bluuebunny/crossref_metadata_2025 huggingface dataset:
Optimal enzyme profiles in unbranched metabolic pathways
Authors: Noor Elad , Liebermeister Wolfram
Published: February 2024

How to optimize the allocation of enzymes in metabolic pathways has been a topic of study for many decades. Although the general problem is complex and nonlinear, we have previously shown that it can be solved by convex optimization. In this paper, we focus on unbranched metabolic pathways with simplified enzymatic rate laws and derive analytic solutions to the optimization problem. We revisit existing solutions based on the limit of mass-action rate laws and present new solutions for other rate laws. Furthermore, we revisit a known relationship between flux control coefficients and enzyme abundances in optimal metabolic states. We generalize this relationship to models with density constraints on enzymes and metabolites, and present a new local relationship between optimal reaction elasticities and enzyme amounts. Finally, we apply our theory to derive simple kinetics-based formulae for protein allocation during bacterial growth.
Paenibacillus baimaensis sp. nov., a bacterium isolated from mountain soil in the habitat of Rhinopithecus bieti
Authors: Wang Qiong , Han Xiu-Lin , Shen Jian-Qiang , Lai Jian-Dong , Zhang Chen-Lu , Fang Zhi-Qin , Lu Tao
Published: February 2024

A novel Gram-positive strain WQ 127069T that was isolated from the soil of Baima Snow Mountain, a habitat of highly endangered Yunnan snub-nosed monkeys (Rhinopithecus bieti), was subjected to a polyphasic taxonomic study. Phylogenetic analysis based on the 16S rRNA gene sequences showed that the isolate belongs to the genus Paenibacillus, showing 98.4 and 96.08 % sequence similarity to the type strains Paenibacillus periandrae PM10T and Paenibacillus foliorum LMG 31456T, respectively. The G+C content of the genomic DNA of strain WQ127069T was 45.6 mol%. The predominant isoprenoid quinone was MK-7, and meso-diaminopimelic acid was present in peptidoglycan. The major cellular fatty acids were antiiso-C15 : 0, iso-C15 : 0 and C16 : 0. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and phosphatidylmonomethylethanolamine. The whole genome average nucleotide identity and digital DNA–DNA hybridization values between strain WQ 127069T and strain PM10T were 93.2 and 52.5 %, respectively. Growth occurred at 5–40 °C (optimally at 20–35 °C), pH 6–8 (optimally at pH7.0) and with 0.5–2 % (w/v) NaCl (optimally at 0.5 %). On the basis of the taxonomic evidence, a novel species, Paenibacillus baimaensis sp. nov., is proposed. The type strain is WQ 127069T (=KCTC 43480T=CCTCC AB 2022381T).
Sex-specific genetic determinants of right ventricular structure and function
Authors: Harbaum Lars , Hennigs Jan K , Pott Julian , Ostermann Jonna , Sinning Christoph R , Sau Arunashis , Sieliwonczyk Ewa , Ng Fu Siong , Rhodes Christopher J , Tello Khodr , Klose Hans , Gräf Stefan , Wilkins Martin R
Published: February 2024

Background

While sex differences in right heart phenotypes have been observed, the molecular drivers remain unknown. We used common genetic variation to provide biological insights into sex differences in the structure and function of the right ventricle (RV).

Methods

RV phenotypes were obtained from cardiac magnetic resonance imaging in 18,156 women and 16,171 men from the UK Biobank, based on a deep-learning approach, including end-diastolic, end-systolic, and stroke volumes, as well as ejection fraction. Observational analyses and sex-stratified genome-wide association studies were performed. Candidate female-specific loci were evaluated against invasively measured hemodynamics in 479 female patients with idiopathic or heritable pulmonary arterial hypertension (PAH), recruited to the UK National Institute for Health Research BioResource Rare Diseases study.

Results

Sex was associated with differences in RV volumes and ejection fraction in models adjusting for left heart counterparts and lung function. Six genome-wide significant loci (13%) revealed heterogeneity of allelic effects between women and men. These included two sex-specific candidate loci present in women only; namely, a locus for RV ejection fraction inBMPR1Aand a locus for RV end-systolic volume nearDMRT2. Epigenetic data indicate that variation at theBMPR1Alocus likely alters transcriptional regulation in RV tissue. In female patients with PAH, a variant located in the promoter ofBMPR1Awas significantly associated with cardiac index (effect size 0.16 l/min/m2), despite similar RV afterload among genotypic groups.

Conclusions

We report sex-specific genetic loci for RV structure and function.BMPR1Ahas emerged as a biologically plausible candidate gene for female-specific genetic determination of RV function, showing associations with cardiac performance under chronically increased afterload in female patients with PAH. Further studies are needed to explore the underlying biological pathways.
Development of the ADFICE_IT clinical decision support system to assist deprescribing of fall-risk increasing drugs: A user-centered design approach
Authors: Groos Sara S. , de Wildt Kelly K. , van de Loo Bob , Linn Annemiek J. , Medlock Stephanie , Shaw Kendrick M. , Herman Eric K. , Seppala Lotta J. , Ploegmakers Kim J. , van Schoor Natasja M. , van Weert Julia C. M. , van der Velde Nathalie
Published: February 2024

Introduction

Deprescribing fall-risk increasing drugs (FRIDs) is a promising intervention for reducing the risk of falling in older adults. Applying appropriate deprescribing in practice can be difficult due to the outcome uncertainties associated with stopping the use of FRIDs. The ADFICE_IT study addresses this complexity with a clinical decision support system (CDSS) that facilitates optimum deprescribing of FRIDs through use of a fall-risk prediction model, aggregation of deprescribing guidelines, and joint medication management. The development process of the CDSS is described in this paper.

Methods

Development followed a user-centered design approach in which users and experts were involved throughout each phase. In phase I, a prototype of the CDSS was developed which involved a scoping and systematic literature review, European survey (n= 581), and semi-structured interviews with physicians (n= 19), as well as the aggregation and testing of deprescribing guidelines and the development of the fall-risk prediction model. In phase II, the feasibility of the CDSS was tested by means of two usability testing rounds with end users (n= 11).

Results

The final CDSS consists of five web pages. A connection between the Electronic Health Record allows for the retrieval of patient data into the CDSS. Key design requirements for the CDSS include easy-to-use features for fast-paced clinical practice environments, actionable deprescribing recommendations, information transparency, and visualization of the patient’s fall-risk estimation. Key elements for the software include a modular architecture, open source, and good security.

Conclusion

The ADFICE_IT CDSS supports physicians in deprescribing FRIDs optimally to prevent falls in older patients. Due to continuous user and expert involvement, each new feedback round led to an improved version of the system. Currently, a multicenter, cluster-randomized controlled trial with process evaluation at hospitals in the Netherlands is being conducted to test the effect of the CDSS on falls.
Peptide-based capture-and-release purification of extracellular vesicles and statistical algorithm enabled quality assessment
Authors: Greenberg Zachary F. , Ali Samantha , Schmittgen Thomas D. , Han Song , Hughes Steven J. , Graim Kiley S. , He Mei
Published: February 2024

Circulating extracellular vesicles (EVs) have gained significant attention for discovering tumor biomarkers. However, isolating EVs with well-defined homogeneous populations from complex biological samples is challenging. Different isolation methods have been found to derive different EV populations carrying different molecular contents, which confounds current investigations and hinders subsequent clinical translation. Therefore, standardizing and building a rigorous assessment of isolated EV quality associated with downstream molecular analysis is essential. To address this need, we introduce a statistical algorithm (ExoQuality Index, EQI) by integrating multiple EV characterizations (size, particle concentration, zeta potential, total protein, and RNA), enabling direct EV quality assessment and comparisons between different isolation methods. We also introduced a novel capture-release isolation approach using a pH-responsive peptide conjugated with NanoPom magnetic beads (ExCy) for simple, fast, and homogeneous EV isolation from various biological fluids. Bioinformatic analysis of next-generation sequencing (NGS) data of EV total RNAs from pancreatic cancer patient plasma samples using our novel EV isolation approach and quality index strategy illuminates how this approach improves the identification of tumor associated molecular markers. Results showed higher human mRNA coverage compared to existing isolation approaches in terms of both pancreatic cancer pathways and EV cellular component pathways using gProfiler pathway analysis. This study provides a valuable resource for researchers, establishing a workflow to prepare and analyze EV samples carefully and contributing to the advancement of reliable and rigorous EV quality assessment and clinical translation.
Arbuscular mycorrhizal fungal diversity and association networks in African tropical rainforest trees
Authors: Olanipon Damilola , Boeraeve Margaux , Jacquemyn Hans
Published: February 2024

Tropical rainforests represent one of the most diverse and productive ecosystems on Earth. High productivity is sustained by efficient and rapid cycling of nutrients through decomposing organic matter, which is for a large part made possible by symbiotic associations between plants and mycorrhizal fungi. In this association, an individual plant typically associates simultaneously with multiple fungi and the fungus associates with multiple plants, creating complex networks between fungi and plants. However, there are still very few studies that have investigated mycorrhizal fungal composition and diversity in tropical rainforest trees, particularly in Africa, and assessed the structure of the network of associations between fungi and rainforest trees. In this study, we collected root and rhizosphere soil samples from Ise Forest Reserve (Southwest Nigeria), and employed a metabarcoding approach to identify the dominant arbuscular mycorrhizal (AM) fungal taxa associating with ten co-occurring tree species and to assess variation in AM communities. Network analysis was used to elucidate the architecture of the network of associations between fungi and tree species. A total of 194 AM fungal Operational Taxonomic Units (OTUs) belonging to six families were identified, with 68% of all OTUs belonging to Glomeraceae. While AM fungal diversity did not differ between tree species, AM fungal community composition did. Network analyses showed that the network of associations was not significantly nested and showed a relatively low level of specialization (H2= 0.43) and modularity (M= 0.44). We conclude that, although there were some differences in AM fungal community composition, the studied tree species associate with a large number of AM fungi. Similarly, most AM fungi had a large host breadth and connected most tree species to each other, thereby potentially working as interaction network hubs.

Graphical Abstract
Differential mitochondrial bioenergetics and cellular resilience in astrocytes, hepatocytes, and fibroblasts from aging baboons
Authors: Adekunbi Daniel A. , Huber Hillary F. , Li Cun , Nathanielsz Peter W. , Cox Laura A. , Salmon Adam B.
Published: February 2024

Biological resilience, broadly defined as ability to recover from acute challenge and return to homeostasis, is of growing importance to the biology of aging. At the cellular level, there is variability across tissue types in resilience and these differences likely to contribute to tissue aging rate disparities. However, there are challenges in addressing these cell-type differences at regional, tissue and subject level. To address this question, we established primary cells from aged male and female baboons between 13.3-17.8 years spanning across different tissues, tissue regions, and cell types including: (1) fibroblasts from skin and from heart separated into left ventricle (LV), right ventricle (RV), left atrium (LA) and right atrium (RA), (2) astrocytes from the prefrontal cortex and hippocampus and (3) hepatocytes. Primary cells were characterized by their cell surface markers and their cellular respiration assessed with Seahorse XFe96. Cellular resilience was assessed by modifying a live-cell imaging approach we previously reported that monitors proliferation of dividing cells following response and recovery to oxidative (50µM-H2O2), metabolic (1mM-glucose) and proteostasis (0.1µM-thapsigargin) stress. We noted significant differences even among similar cell types that are dependent on tissue source and the diversity in cellular response is stressor specific. For example, astrocytes were more energetic and exhibited greater resilience to oxidative stress (OS) than both fibroblasts and hepatocytes. RV and RA fibroblasts were less resilient to OS compared with LV and LA respectively. Skin fibroblasts were less impacted by proteostasis stress compared to astrocytes and cardiac fibroblasts. Future studies will test the functional relationship of these outcomes to age and developmental status of donors as potential predictive markers.
Peptide-stimulation of autologous T cells reverts immune checkpoint inhibitor resistance of hypermutated colorectal cancer cells
Authors: Schwarz Sandra , Zhaoran Su , Krohn Mathias , Löffler Markus W. , Schlosser Andreas , Linnebacher Michael
Published: February 2024

Two hypermutated colon cancer cases with patient-derived cell lines, peripheral and tumor-infiltrating T cells available were selected for detailed investigation of immunological response.

T cells co-cultured with autologous tumor cells showed only low levels of pro-inflammatory cytokines and failed at tumor recognition. Similarly, treatment of co-cultures with immune checkpoint inhibitors (ICI) did not boost antitumor immune responses. Since proteinase inhibitor 9 (PI-9) was detected in tumor cells, a specific inhibitor (PI-9i) was used in addition to ICI in T cell cytotoxicity testing. However, only pre-stimulation with tumor-specific peptides (cryptic and neoantigenic) significantly increased recognition and elimination of tumor cells by T cells independently of ICI or PI-9i.

We showed, that ICI resistant tumor cells can be targeted by tumor-primed T cells and also demonstrated the superiority of tumor-naïve peripheral blood T cells compared to highly exhausted tumor-infiltrating T cells. Future precision immunotherapeutic approaches should include multimodal strategies to successfully induce durable anti-tumor immune responses.
Transcription Factor Condensates Mediate Clustering ofMETRegulon and Enhancement in Gene Expression
Authors: Lee James , Simpson Leman , Li Yi , Becker Samuel , Zou Fan , Zhang Xin , Bai Lu
Published: February 2024

Some transcription factors (TFs) can form liquid-liquid phase separated (LLPS) condensates. However, the functions of these TF condensates in 3D genome organization and gene regulation remain elusive. In response to methionine (met) starvation, budding yeast TF Met4 and a few co-activators, including Met32, induce a set of genes involved in met biosynthesis. Here, we show that the endogenous Met4 and Met32 form co-localized puncta-like structures in yeast nuclei upon met depletion. Recombinant Met4 and Met32 form mixed droplets with LLPS propertiesin vitro. In relation to chromatin, Met4 puncta co-localize with target genes, and at least a subset of these target genes are clustered in 3D in a Met4-dependent manner. AMET3pr-GFP reporter inserted near several native Met4 binding sites becomes co-localized with Met4 puncta and displays enhanced transcriptional activity. A Met4 variant with a partial truncation of an intrinsically disordered region (IDR) shows less puncta formation, and this mutant selectively reduces the reporter activity near Met4 binding sites to the basal level. Overall, these results support a model where Met4 and co-activators form condensates to bring multiple target genes into a vicinity with higher local TF concentrations, which facilitates a strong response to methionine depletion.
DNA methylation modulates nucleosome retention in sperm and H3K4 methylation deposition in early mouse embryos
Authors: Fanourgakis Grigorios , Gaspa-Toneu Laura , Komarov Pavel A. , Ozonov Evgeniy A. , Smallwood Sebastien A. , Peters Antoine H.F.M.
Published: February 2024

DNA methylation (DNAme) serves a stable gene regulatory function in somatic cells (1). In the germ line and during early embryogenesis, however, DNAme undergoes global erasure and re-establishment to support germ cell and embryonic development (2). Whilede novoDNAme acquisition during male germ cell development is essential for setting genomic DNA methylation imprints, other intergenerational roles for paternal DNAme in defining embryonic chromatin after fertilization are unknown. To approach this question, we reduced levels of DNAme in developing male germ cells through conditional gene deletion of thede novoDNA methyltransferases DNMT3A and DNMT3B in undifferentiated spermatogonia. We observed that DNMT3A serves a DNAme maintenance function in undifferentiated spermatogonia while DNMT3B catalyzesde novoDNAme during spermatogonial differentiation. Mutant male germ cells nevertheless completed their differentiation to sperm. Failingde novoDNAme inDnmt3a/Dnmt3bdouble deficient spermatogonia is associated with increased nucleosome occupancy in mature sperm, preferentially at sites with higher CpG content, supporting the model that DNAme modulates nucleosome retention in sperm (3). To assess the impact of altered sperm chromatin in the formation of embryonic chromatin, we measured H3K4me3 occupancy at paternal and maternal alleles in 2-cell embryos using a newly developed transposon-based tagging assay for modified chromatin. Our data show that reduced DNAme in sperm renders paternal alleles permissive for H3K4me3 establishment in early embryos, independently of possible paternal inheritance of sperm born H3K4me3. Together, this study provides first evidence that paternally inherited DNAme directs chromatin formation during early embryonic development.
Identification of B cell subsets based on antigen receptor sequences using deep learning
Authors: Lee Hyunho , Shin Kyoungseob , Lee Yongju , Lee Soobin , Lee Seungyoun , Lee Eunjae , Kim Seung Woo , Shin Ha Young , Kim Jong Hoon , Chung Junho , Kwon Sunghoon
Published: February 2024

B cell receptors (BCRs) denote antigen specificity, while corresponding cell subsets indicate B cell functionality. Since each B cell uniquely encodes this combination, physical isolation and subsequent processing of individual B cells become indispensable to identify both attributes. However, this approach accompanies high costs and inevitable information loss, hindering high-throughput investigation of B cell populations. Here, we present BCR-SORT, a deep learning model that predicts cell subsets from their corresponding BCR sequences by leveraging B cell activation and maturation signatures encoded within BCR sequences. Subsequently, BCR-SORT is demonstrated to improve reconstruction of BCR phylogenetic trees, and reproduce results consistent with those verified using physical isolation-based methods or prior knowledge. Notably, when applied to BCR sequences from COVID-19 vaccine recipients, it revealed inter-individual heterogeneity of evolutionary trajectories towards Omicron-binding memory B cells. Overall, BCR-SORT offers great potential to improve our understanding of B cell responses.
The full length BEND2 protein is dispensable for spermatogenesis but required for setting the ovarian reserve in mice
Authors: Huang Yan , Bucevic Nina , Coves Carmen , Felipe-Medina Natalia , Marcet-Ortega Marina , Nikou Nikoleta , Madrid-Sandín Cristina , López-Panadés Maria , Buza Carolina , Miralles Neus Ferrer , Iborra Antoni , Pujol Anna , Pendás Alberto M. , Roig Ignasi
Published: February 2024

Infertility affects up to 12% of couples globally, with genetic factors contributing to nearly half of the cases. Advances in genomic technologies have led to the discovery of genes likeBend2, which plays a crucial role in gametogenesis. In the testis,Bend2expresses two protein isoforms: full-length and a smaller one. Ablation of both proteins results in an arrested spermatogenesis. Because theBend2locus is on the X chromosome, and theBend2−/ymutants are sterile, BEND2’s role in oogenesis remained elusive.

In this study, we employed a novelBend2mutation that blocks the expression of the full-length BEND2 protein but allows the expression of the smaller BEND2 isoform. Interestingly, this mutation does not confer male sterility and mildly affects spermatogenesis. Thus, it allowed us to study the role of BEND2 in oogenesis. Our findings demonstrate that full-length BEND2 is dispensable for male fertility, and its ablation leads to a reduced establishment of the ovarian reserve. These results reveal a critical role for full-length BEND2 in oogenesis and provide insights into the mechanisms underlying the establishment of the ovarian reserve. Furthermore, these findings hold relevance for the diagnostic landscape of human infertility.
Spatially Exploring RNA Biology in Archival Formalin-Fixed Paraffin-Embedded Tissues
Authors: Bai Zhiliang , Zhang Dingyao , Gao Yan , Tao Bo , Bao Shuozhen , Enninful Archibald , Zhang Daiwei , Su Graham , Tian Xiaolong , Zhang Ningning , Xiao Yang , Liu Yang , Gerstein Mark , Li Mingyao , Xing Yi , Lu Jun , Xu Mina L. , Fan Rong
Published: February 2024

Spatial transcriptomics has emerged as a powerful tool for dissecting spatial cellular heterogeneity but as of today is largely limited to gene expression analysis. Yet, the life of RNA molecules is multifaceted and dynamic, requiring spatial profiling of different RNA species throughout the life cycle to delve into the intricate RNA biology in complex tissues. Human disease-relevant tissues are commonly preserved as formalin-fixed and paraffin-embedded (FFPE) blocks, representing an important resource for human tissue specimens. The capability to spatially explore RNA biology in FFPE tissues holds transformative potential for human biology research and clinical histopathology. Here, we present Patho-DBiT combiningin situpolyadenylation and deterministic barcoding for spatial full coverage transcriptome sequencing, tailored for probing the diverse landscape of RNA species even in clinically archived FFPE samples. It permits spatial co-profiling of gene expression and RNA processing, unveiling region-specific splicing isoforms, and high-sensitivity transcriptomic mapping of clinical tumor FFPE tissues stored for five years. Furthermore, genome-wide single nucleotide RNA variants can be captured to distinguish different malignant clones from non-malignant cells in human lymphomas. Patho-DBiT also maps microRNA-mRNA regulatory networks and RNA splicing dynamics, decoding their roles in spatial tumorigenesis trajectory. High resolution Patho-DBiT at the cellular level reveals a spatial neighborhood and traces the spatiotemporal kinetics driving tumor progression. Patho-DBiT stands poised as a valuable platform to unravel rich RNA biology in FFPE tissues to study human tissue biology and aid in clinical pathology evaluation.
Reducing Cathodic Drift during Isoelectric Focusing using Microscale Immobilized pH Gradient Gels
Authors: Lomeli Gabriela , Herr Amy E.
Published: February 2024

Microfluidic analytical tools play an important role in miniaturizing proteomic assays for improved detection sensitivity, throughput, and automation. Microfluidic isoelectric focusing (IEF) can resolve proteoforms in lysate from low-to-single cell numbers. However, IEF assays often use carrier ampholytes (CAs) to establish a pH gradient for protein separation, presenting limitations like pH instability in the form of cathodic drift (migration of focused proteins toward the cathode). Immobilized pH gradient (IPG) gels reduce cathodic drift by covalently immobilizing the pH buffering components to a matrix. To our knowledge, efforts to implement IPG gels at the microscale have been limited to glass microdevices. To adapt IEF using IPGs to widely used microfluidic device materials, we introduce a polydimethylsiloxane (PDMS)-based microfluidic device and compare the microscale pH gradient stability of IEF established with IPGs, CAs, and a hybrid formulation of IPG gels and CAs (mixed-bed IEF). The PDMS-based IPG microfluidic device (μIPG) resolved analytes differing by 0.1 isoelectric point within a 3.5-mm separation lane over a 20-min focusing duration. During the 20-min duration, we observed markedly different cathodic drift velocities among the three formulations: 60.1 µm/min in CA-IEF, 2.5 µm/min in IPG-IEF (∼24-fold reduction versus CA-IEF), and 1.4 µm/min in mixed-bed IEF (∼43-fold reduction versus CA-IEF). Lastly, mixed-bed IEF in a PDMS device resolved green fluorescent protein (GFP) proteoforms from GFP-expressing human breast cancer cell lysate, thus establishing stability in lysate from complex biospecimens. µIPG is a promising and stable technique for studying proteoforms from small volumes.
T-cells specific for KSHV and HIV migrate to Kaposi sarcoma tumors and persist over time
Authors: Ravishankar Shashidhar , Towlerton Andrea M.H. , Tiamiyu Iyabode L. , Mooka Peter , Nankoma Janet , Kafeero James , Mubiru Dennis , Sekitene Semei , Aicher Lauri D. , Miller Chris P. , Coffey David G. , Okoche Lazarus , Atwinirembabazi Prisca , Okonye Joseph , White Jessica , Koelle David M. , Jing Lichen , Phipps Warren T. , Warren Edus H.
Published: February 2024

Kaposi sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi sarcoma (KS), which causes significant morbidity and mortality worldwide, particularly in people living with HIV (PLWH) and in sub-Saharan Africa where KSHV seroprevalence is high. Postulating that T-cells specific for KSHV and HIV would be attracted to KS tumors, we performed transcriptional profiling and T-cell receptor (TCR) repertoire analysis of tumor biopsies from 144 Ugandan adults with KS, 106 of whom were also living with HIV. We show that CD8+T-cells and M2-polarized macrophages are the most common immune cells in KS tumors. The TCR repertoire of T-cells associated with KS tumors is shared across spatially and temporally distinct tumors from the same individual. Clusters of T-cells with predicted shared specificity for uncharacterized antigens, potentially encoded by KSHV or HIV, comprise ∼25% of the T-cells in KS tumors. Single-cell RNA-sequencing of blood from a subset of 9 adults captured 4,283 unique αβ TCRs carried in 14,698 putative KSHV- or HIV-specific T-cells, which carried an antigen-experienced effector phenotype. T-cells engineered to express a representative sample of these TCRs showed high-avidity recognition of KSHV- or HIV-encoded antigens. These results suggest that a polyspecific, high-avidity KSHV- and HIV-specific T-cell response, potentially inhibited by M2 macrophages, migrates to and localizes with KS tumors. Further analysis of KSHV- and HIV-specific T-cells in KS tumors will provide insight into the pathogenesis of KS and could guide the development of specific immune therapy based on adoptive transfer or vaccination.

Author Summary

In this work, we set out to examine Kaposi Sarcoma (KS) tumor tissue, as well as peripheral blood cells from individuals with KS, both living with and without HIV. Our goal was to identify T-cells that specifically recognize antigens encoded by Kaposi sarcoma-associated herpesvirus (KSHV) or HIV. By analyzing the T-cell repertoire in KS tumor biopsies from people in Uganda with different types of KS, we uncovered clusters of T-cells with previously unknown ability to recognize these viruses. Through single-cell sequencing of peripheral blood cells, we also observed that many of these T-cells had cell-killing properties. Notably, they often coexisted with a subset of macrophages with immunosuppressive properties, which we suspect may be suppressing the function of virus-targeting T-cells. Our findings suggest that additional studies of these virus-targeting T-cells and their interaction with immunosuppressive macrophages could significantly advance the development of effective therapeutics against KS.
Invariant set theory for predicting failure of antibiotic cycling
Authors: Anderson Alejandro , Kinahan Matthew W. , Gonzalez Alejandro H. , Udekwu Klas , Hernandez-Vargas Esteban A.
Published: February 2024

The limited availability of antibiotics and the need for prompt decision-making present significant challenges for healthcare practitioners. When faced with this situation, practitioners must prioritize their approach based on several key factors. By leveraging the emergent understanding of collateral sensitivity among antibiotic-exposed pathogens, we demonstrate the utility of control invariant sets to predict treatment failure when antibiotic cycling is applied as a therapeutic strategy aiming to eradicate or prevent emergence of multi-drug resistant pathogens. Our results here pave the way for point-of-care diagnostic technologies to identify infections and select appropriate treatments quickly, reducing unnecessary antibiotic use.
Far-red and sensitive sensor for monitoring real time H2O2dynamics with subcellular resolution and in multi-parametric imaging applications
Authors: Lee Justin Daho , Nguyen Amanda , Jin Zheyu Ruby , Moghadasi Aida , Gibbs Chelsea E. , Wait Sarah J. , Evitts Kira M. , Asencio Anthony , Bremner Samantha B , Zuniga Shani , Chavan Vedant , Williams Andy , Smith Netta , Regnier Michael , Young Jessica E. , Mack David , Nance Elizabeth , Boyle Patrick M. , Berndt Andre
Published: February 2024

H2O2is a key oxidant in mammalian biology and a pleiotropic signaling molecule at the physiological level, and its excessive accumulation in conjunction with decreased cellular reduction capacity is often found to be a common pathological marker. Here, we present a red fluorescent Genetically Encoded H2O2Indicator (GEHI) allowing versatile optogenetic dissection of redox biology. Our new GEHI, oROS-HT, is a chemigenetic sensor utilizing a HaloTag and Janelia Fluor (JF) rhodamine dye as fluorescent reporters. We developed oROS-HT through a structure-guided approach aided by classic protein structures and recent protein structure prediction tools. Optimized with JF635, oROS-HT is a sensor with 635 nm excitation and 650 nm emission peaks, allowing it to retain its brightness while monitoring intracellular H2O2dynamics. Furthermore, it enables multi-color imaging in combination with blue-green fluorescent sensors for orthogonal analytes and low auto-fluorescence interference in biological tissues. Other advantages of oROS-HT over alternative GEHIs are its fast kinetics, oxygen-independent maturation, low pH sensitivity, lack of photo-artifact, and lack of intracellular aggregation. Here, we demonstrated efficient subcellular targeting and how oROS-HT can map inter and intracellular H2O2diffusion at subcellular resolution. Lastly, we used oROS-HT with the green fluorescent calcium indicator Fluo-4 to investigate the transient effect of the anti-inflammatory agent auranofin on cellular redox physiology and calcium levels via multi-parametric, dual-color imaging.
Expression of elastin, FBXW2, fibrillin-1, and α-smooth muscle actin in damaged blood vessels of bovine legs
Authors: Akiyama Mari
Published: February 2024

Elastic fibers present within the internal and external elastic laminae play a vital role in maintaining the health of blood vessels. The impairment of smooth muscle cell function results in the development of vascular diseases. In this study, blood vessels from bovine legs were carefully examined in culture dishes for 5 weeks. Examination of elastin, F-box and WD-40 domain-containing protein 2 (FBXW2), and fibrillin-1 in serial sections revealed that elastin and FBXW2 were present in the internal and external elastic laminae, whereas fibrillin-1 was present outside of them. After 5 weeks, elastin and FBXW2 maintained the shape of the elastic lamina, whereas the fibrillin-1-rich layer spread and migrated from the elastic lamina. When the blood vessel lumen was blocked by tissue rich in fibrillin-1, α-smooth muscle actin (SMA) expression weakened. Although elastin and fibrillin-1 are components of elastic fibers, this study revealed that fibrillin-1 has a role different from that of elastin. This in vitro model is useful for identifying the mechanisms underlying vascular degradation.
BootCellNet, a resampling-based procedure, promotes unsupervised identification of cell populations via robust inference of gene regulatory networks
Authors: Kumagai Yutaro
Published: February 2024

Recent advances in measurement technologies, particularly single-cell RNA sequencing (scRNA-seq), have revolutionized our ability to acquire large amounts of omics-level data on cellular states. As measurement techniques evolve, there has been an increasing need for data analysis methodologies, especially those focused on cell-type identification and inference of gene regulatory networks (GRNs). We have developed a new method named BootCellNet, which employs smoothing and resampling to infer GRNs. Using the inferred GRNs, BootCellNet further infers the minimum dominating set (MDS), a set of genes that determines the dynamics of the entire network. We have demonstrated that BootCellNet robustly infers GRNs and their MDSs from scRNA-seq data and facilitates unsupervised identification of cell clusters using scRNA-seq datasets of peripheral blood mononuclear cells and hematopoiesis. It has also identified COVID-19 patient-specific cells and their potential regulatory transcription factors. BootCellNet not only identifies cell types in an unsupervised and explainable way but also provides insights into the characteristics of identified cell types through the inference of GRNs and MDS.

Author Summary

Single-cell omics technologies, such as single-cell RNA-seq (scRNA-seq), are instrumental in identifying novel cell subsets that are involved in various biological processes and diseases. These technologies, however, require further development in data analysis, especially in areas focused on cell-type identification and inference of interactions between genes. The problem of cell-type identification essentially involves clustering, which necessitates a balance between distinguishing different cell states and grouping similar ones together. Current clustering methods still suffer from uncertainty in determining the appropriate number of clusters and in explaining why some cells are clustered together but others are separated. The inference of interactions between genes, gene regulatory network (GRN), remains challenging due to the noisy nature of scRNA-seq. We have developed BootCellNet, a method that infers GRNs and a set of genes dominating the network dynamics and utilizes the set to cluster cells and identify cell types. The method addresses challenges in GRN identification and clustering methods simultaneously and will facilitate the generation of the working hypotheses from a large amount of scRNA-seq data.
Disentangling competitive and cooperative components of the interactions between microbial species
Authors: Ansari Aamir Faisal , Sambamoorthy Gayathri , Alexander Thrisha C , Reddy Yugandhar B.S. , Raut Janhavi , Dixit Narendra M.
Published: February 2024

Interactions between microbial species have been characterized by the net influences, positive or negative, that each species in a pair exerts on the other. This conventional view of interactions being either positive or negative proves restrictive in predicting the behaviour of microbial communities and, more importantly, influencing them towards desired community structures. Here, we propose a more fundamental characterization of the interactions. The net interactions typically comprise positive and negative underlying components. Yet, the conventional view prevails because the components have been difficult to disentangle. We have developed a methodology to disentangle them when metabolic interactions predominate. We conceived a theoretical resource partitioning between species that helps estimate the positive components. The negative components then follow from knowledge of the net interactions. The interactions between two species are then characterized by the ‘quartet’ of these components. We applied the methodologyin silicoto 28 species pairs from the human oral microbiome, yielding 56 net interactions and their 112 quartet components. We found that on average the net interactions comprised positive and negative components of comparable strengths. Interestingly, weak net interactions often arose from the cancellation of strong underlying components. Furthermore, we found species pairs with similar net interactions but vastly different underlying components. Extant community ecology theories, based on net interactions, cannot distinguish between such pairs. The quartet explained several confounding experimental observations and offered new insights into microbial community ecology. We envision its implications in the construction of more refined ecological theories and the engineering of synthetic microbial communities.
Whole Genome Sequencing of DENV-2 isolated fromAedes aegyptimosquitoes in Esmeraldas, Ecuador. Genomic epidemiology of genotype III Southern Asian-American in the country
Authors: Carrazco-Montalvo Andrés , Gutiérrez-Pallo Diana , Arévalo Valentina , Ponce Patricio , Rodríguez-Polit Cristina , Alarcón Damaris , Echeverría-Garcés Gabriela , Coloma Josefina , Nipaz Victoria , Cevallos Varsovia
Published: February 2024

Ecuador is a tropical country reporting Dengue virus (DENV) outbreaks with areas of hyperendemic viral transmission. Entomo-virological surveillance and monitoring effort conducted in the Northwestern border province of Esmeraldas in April 2022, five pools of femaleAedes aegyptimosquitoes from a rural community tested positive for DENV serotype 2 by RT-qPCR. One pool was sequenced by Illumina MiSeq, and it corresponded to genotype III Southern Asian-American. Comparison with other genomes revealed genetic similarity to a human DENV genome sequenced in 2021, also from Esmeraldas. Potential introduction events to the country could have originated from Colombia, considering the vicinity of the collection sites to the neighboring country and high human movement. The inclusion of genomic information complements entomo-virological surveillance, providing valuable insights into genetic variants. This contribution enhances our understanding of Dengue virus (DENV) epidemiology in rural areas and guides evidence-based decisions for surveillance and interventions.

Author Summary

In this study, the complete genetic information of the dengue virus isolated from the mosquito vectorAedes aegyptiwas characterized using molecular biology, sequencing, and bioinformatics analysis. The isolation of the virus from the vector has contributed significantly to our understanding of the transmission dynamics of the disease and its genetic relationship with human cases. Identifying the serotypes and genotypes circulating in Ecuador has enabled us to comprehend the evolution of the virus from 2014 to 2023. Therefore, surveillance of genetic variability would aid in adapting precise prevention and control strategies to the genetic variants present in a specific geographical area. Entomo-virological surveillance is essential for assessing changes in risk and the impact of control measures.
Comparative analysis of 136,401 Admixed Americans and 419,228 Europeans reveals ancestry-specific genetic determinants of clonal haematopoiesis
Authors: Wen Sean , Kuri-Morales Pablo , Hu Fengyuan , Nag Abhishek , Tachmazidou Ioanna , Vishnu Vardhan Deevi Sri , Taiy Haeyam , Smith Katherine , Loesch Douglas P. , Burren Oliver S. , Dhindsa Ryan S. , Wasilewski Sebastian , Alegre-Díaz Jesus , Berumen Jaime , Emberson Jonathan , Torres Jason M. , Collins Rory , Carss Keren , Wang Quanli , Petrovski Slavé , Tapia-Conyer Roberto , Fabre Margarete A. , Harper Andrew R. , Vassiliou George , Mitchell Jonathan
Published: February 2024

The development of clonal haematopoiesis (CH), the age-related expansion of mutated haematopoietic stem cell (HSC) clones, is influenced by genetic and non-genetic factors. To date, large-scale studies of CH have focused on individuals of European descent, such that the impact of genetic ancestry on CH development remains incompletely understood. Here, we investigate this by studying CH in 136,401 admixed participants from the Mexico City Prospective Study (MCPS) and 419,228 European participants from the UK Biobank (UKB). We observe that CH was significantly less common in MCPS compared to UKB (adjusted odds ratio (OR) = 0.56 [95% Cl = 0.55-0.59],P= 1.60 x 10-206), a difference that persisted when comparing MCPS participants whose genomes were >50% ancestrally Indigenous American to those whose genomes were >50% ancestrally European (adjusted OR = 0.76 [0.70-0.83],P= 1.78 x 10-10). Genome- and exome-wide association analyses in MCPS participants identified two novel loci associated with CH (CSGALNACT1andDIAPH3), and ancestry-specific variants in theTCL1Blocus with opposing effect onDNMT3A-versus non-DNMT3A-CH. Meta-analysis of the MCPS and UKB cohorts identified another five novel loci associated with overall or gene specific CH, including polymorphisms atPAPR11/CCND2,MEIS1andUBE2G1/SPNS3. Our CH study, the largest in a non-European population to date, demonstrates the profound impact of ancestry on CH development and reveals the power of cross-ancestry comparisons to derive novel insights into CH pathogenesis and advance health equity amongst different human populations.
Cytoplasmic protein-free mRNA induces stress granules by two G3BP1/2-dependent mechanisms
Authors: Ihn Sean J. , Farlam-Williams Laura , Palazzo Alexander F. , Lee Hyun O.
Published: February 2024

Stress granules are cytoplasmic membraneless organelles that sequester proteins and non-translating mRNAs in response to various stressors. To assess the contributions of mRNA and RNA-binding proteins to stress granule formation, we use microinjection to deliver protein-free mRNA into the cytoplasm in a controlled manner. We demonstrate that mRNAs trigger stress granule formation through two mechanisms that are enhanced by the presence of G3BP1 and G3BP2. Low concentrations ofin vitrotranscribed mRNA activated protein kinase R (PKR), leading to phosphorylation and inhibition of the eukaryotic translation initiation factor eIF2α and stress granule formation. This was inhibited by replacing uridine with pseudouridine in the mRNA or by treating it with RNase III, which cleaves double-stranded RNA. High concentrations of mRNA triggered stress granule formation by a mechanism that was independent of PKR and enhanced by G3BP1/2, highlighting the importance of both protein-free mRNA and RNA-binding proteins in stress granule formation.

Graphical Abstract/Model

Microinjected mRNA induces stress granules in mammalian cells by two G3BP1/2-dependent mechanisms: one requires the stress-sensing protein kinase PKR to phosphorylate the translation initiation factor eIF2α, and the other is independent of PKR and phospho-eIF2α and acts when the cytoplasmic concentration of ribosome-free mRNA is increased acutely.
MS-DIAL 5 multimodal mass spectrometry data mining unveils lipidome complexities
Authors: Takeda Hiroaki , Matsuzawa Yuki , Takeuchi Manami , Takahashi Mikiko , Nishida Kozo , Harayama Takeshi , Todoroki Yoshimasa , Shimizu Kuniyoshi , Sakamoto Nami , Oka Takaki , Maekawa Masashi , Chung Mi Hwa , Kurizaki Yuto , Kiuchi Saki , Tokiyoshi Kanako , Buyantogtokh Bujinlkham , Kurata Misaki , Kvasnička Aleš , Takeda Ushio , Uchino Haruki , Hasegawa Mayu , Miyamoto Junki , Tanabe Kana , Takeda Shigenori , Mori Tetsuya , Kumakubo Ryota , Tanaka Tsuyoshi , Yoshino Tomoko , Arita Makoto , Tsugawa Hiroshi
Published: February 2024

Lipidomics and metabolomics communities comprise various informatics tools; however, software programs that can handle multimodal mass spectrometry (MS) data with structural annotations guided by the Lipidomics Standards Initiative are limited. Here, we provide MS-DIAL 5 to facilitate the in-depth structural elucidation of lipids through electron-activated dissociation (EAD)-based tandem MS, as well as determine their molecular localization through MS imaging (MSI) data using a species/tissue-specific lipidome database containing the predicted collision-cross section (CCS) values. With the optimized EAD settings using 14 eV kinetic energy conditions, the program correctly delineated the lipid structures based on EAD-MS/MS data from 96.4% of authentic standards. Our workflow was showcased by annotating thesn- and double-bond positions of eye-specific phosphatidylcholine molecules containing very-long-chain polyunsaturated fatty acids (VLC-PUFAs), characterized as PC n-3-VLC-PUFA/FA. Using MSI data from the eye and HeLa cells supplemented with n-3-VLC-PUFA, we identified glycerol 3-phosphate (G3P) acyltransferase (GPAT) as an enzyme candidate responsible for incorporating n-3 VLC-PUFAs into thesn-1 position of phospholipids in mammalian cells, which was confirmed using recombinant proteins in a cell-free system. Therefore, the MS-DIAL 5 environment, combined with optimized MS data acquisition methods, facilitates a better understanding of lipid structures and their localization, offering novel insights into lipid biology.
Measurement of adhesion and traction of cells at high yield (MATCHY) reveals an energetic ratchet driving nephron condensation
Authors: Liu Jiageng , Prahl Louis S. , Huang Aria , Hughes Alex J.
Published: February 2024

Engineering of embryonic strategies for tissue-building has extraordinary promise for regenerative medicine. This has led to a resurgence in interest in the relationship between cell biophysical properties and morphological transitions. However, mapping gene or protein expression data to cell biophysical properties to physical morphogenesis remains challenging with current techniques. Here we present MATCHY (multiplexedadhesion and traction ofcells athighyield). MATCHY advances the multiplexing and throughput capabilities of existing traction force and cell-cell adhesion assays using microfabrication and an automated computation scheme with machine learning-driven cell segmentation. Both biophysical assays are coupled with serial downstream immunofluorescence to extract cell type/signaling state information. MATCHY is especially suited to complex primary tissue-, organoid-, or biopsy-derived cell mixtures since it does not rely ona prioriknowledge of cell surface markers, cell sorting, or use of lineage-specific reporter animals. We first validate MATCHY on canine kidney epithelial cells engineered for RET tyrosine kinase expression and quantify a relationship between downstream signaling and cell traction. We go on to create a biophysical atlas of primary cells dissociated from the mouse embryonic kidney and use MATCHY to identify distinct biophysical states along the nephron differentiation trajectory. Our data complement expression-level knowledge of adhesion molecule changes that accompany nephron differentiation with quantitative biophysical information. These data reveal an ‘energetic ratchet’ that explains spatial nephron progenitor cell condensation from the niche as they differentiate, which we validate through agent-based computational simulation. MATCHY offers automated cell biophysical characterization at >104-cell throughput, a highly enabling advance for fundamental studies and new synthetic tissue design strategies for regenerative medicine.