIT transit times in birds relative to other vertebrates—but GIT transit has never been collectively quantified for bats. Unique among mammals, bat GIT microbiomes are dominated by Pseudomonadota bacteria (previously Proteobacteria), which also dominate the microbiomes of flying birds – we hypothesized this convergence to result from rapid GIT transit times for both volant taxa. We conducted a meta-analysis of vertebrate GIT transit times which showed that bats and flying birds have significantly faster transit times relative to nonvolant vertebrates. Additionally, within the bat order (Chiroptera), we demonstrated decreasing transit times associated with increasing body mass, a pattern contrasting other vertebrates (including volant birds) and possibly influencing GIT microbiome composition. This inverted mass-transit association is likely driven by diet as fruit- and nectar-consuming Pteropodids are the largest of all bats.
Do transformers and CNNs learn different concepts of brain age?
Authors: Siegel Nys Tjade , Kainmueller Dagmar , Deniz Fatma , Ritter Kerstin , Schulz Marc-Andre
Published: August 2024

“Predicted brain age” refers to a biomarker of structural brain health derived from machine learning analysis of T1-weighted brain magnetic resonance (MR) images. A range of machine learning methods have been used to predict brain age, with convolutional neural networks (CNNs) currently yielding state-of-the-art accuracies. Recent advances in deep learning have introduced transformers, which are conceptually distinct from CNNs, and appear to set new benchmarks in various domains of computer vision. However, transformers have not yet been applied to brain age prediction. Thus, we address two research questions: First, are transformers superior to CNNs in predicting brain age? Second, do conceptually different deep learning model architectures learn similar or different “concepts of brain age”? We adapted a Simple Vision Transformer (sViT) and a Shifted Window Transformer (SwinT) to predict brain age, and compared both models with a ResNet50 on 46,381 T1-weighted structural MR images from the UK Biobank. We found that SwinT and ResNet performed on par, while additional training samples will most likely give SwinT the edge in prediction accuracy. We identified that different model architectures may characterize different (sub-)sets of brain aging effects, representing diverging concepts of brain age. Thus, we systematically tested whether sViT, SwinT and ResNet focus on different concepts of brain age by examining variations in their predictions and clinical utility for indicating deviations in neurological and psychiatric disorders. Reassuringly, we did not find substantial differences in the structure of brain age predictions between model architectures. Based on our results, the choice of deep learning model architecture does not appear to have a confounding effect on brain age studies.
The Evolution of Extreme Genetic Variability in a Parasite-Resistance Complex in a Planktonic Crustacean
Authors: Naser-Khdour Suha , Scheuber Fabian , Fields Peter D. , Ebert Dieter
Published: August 2024

Genomic regions that play a role in parasite defense are often found to be highly variable, with the MHC serving as an iconic example. Single nucleotide polymorphisms may represent only a small portion of this variability, with Indel polymorphisms and copy number variation further contributing. In extreme cases, haplotypes may no longer be recognized as homologs. Understanding the evolution of such highly divergent regions is challenging because the most extreme variation is not visible using reference-assisted genomic approaches. Here we analyze the case of the Pasteuria Resistance Complex (PRC) in the crustacean Daphnia magna, a defense complex in the host against the common and virulent bacterium Pasteuria ramosa. Two haplotypes of this region have been previously described, with parts of it being non-homologous, and the region has been shown to be under balancing selection. Using pan-genome analysis and tree reconciliation methods to explore the evolution of the PRC and its characteristics within and between species of Daphnia and other Cladoceran species, our analysis revealed a remarkable diversity in this region even among host species, with many non-homologous hyper-divergent-haplotypes. The PRC is characterized by extensive duplication and losses of Fucosyltransferase (FuT) and Galactosyltransferase (GalT) genes that are believed to play a role in parasite defense. The PRC region can be traced back to common ancestors over 250 million years. The unique combination of an ancient resistance complex and a dynamic, hyper-divergent genomic environment presents a fascinating opportunity to investigate the role of such regions in the evolution and long-term maintenance of resistance polymorphisms. Our findings offer valuable insights into the evolutionary forces shaping disease resistance and adaptation, not only in the genus Daphnia, but potentially across the entire Cladocera class.
Hypoxia promotes airway differentiation in the human lung epithelium
Authors: Dong Ziqi , Wit Niek , Agarwal Aastha , Dubal Dnyanesh , van den Ameele Jelle , Reid Adam James , Nathan James A. , Rawlins Emma L.
Published: August 2024

Human early embryos develop under physiological hypoxia, but how hypoxia regulates human organogenesis remains little known. We have investigated oxygen availability effects on the human lung epithelium using organoids. We find first-trimester lung epithelial progenitors remain undifferentiated under normoxia, but spontaneously differentiate towards multiple airway cell types and inhibit alveolar differentiation under hypoxia. Using chemical and genetic tools, we demonstrate that hypoxia-induced airway differentiation is dependent on HIF (Hypoxia-Inducible Factor) pathways, with HIF1α and HIF2α differentially regulating fate decisions. Transcription factors KLF4 and KLF5 are direct targets of the HIF pathway and promote progenitor differentiation to basal and secretory cells. Chronic hypoxia also induces transdifferentiation of human alveolar type 2 cells to airway cells via the HIF pathway, mimicking alveolar bronchiolization processes in lung disease. Our results reveal roles for hypoxia and HIF signalling during human lung development and have implications for aberrant cell fate decisions in chronic lung diseases.
Making the effect visible – OX40 targeting nanobodies forin vivoimaging of activated T cells
Authors: Frecot Desiree I. , Blaess Simone , Wagner Teresa R. , Kaiser Philipp D. , Traenkle Bjoern , Fandrich Madeleine , Jakobi Meike , Scholz Armin M. , Nueske Stefan , Schneiderhan-Marra Nicole , Gouttefangeas Cécile , Kneilling Manfred , Pichler Bernd J. , Sonanini Dominik , Rothbauer Ulrich
Published: August 2024

Purpose

Human OX40 (hOX40/CD134), a member of the TNF receptor superfamily, is mainly expressed on activated T lymphocytes. Triggered by its ligand OX40L (CD252), it provides costimulatory signals that support the differentiation, proliferation and long-term survival of T cells. Besides being a relevant therapeutic target, hOX40 is also an important biomarker for monitoring the presence or infiltration of activated T cells within the tumor microenvironment (TME), the inflammatory microenvironment (IME) in immune-mediated diseases (IMIDs) and the lymphatic organs. Here, we developed novel single domain antibodies (nanobodies, Nbs) targeting hOX40 to monitor the activation status of T cells byin vivomolecular imaging.

Methods

Nbs against hOX40 (hOX40-Nbs) were selected from an immunized Nb-library by phage display. The identified hOX40-Nbs were characterizedin vitro, including determination of their specificity, affinity, stability, epitope recognition and their impact on OX40 signaling and T cell function. A lead candidate was site-specifically conjugated with a fluorophore via sortagging and applied for noninvasivein vivooptical imaging (OI) of hOX40-expressing cells in a xenograft mouse model.

Results

Our selection campaign revealed four unique Nbs that exhibit strong binding affinities and high stabilities under physiological conditions. Epitope binning and domain mapping indicated the targeting of at least two different epitopes on hOX40. When analyzing their impact on OX40 signaling, an agonistic effect was excluded for all validated Nbs. Incubation of activated T cells with hOX40-Nbs did not affect cell viability or proliferation patterns, whereas differences in cytokine release were observed.In vivoOI with a fluorophore-conjugated lead candidate in experimental mice with hOX40-expressing xenografts demonstrated its specificity and functionality as an imaging probe.

Conclusion

Considering the need for advanced probes for noninvasivein vivomonitoring of T cell activation dynamics, we propose, that our hOX40-Nbs have a great potential as imaging probes for noninvasive and longitudinalin vivodiagnostics. Quantification of OX40+T cells in TME or IME will provide crucial insights into the activation state of infiltrating T cells, offering a valuable biomarker for assessing immune responses, predicting treatment efficacy, and guiding personalized immunotherapy strategies in patients with cancer or IMIDs.
dia-PASEF Proteomics of Tumor and Stroma LMD Enriched from Archived HNSCC Samples
Authors: Panigrahi Aswini , Hunt Allison L , Assis Diego , Willetts Matthew , Kallakury Bhaskar V , Davidson Bruce , Conrads Thomas P , Goldman Radoslav
Published: August 2024

We employed laser microdissection to selectively harvest tumor cells and stroma from the microenvironment of formalin-fixed, paraffin-embedded head and neck squamous cell carcinoma (HNSCC) tissues. The captured HNSCC tissue fractions were analyzed by quantitative mass spectrometry-based proteomics using a data independent analysis approach. In paired samples, we achieved excellent proteome coverage having quantified 6,668 proteins with a median quantitative coefficient of variation under 10%. We observed significant differences in relevant functional pathways between the spatially resolved tumor and stroma regions. Our results identified extracellular matrix (ECM) as a major component enriched in the stroma, including many cancer associated fibroblast signature proteins in this compartment. We demonstrate the potential for comparative deep proteome analysis from very low starting input in a scalable format that is useful to decipher the alterations in tumor and the stromal microenvironment. Correlating such results with clinical features or disease progression will likely enable identification of novel targets for disease classification and interventions.
Somatic mutation phasing and haplotype extension using linked-reads in multiple myeloma
Authors: Foltz Steven M. , Li Yize , Yao Lijun , Terekhanova Nadezhda V. , Weerasinghe Amila , Gao Qingsong , Dong Guanlan , Schindler Moses , Cao Song , Sun Hua , Jayasinghe Reyka G. , Fulton Robert S. , Fronick Catrina C. , King Justin , Kohnen Daniel R. , Fiala Mark A. , Chen Ken , DiPersio John F. , Vij Ravi , Ding Li
Published: August 2024

Somatic mutation phasing informs our understanding of cancer-related events, like driver mutations. We generated linked-read whole genome sequencing data for 23 samples across disease stages from 14 multiple myeloma (MM) patients and systematically assigned somatic mutations to haplotypes using linked-reads. Here, we report the reconstructed cancer haplotypes and phase blocks from several MM samples and show how phase block length can be extended by integrating samples from the same individual. We also uncover phasing information in genes frequently mutated in MM, includingDIS3,HIST1H1E,KRAS,NRAS, andTP53, phasing 79.4% of 20,705 high-confidence somatic mutations. In some cases, this enabled us to interpret clonal evolution models at higher resolution using pairs of phased somatic mutations. For example, our analysis of one patient suggested that twoNRAShotspot mutations occurred on the same haplotype but were independent events in different subclones. Given sufficient tumor purity and data quality, our framework illustrates how haplotype-aware analysis of somatic mutations in cancer can be beneficial for some cancer cases.
Deep skin fibroblast-mediated macrophage recruitment supports acute wound healing
Authors: Amuso Veronica M. , Haas MaryEllen R. , Cooper Paula O. , Chatterjee Ranojoy , Hafiz Sana , Salameh Shatha , Gohel Chiraag , Mazumder Miguel F. , Josephson Violet , Khorsandi Khatereh , Horvath Anelia , Rahnavard Ali , Shook Brett A.
Published: August 2024

Epithelial and immune cells have long been appreciated for their contribution to the early immune response after injury; however, much less is known about the role of mesenchymal cells. Using single nuclei RNA-sequencing, we defined changes in gene expression associated with inflammation at 1-day post-wounding (dpw) in mouse skin. Compared to keratinocytes and myeloid cells, we detected enriched expression of pro-inflammatory genes in fibroblasts associated with deeper layers of the skin. In particular, SCA1+ fibroblasts were enriched for numerous chemokines, including CCL2, CCL7, and IL33 compared to SCA1-fibroblasts. Genetic deletion ofCcl2in fibroblasts resulted in fewer wound bed macrophages and monocytes during injury-induced inflammation with reduced revascularization and re-epithelialization during the proliferation phase of healing. These findings highlight the important contribution of deep skin fibroblast-derived factors to injury-induced inflammation and the impact of immune cell dysregulation on subsequent tissue repair.
Image-Guided Targeting of Mitochondrial Metabolism Sensitizes Pediatric Malignant Rhabdoid Tumors to Low Dose Radiotherapy
Authors: Xia Wenxi , Goff Matthew , Schiavone Carmine , Singh Neetu , Huang Jiemin , Need Esther , Cave Joseph , Gillespie David L. , Jensen Randy L. , Pagel Mark D. , Dogra Prashant , Shi Sixiang , Goel Shreya
Published: August 2024

Tumor hypoxia leads to radioresistance and markedly worse clinical outcomes for pediatric malignant rhabdoid tumors (MRT). Our transcriptomics and bioenergetic profiling data reveal that mitochondrial oxidative phosphorylation (OXPHOS) is a metabolic vulnerability of MRT and can be exploited to overcome consumptive hypoxia by repurposing an FDA-approved anti-malarial drug, Atovaquone (AVO). We then establish the utility of Oxygen-Enhanced-Multispectral Optoacoustic Tomography (OE-MSOT), a label-free, ionizing radiation-free imaging modality, to visualize and quantify spatiotemporal changes in tumor hypoxia in response to AVO. We show a potent but transient increase in tumor oxygenation upon AVO treatment which results in complete elimination of tumors in all tested mice when combined with 10 Gy radiotherapy, a dose several times lower than the current clinic standard. Finally, we use translational mathematical modeling for systematic evaluation of dosing regimens, administration timing, and therapeutic synergy in a virtual clinical patient population. Together, our work establishes a framework for safe and pediatric patient-friendly image-guided metabolic radiosensitization of rhabdoid tumors.
Physiological oxygen concentration during sympathetic primary neuron culture improves neuronal health and reduces HSV-1 reactivation
Authors: Dochnal Sara A. , Krakowiak Patryk A. , Whitford Abigail L. , Cliffe Anna R.
Published: August 2024

Herpes simplex virus-1 (HSV-1) establishes a latent infection in peripheral neurons and periodically reactivates in response to a stimulus to permit transmission.In vitromodels using primary neurons are invaluable to studying latent infection because they use bona fide neurons that have undergone differentiation and maturationin vivo. However, culture conditionsin vitroshould remain as close to thosein vivoas possible. This is especially important when considering minimizing cell stress, as it is a well-known trigger of HSV reactivation. We recently developed an HSV-1 model system that requires neurons to be cultured for extended lengths of time. Therefore, we sought to refine culture conditions to optimize neuronal health and minimize secondary effects on latency and reactivation. Here, we demonstrate that culturing primary neurons under conditions closer to physiological oxygen concentrations (5% oxygen) results in cultures with features consistent with reduced stress. Furthermore, culture in these lower oxygen conditions diminishes the progression to full HSV-1 reactivation despite minimal impacts on latency establishment and earlier stages of HSV-1 reactivation. We anticipate that our findings will be useful for the broader microbiology community as they highlight the importance of considering physiological oxygen concentration in studying host-pathogen interactions.
Predicting metal-binding proteins and structures through integration of evolutionary-scale and physics-based modeling
Authors: Dai Xin , Henderson Max , Yoo Shinjae , Liu Qun
Published: August 2024

Metals are essential elements in all living organisms, binding to approximately 50% of proteins. They serve to stabilize proteins, catalyze reactions, regulate activities, and fulfill various physiological and pathological functions. While there have been many advancements in determining the structures of protein-metal complexes, numerous metal-binding proteins still need to be identified through computational methods and validated through experiments. To address this need, we have developed the ESMBind workflow, which combines evolutionary scale modeling (ESM) for metal-binding prediction and physics-based protein-metal modeling. Our approach utilizes the ESM-2 and ESM-IF models to predict metal-binding probability at the residue level. In addition, we have designed a metal-placement method and energy minimization technique to generate detailed 3D structures of protein-metal complexes. Our workflow outperforms other models in terms of residue and 3D-level predictions. To demonstrate its effectiveness, we applied the workflow to 142 uncharacterized fungal pathogen proteins and predicted metal-binding proteins involved in fungal infection and virulence.
Bayesian Estimation of Allele-Specific Expression in the Presence of Phasing Uncertainty
Authors: Zou Xue , Gomez Zachary W. , Reddy Timothy E. , Allen Andrew S. , Majoros William H.
Published: August 2024

Motivation

Allele-specific expression (ASE) analyses aim to detect imbalanced expression of maternal versus paternal copies of an autosomal gene. Such allelic imbalance can result from a variety of cis-acting causes, including disruptive mutations within one copy of a gene that impact the stability of transcripts, as well as regulatory variants outside the gene that impact transcription initiation. Current methods for ASE estimation suffer from a number of shortcomings, such as relying on only one variant within a gene, assuming perfect phasing information across multiple variants within a gene, or failing to account for alignment biases and possible genotyping errors.

Results

We developed BEASTIE, a Bayesian hierarchical model designed for precise ASE quantification at the gene level, based on given genotypes and RNA-Seq data. BEASTIE addresses the complexities of allelic mapping bias, genotyping error, and phasing errors by incorporating empirical phasing error rates derived from Genome-in-a-Bottle individual NA12878. BEASTIE surpasses existing methods in accuracy, especially in scenarios with high phasing errors. This improvement is critical for identifying rare genetic variants often obscured by such errors. Through rigorous validation on simulated data and application to real data from the 1000 Genomes Project, we establish the robustness of BEASTIE. These findings underscore the value of BEASTIE in revealing patterns of ASE across gene sets and pathways.

Availability and Implementation

The software is freely available fromhttps://github.com/x811zou/BEASTIE. BEASTIE is available as Python source code and as a Docker image.

Supplementary information

Additional information is available online.
microRNA-1 Regulates Metabolic Flexibility in Skeletal Muscle via Pyruvate Metabolism
Authors: Ismaeel Ahmed , Peck Bailey D. , Montgomery McLane M. , Burke Benjamin I. , Goh Jensen , Kang Gyumin , Franco Abigail B. , Xia Qin , Goljanek-Whysall Katarzyna , McDonagh Brian , McLendon Jared M. , Koopmans Pieter J. , Jacko Daniel , Schaaf Kirill , Bloch Wilhelm , Gehlert Sebastian , Wen Yuan , Murach Kevin A. , Peterson Charlotte A. , Boudreau Ryan L. , Fisher-Wellman Kelsey H. , McCarthy John J.
Published: August 2024

MicroRNA-1 (miR-1) is the most abundant miRNA in adult skeletal muscle. To determine the function of miR-1 in adult skeletal muscle, we generated an inducible, skeletal muscle-specific miR-1 knockout (KO) mouse. Integration of RNA-sequencing (RNA-seq) data from miR-1 KO muscle with Argonaute 2 enhanced crosslinking and immunoprecipitation sequencing (AGO2 eCLIP-seq) from human skeletal muscle identified miR-1 target genes involved with glycolysis and pyruvate metabolism. The loss of miR-1 in skeletal muscle induced cancer-like metabolic reprogramming, as shown by higher pyruvate kinase muscle isozyme M2 (PKM2) protein levels, which promoted glycolysis. Comprehensive bioenergetic and metabolic phenotyping combined with skeletal muscle proteomics and metabolomics further demonstrated that miR-1 KO induced metabolic inflexibility as a result of pyruvate oxidation resistance. While the genetic loss of miR-1 reduced endurance exercise performance in mice and inC. elegans,the physiological down-regulation of miR-1 expression in response to a hypertrophic stimulus in both humans and mice causes a similar metabolic reprogramming that supports muscle cell growth. Taken together, these data identify a novel post-translational mechanism of adult skeletal muscle metabolism regulation mediated by miR-1.
Molecular dynamics simulations illuminate the role of sequence context in the ELF3-PrD-based temperature sensing mechanism in plants
Authors: Lindsay Richard J. , Viegas Rafael Giordano , Leite Vitor B. P. , Wigge Philip A. , Hanson Sonya M.
Published: August 2024

The evening complex (EC) is a tripartite DNA repressor and a core component of the circadian clock that provides a mechanism for temperature-responsive growth and development of many plants. ELF3, a component of the EC, is a disordered scaffolding protein that blocks transcription of growth genes at low temperature. At increased temperature EC DNA binding is disrupted and ELF3 is sequestered in a reversible nuclear condensate, allowing transcription and growth to proceed. The condensation is driven by a low complexity prion-like domain (PrD), and the sensitivity of the temperature response is modulated by the length of a variable polyQ tract, with a longer polyQ tract corresponding to enhanced condensate formation and hypocotyl growth at increased temperature. Here, a series of computational studies provides evidence that polyQ tracts promote formation of temperature-sensitive helices in flanking residues with potential impacts for EC stability under increasing temperature. REST2 simulations uncover a heat-induced population of condensation-prone conformations that results from the exposure of ‘sticky’ aromatic residues by temperature-responsive breaking of long-range contacts. Coarse-grained Martini simulations reveal both polyQ tract length and sequence context modulate the temperature dependence of cluster formation. Understanding the molecular mechanism underlying the ELF3-PrD temperature response in plants has implications for technologies including modular temperature-response elements for heat-responsive protein design and agricultural advances to enable optimization of crop yields and allow plants to thrive in increasingly inhospitable environments.
Different folding mechanisms in prion proteins from mammals with different disease susceptibility observed at the single-molecule level
Authors: Anand Uttam , Patra Shubhadeep , Sekar Rohith Vedhthaanth , Garen Craig R. , Woodside Michael T.
Published: August 2024

Misfolding of the protein PrP causes prion diseases in mammals. Disease susceptibility varies widely among species, despite PrP sequences differing by only a few amino acids. How these differences alter PrP folding and misfolding remains unclear. We compared the folding dynamics of single PrP molecules from three species with different disease susceptibility: dogs (immune), hamsters (susceptible), and bank voles (extremely susceptible). Measurements with optical tweezers revealed important differences between the folding cooperativity, pathways, energy barriers, and kinetics of these proteins. In contrast to the two-state folding of hamster PrP, dog PrP always folded through multiple intermediates. However, both featured rapid native folding, homogeneous energy barriers, and no readily observable misfolding. Bank vole PrP also folded via intermediates, but more slowly and via inhomogeneous barriers. Most notably, it formed several metastable misfolded states starting from the unfolded state. Analyzing the sequence of intermediates seen in pulling curves, we found significant differences in the folding pathways for dog and bank vole PrP, implying that sequence mutations altered energy barriers so as to redirect folding pathways. These results show that subtle differences in PrP sequence between species produce profound changes in folding behavior, providing insight into the factors underlying misfolding propensity.