bluuebunny/crossref_metadata_2025 huggingface dataset:
Single Cell Resolution Tracking of Cutaneous T-Cell Lymphoma Reveals Clonal Evolution in Disease Progression
Authors: Dorando Hannah K. , Andrews Jared M. , Borcherding Nicholas C. , Quinn Chaz C. , Schmidt Jennifer A. , Khatavkar Oam U. , Aluri Jahnavi , Harmon Michael T. , Watkins Marcus P. , Frank Anastasia , Cooper Megan A. , Musiek Amy C. , Mehta-Shah Neha , Payton Jacqueline E.
Published: February 2025

Cutaneous T-cell lymphoma (CTCL) remains a challenging disease due to its significant heterogeneity, therapy resistance, and relentless progression. Multi-omics technologies offer the potential to provide uniquely precise views of disease progression and response to therapy. We present here a comprehensive multi-omics view of CTCL clonal evolution, incorporating exome, whole genome, epigenome, bulk-, single cell (sc) VDJ-, and scRNA-sequencing of 114 clinically annotated serial skin, peripheral blood, and lymph node samples from 35 CTCL patients. We leveraged this extensive dataset to define the molecular underpinnings of CTCL progression in individual patients at single cell resolution with the goal of identifying clinically useful biomarkers and therapeutic targets. Our studies identified a large number of recurrent progression-associated clonal genomic alterations; we highlight mutation of CCR4, PI3K signaling, and PD-1 checkpoint pathways as evasion tactics deployed by malignant T cells. We also identified a gain of function mutation in STAT3 (D661Y) and demonstrated by CUT&RUN-seq that it enhances binding to transcription start sites of genes in Rho GTPase pathways, which we previously reported to have activated chromatin and increased expression in HDACi-resistant CTCL. These data provide further support for a previously unrecognized role for Rho GTPase pathway dysregulation in CTCL progression. A striking number of progression-associated mutations occurred in chromatin methylation modifiers, including EZH2, suggesting that EZH1/2 inhibition may also benefit patients with CTCL. Knowledge of these molecular changes should be leveraged for improved disease monitoring, biomarker-informed clinical trial design, and new therapeutic strategies in this challenging and incurable cancer.
Pedigree-based genome-wide imputation using a low-density amplicon panel for the highly polymorphic Pacific oysterCrassostrea(Magallana)gigas
Authors: Sutherland Ben J. G. , Divilov Konstantin , Thompson Neil F. , Delomas Thomas A. , Lunda Spencer L. , Langdon Christopher J. , Green Timothy J.
Published: February 2025

High-density genomic data are instrumental for selective breeding, but the high costs associated with these approaches can hinder progress, as is the case for most aquaculture species. A strategy to reduce genotyping costs is to genotype a few select animals at high-density (e.g., parents, grandparents), and large numbers of other animals at low density (e.g., offspring), then impute genotypes. This has been demonstratedin silicofor Pacific oysterCrassostrea(Magallana)gigasbut was particularly challenging relative to other species and has never been empirically tested. Here, four families of Pacific oysters, bred via marker-assisted selection for variation at a locus for ostreid herpesvirus 1 (OsHV-1) survivorship, were exposed to OsHV-1 then genotyped using a low-density amplicon panel (n = 240). Parents were genotyped with the amplicon panel and whole-genome resequencing data. Offspring genotypes were imputed, and accuracy was characterized against a hold-out dataset. Imputation reduced minor allele frequencies and enriched homozygosity. Anin silicothree-generation analysis was used to investigate the effect of deepening the pedigree, which generated superior results (genotype concordance = 84.5%, allelic dosage r = 0.73), although the two-generation imputation is likely sufficient for selection decisions (GC = 75.3%; r = 0.63). Genome-wide associations to OsHV-1 survivorship with imputed data identified major effect loci on the expected chromosome 8, but in a different region than expected, pointing to a potentially more complex genetic architecture for the trait. These methods demonstrate the empirical utility of amplicon panel-based genome-wide imputation in shellfish, and thus enable low-cost selective breeding techniques.
Clonal Heterogeneity in Human Pancreatic Ductal Adenocarcinoma and Its Impact on Tumor Progression
Authors: Kalfakakou Despoina , Cameron Daniel C. , Kawaler Emily A. , Tsuda Motoyuki , Wang Lidong , Jing Xiaohong , Hajdu Cristina , Tamayo Dylan L. , Shim Yoona , Ackermann Amanda , Weissinger Daniel , Zimny Hayley , Hernandez Rosmel , Beier Matthew , Dimartino Dacia , Meyn Peter , Rice Kalina , Selvaraj Shanmugapriya , Loomis Cynthia , Heguy Adriana , Lund Amanda W. , Sears Rosalie C. , Welling Theodore H. , Dolgalev Igor , Tsirigos Aristotelis , Simeone Diane M.
Published: February 2025

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer characterized by profound desmoplasia and cellular heterogeneity, which cannot be fully resolved using traditional bulk sequencing approaches. To understand the contribution of this heterogeneity to PDAC biology, we analyzed a large cohort of primary human PDAC samples (n = 62), profiling 443,451 single cells and 53,236 spatial transcriptomic spots using a combined single-cell RNA sequencing and spatial transcriptomics approach. Our analysis revealed significant intratumoral heterogeneity, with multiple genetically distinct neoplastic clones co-existing within individual tumors. These clones exhibited diverse transcriptional states and subtype profiles, challenging the traditional binary classification of PDAC into basal and classical subtypes; instead, our findings support a transcriptional continuum influenced by clonal evolution and spatial organization. Additionally, these clones each interacted uniquely with surrounding cell types in the tumor microenvironment. Phylogenetic analysis uncovered a rare but consistent classical-to-basal clonal transition associated withMYCamplification and immune response depletion, which were validated experimentally, suggesting a mechanism driving the emergence of a more aggressive basal clonal phenotype. Spatial analyses further revealed dispersed clones enriched for epithelial-to-mesenchymal transition (EMT) activity and immune suppression, correlating with metastatic potential and colonization of lymph node niches. These dispersed clones tended to transition toward a basal phenotype, contributing to disease progression. Our findings highlight the critical role of clonal diversity, transcriptional plasticity, and TME interactions in shaping human PDAC biology. This work provides new insights into the molecular and spatial heterogeneity of PDAC and offers potential avenues for therapeutic intervention targeting clonal evolution and the mechanisms driving metastasis.
Stem Cell-Parenchymal Fusion: Communication and Gene Regulation
Authors: Huang JoAnne , Nazaryabrbekoh Fateme , Kim Joohyun , Jung Jangwook P.
Published: February 2025

We investigated the cellular and molecular consequences of accidental cell fusion between mHL1 and mMSC cell lines using previously published single-cell RNA sequencing (scRNA-seq) data. We characterized the resulting cell types, their communication patterns, and underlying gene regulatory networks. Initial analyses identified four distinct cell types (mHL1, mHL1 fusion, mMSC, and mMSC fusion), which were also consolidated into three clusters via unsupervised learning. Differential gene expression analysis revealed diverse cell type associations with shared and exclusive gene expression. Single-cell compatible weighted gene co-expression network analysis (WGCNA) linked specific gene modules to distinct cell types and associated biological processes, including muscle contraction and metabolism in mHL1/mHL1 fusion, and extracellular matrix (ECM) formation and lipid metabolism in mMSC fusion. Cell-cell communication analysis revealed distinct intercellular communication patterns, with mMSC primarily acting as a ligand and mHL1 as both ligand and receptor. Reducing cell type complexity progressively simplified communication networks and altered pathway enrichment, while ECM-receptor pathways remained largely unchanged, with only minor shifts in ligand-receptor pairs. Interestingly, removing parental cells from the analysis altered cell type assignments, underscoring the influence of parental cell presence on fusion outcomes. Finally, gene regulatory network analysis identified key transcription factors (TFs) driving cell identity, distinguishing between master regulators and TFs with high cell-type specificity. These findings demonstrate the power of integrating multiple inter- and intra-cellular communication analysis approaches to dissect the complex interplay between cell type, communication, and gene regulation in cell fusion.
Germline and somatic variation influence nuclear-mitochondrial crosstalk in tumourigenesis
Authors: Daunesse Maëlle , Sundaram Vasavi , Kentepozidou Elissavet , Connelly John , Anderson Craig J. ,   Liver Cancer Evolution Consortium, López-Bigas Núria , Semple Colin A. , Odom Duncan T. , Taylor Martin S. , Roller Maša , Aitken Sarah J. , Flicek Paul
Published: February 2025

Hepatocellular carcinoma is the most prevalent primary liver cancer and arises from hepatocytes, which are very metabolically active and contain abundant mitochondria. Despite extensive characterisation of nuclear driver genes and pathways, the role of crosstalk between the mitochondrial and nuclear genomes in mitochondrial mutagenesis remains poorly understood. To address this, we leveraged a chemical carcinogenesis model of liver cancer in four mouse strains. We analysed hundreds of tumours with paired whole-genome sequencing and total RNA sequencing to comprehensively characterise mitochondrial mutations and expression. Followingde novoassembly of three new mitochondrial genomes, we devised an accurate and efficient heteroplasmy detection approach and analysed mitochondrial DNA (mtDNA) content and transcription. Across strains, there was high concordance of the number of heteroplasmies, mutation signatures, and variant allele frequencies. In contrast, the heteroplasmy rate and genic locations differed between strains, suggesting strain-specificity in DNA repair mechanisms in early tumour development. Independent of age or environmental factors, tumours had lower mtDNA content than adjacent normal liver, suggesting mitochondrial loss during tumourigenesis. Additionally, within individual strains, mtDNA content differences were associated with nuclear driver gene choice. Our comprehensive characterisation of mitochondrial genomes and expression patterns demonstrates that both germline and somatic genetic variation influence tumour mtDNA content, mutation patterns, and gene expression.
Triterpenoid CDDO-EA Protects from Hyperglycemia, Hyperinsulinemia, and Obesity by Decreasing Energy Intake
Authors: Cantu Austin E , Rasa Cordelia , Mito Shizue , Cantu Denae , Lopez-Alvarenga Juan Carlos , Rivera-Lopez Leslie Lynnette , Rios Israel , Abrego-Gonzalez Ashley , Reyna Sara M
Published: February 2025

Obesity is a significant factor in the development of type 2 diabetes (T2D). Treatment of obesity is pivotal in the prevention and management of T2D, and the development of new pharmacological therapies are studied for improving insulin resistance and glucose intolerance. Oleanolic acid derived triterpenoids, 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acids (CDDOs), are studied to elucidate the mechanisms by which they protect against obesity. However, there remain fundamental gaps in knowledge regarding the physiological and molecular mechanisms by which CDDOs protect against obesity. Our recently published studies showed that CDDO-ethyl amide (CDDO-EA) prevents skeletal muscle inflammation by inhibiting activation of nuclear factor kappa B (NF-κB) signaling. Moreover, CDDO-EA induced translocation of the glucose transporter, GLUT4, in skeletal muscle cells. We hypothesized that CDDO-EA protects from obesity-induced hyperglycemia in mice fed a high fat diet (HFD). Our results show that CDDO-EA protects from HFD-induced obesity but has no effect on body weight in mice fed a low-fat diet (LFD). Our data show that CDDO-EA inhibition of weight gain is associated with reduced caloric intake and glucose and insulin levels in mice fed a HFD. This highlights the potential of CDDO-EA as a therapeutic agent for obesity treatment and the protection against the development of T2D.
MicroRNA-34a suppresses KLF2 to promote pathological angiogenesis through the CXCR4/CXCL12 pathway in age-related macular degeneration
Authors: Colasanti Jason J. , Lin Joseph B. , Terao Ryo , Lee Tae Jun , Santeford Andrea , Apte Rajendra S.
Published: February 2025

Age-related macular degeneration (AMD), characterized by pathologic choroidal neovascularization (CNV), is a leading cause of vision loss in the elderly. Vascular endothelial growth factor A (VEGFa) antagonists can prevent acute vision loss, but high treatment burden and loss of efficacy with chronic therapy highlight the need to explore alternative mechanisms. Recently, microRNA-34a (miR-34a) has emerged as a key regulator in aging and age-related diseases, but its role in neovascular AMD is unclear. In an injury-induced murine CNV model, we discovered miR-34a promoted pathological angiogenesis, without altering expression of Vegfa or its receptor Kdr, the canonical regulators of CNV. Mechanistically, miR-34a directly targets and inhibits the transcription factor KLF2 thereby upregulating the pro-angiogenic factors CXCR4 and CXCL12. Finally, we show miR-34a exacerbates CNV in aged mice and is expressed in CNV lesions excised from wet AMD patients. These findings establish a causal link between the age-related miR-34a and neovascularization in AMD.

Teaser

Identification of a molecular mechanism involved in the pathogenesis of a prevalent and debilitating age-related ocular disease.
Wnt-dependent spatiotemporal reprogramming of bone marrow niches drives fibrosis
Authors: Banjanin Bella , Nagai James , Mun YeVin , Fuchs Stijn , Snoeren Inge , Boers Joachim , Ruiz Tejada Segura Mayra L. , Mora Hector Tejeda , Galyga Anna Katharina , Benabid Adam , Sarkis Rita , Naveiras Olaia , Rizk Marta , Wolf Michael , Craveiro Rogerio B. , Peisker Fabian , Stalmann Ursula , Pritchard Jessica E. , Ryou Hosuk , Alham Nasullah Khalid , Weiler Marek , Kiessling Fabian , Lammers Twan , Migliaccio Anna Rita , Sivaraj Kishor Kumar , Adams Ralf H. , Bindels Eric , Gribnau Joost , Royston Daniel , Gleitz Hélène F.E , Kramann Rafael , Nombela-Arrieta César , Costa Ivan G. , Schneider Rebekka K.
Published: February 2025

Bone marrow fibrosis is the most extensive matrix remodeling of the microenvironment and can includede novoformation of bone (osteosclerosis). Spatiotemporal information on the contribution of distinct bone marrow niche populations to this process is incomplete. We demonstrate that fibrosis-inducing hematopoietic cells cause profibrotic reprogramming of perivascular CXCL12 abundant reticular (CAR) progenitor cells resulting in loss of their hematopoiesis-support and upregulation of osteogenic and pro-apoptotic programs. In turn, peritrabecular osteolineage cells (OLCs) are activated in an injury-specific, Wnt-dependent manner, comparable to skeletal repair. OLCs fuel bone marrow fibrosis through their expansion and skewed differentiation, resulting in osteosclerosis and expansion of Ly6a+ fibroblasts. NCAM1 expression marks peritrabecular OLCs and their expansion into the central marrow is specific for fibrosis in mice and patients. Peritrabecular stromal b-catenin expression is linked to fibrosis in patients and inhibition of Wnt signaling reduces bone marrow fibrosis and osteosclerosis, possibly being a clinically relevant therapeutic target.
Evaluation of the Safety and Efficacy of the Respiratory Syncytial Virus FG Chimeric Vaccine KD-409 in Rodent Models for Maternal and Pediatric Vaccination
Authors: Yamaue Ryo , Terashima Madoka , Soejima Kenji , Torikai Masaharu
Published: February 2025

Respiratory syncytial virus (RSV) causes severe infection in neonates and infants. However, a suitable RSV vaccine for children has yet to be approved. We developed an effective and safe RSV vaccine for newborns and children. Accordingly, we evaluated the safety and efficacy of the RSV FG chimeric protein KD-409, which incorporates a highly conserved region of the RSV G protein into the RSV F protein, in several rodent models. We confirmed the effect of our vaccine-induced antibody transfer using a guinea pig model. Subsequently, we evaluated the exacerbation of infection in a BALB/c mouse model of passive immunity, which was designed to mimic the vaccination of pregnant women. Notably, KD-409 in the presence of alum did not exacerbate infection, which occurred upon administering pre-F with alum. Our active immunization model of BALB/c mice, which simulated vaccination with a pediatric vaccine, suggested that KD-409 with alum was less likely to exacerbate inflammation than FI-RSV or pre-F with alum. The efficacy was evaluated in a cotton rat model, in which KD-409 demonstrated better protection against infection than pre-F without adjuvant, the only currently approved formulation for immunizing pregnant women. Collectively, antibody transfer in pregnant guinea pigs during immunization, safety in mice during passive and active immunization, and efficacy in cotton rats demonstrate the high potential of KD-409 as a novel vaccine candidate. Our results provide insights into the potential of KD-409 as a safe and effective next-generation RSV vaccine that can cover the neonatal-to-pediatric age range.

IMPORTANCE

The safety and efficacy of KD-409, an RSV FG chimeric protein, were evaluated in appropriate rodents. We found that KD-409 was less likely to exacerbate infections and symptoms, and exhibited superior safety and efficacy to pre-F without adjuvant, which is the only approved vaccine for pregnant women. Thus, KD-409 represents a valuable immunization modality for maternal and pediatric immunization.
Putative interfollicular stem cells of skin epidermis possess a specific mechanical signature that evolves during aging
Authors: Miny Sarah , Runel Gaël , Chlasta Julien , Lapart Jean-André , Bonod Christelle
Published: February 2025

Skin homeostasis and self-renewal are partially maintained by interfollicular stem cells (ISCs), located in the basal layer above the dermal papillae of the dermo-epidermal junction (DEJ). Aging leads to a decline in skin renewal and a concurrent reduction in stem cell potential. It is also marked by disorganization of the extracellular matrix in both the DEJ and dermis, and flattening of the DEJ. To better understand ISC aging, new methods are needed to characterize ISCs and their environment. Since mechanical properties of cells and their substrate influence cell fate, we employed atomic force microscopy to explore whether ICSs niches and the DEJ exhibit distinct mechanical properties. Our findings reveal that ISCs possess greater stiffness than other basal cells, a mechanical signature that diminishes with age. Additionally, the DEJ beneath ISCs shows higher stiffness than under other basal cells, providing ISCs with a specific mechanical environment, which also deteriorates during aging.In vitro, sorting of ISCs based on MCSP expression effectively isolates ISCs beneath the dermal papillae, allowing the measurement of their mechanical signature and stemness potential under varying mechanical conditions. The study of ISC mechanical signatures offers a promising approach for characterizing 3D skin models and understanding defects in skin renewal and wound healing.
The response of mitochondrial position to glucose stimulation in a model system of the pancreatic beta cell
Authors: Perez Luis , Ng Xue Wen , Piston David W. , Mukherji Shankar
Published: February 2025

The compartmentalization of eukaryotic cells into membrane-bound organelles with specific subcellular positioning enables precise spatial and temporal control of cellular functions. While functionally significant mitochondrial localization has been demonstrated in cells such as neurons, it remains unclear how general these cell principles are. Here, we examine the spatial organization of mitochondria within MIN6 pancreatic beta cells under variable glucose conditions. We observe glucose-dependent redistributions of mitochondria, favoring peripheral localization at elevated glucose levels when insulin secretion is also elevated. Our results suggest that active mitochondrial transport along microtubules and calcium activity, but not ATP synthesis, are critical regulators of this redistribution. We derived a mathematical model that reveals a putative affinity of the mitochondria for cellular membranes competes with mitochondrial microtubule attachment to play an important role in establishing the mitochondrial spatial patterns we observe. These results suggest that mitochondrial positioning may contribute to optimizing energy delivery in response to local demand, potentially representing a general regulatory mechanism across various cell types.
Small and large extracellular vesicles from human preovulatory follicular fluid display distinct ncRNA cargo profile and differential effect on granulosa cell line KGN
Authors: Varik Inge , Saretok Katariina Johanna , Rosenberg Kristine , Quintero Ileana , Puhka Maija , Volkova Nataliia , Trošin Aleksander , Guazzi Paolo , Velthut-Meikas Agne
Published: February 2025

Follicular fluid extracellular vesicles (FF EVs) facilitate communication between oocytes and somatic cells within the ovarian follicle, playing a pivotal role in follicular development. This study highlights the molecular and functional distinctions between small (SEV) and large (LEV) FF EV subpopulations, revealing their specialized regulatory roles in granulosa cell (GC) biology and their consequential impact on ovarian function. Single-EV profiling uncovered distinct tetraspanin distributions, with LEVs containing a lower proportion of CD9/CD63/CD81-positive particles compared to SEVs. Functionally, SEVs reduced estradiol secretion by GCs, whereas LEVs enhanced progesterone production, demonstrating their differential effects on steroidogenesis. Transcriptomic analysis revealed extensive SEV-induced changes in GC gene expression, affecting pathways involved in transcription, TGF-β signaling, extracellular matrix (ECM) remodeling, and cell cycle regulation. In contrast, LEVs elicited minimal transcriptional changes, primarily modulating genes associated with immune regulation and oxidative stress defense. Small RNA sequencing further revealed distinct non-coding RNA (ncRNA) profiles, with SEVs enriched in miRNAs targeting pathways critical for GC differentiation, while LEVs carried higher levels of piRNAs implicated in maintaining genomic stability. These findings advance our understanding of FF EV-mediated intercellular communication and underscore the importance of investigating EV subpopulations independently.
TRPV2 and TRPM2 channels facilitate pulmonary endothelial barrier recovery after ROS-induced permeability
Authors: Schaller Lena , Gudermann Thomas , Dietrich Alexander
Published: February 2025

Reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), are known signaling molecules that increase endothelial barrier permeability. In this study, we investigated the roles of two redox-sensitive transient receptor potential (TRP) ion channels, TRPM2 and TRPV2, in H2O2- induced endothelial barrier dysfunction. Using primary human pulmonary microvascular endothelial cells (HPMEC), we employed impedance-based resistance measurements, Western blot, and immunofluorescence staining to assess the effects of H2O2on the endothelial barrier. Exposure to sublytic concentrations of H2O2caused an acute loss of endothelial barrier integrity, accompanied by the cleavage of vascular endothelial cadherin (VE-cadherin), which was also apparent after application of the TRPV2 activator cannabidiol. The inhibition of either TRPV2 with tranilast or a disintegrin and metalloprotease domain-containing protein 10 (ADAM10) with GI254023X significantly reduced H2O2-induced VE-cadherin cleavage, while TRPM2 inhibition by econazole significantly increased H2O2-driven VE-cadherin cleavage. Although inhibition of either TRP channel did not prevent the initial loss of barrier resistance upon H2O2exposure, both were essential for the subsequent recovery of barrier integrity. Time-course immunofluorescence stainings revealed that HPMEC barrier recovery involved a transient localization of N-cadherin proteins at cell-cell junctions, which were replaced by VE-cadherin within 90 minutes. This process of cadherin-switching did not occur upon inhibition of TRPV2 or ADAM10. Our results highlight complementary roles for TRPM2 and TRPV2 as redox sensitive ion channels in the microvascular endothelium and provide insight into the mechanisms underlying pulmonary microvascular endothelial barrier recovery.

Graphical Abstract: See text for more details
Minimizing detection bias of somatic mutations in a highly heterozygous oak genome
Authors: Xian Wenfei , Carbonell-Bejerano Pablo , Rabanal Fernando A. , Bezrukov Ilja , Reymond Philippe , Weigel Detlef
Published: February 2025

Somatic mutations are particularly relevant for long-lived organisms. Sources of somatic mutations include imperfect DNA repair, replication errors, and exogenous damage such as ultraviolet radiation. A previous study estimated a surprisingly low number of somatic mutations in a 234-year-old individual of the pedunculate oak (Quercus robur), known as the Napoleon Oak. It has been suggested that the true number of somatic mutations was underestimated due to gaps in the reference genome and too conservative filtering of potential mutations. We therefore generated new high-fidelity long-read data for the Napoleon Oak (n= 12) to produce both a pseudo-haploid genome assembly and a partially phased diploid assembly. The high heterozygosity allowed for complete reconstruction of phased and gapless centromeres for 22 of the 24 chromosomes. On the other hand, the high heterozygosity posed challenges for short-read alignments. Use of only the pseudo-haploid assembly as a reference led to potential misalignments, while use of only the diploid assembly reduced variant detection sensitivity. Since most somatic mutations are layer-specific, the observed frequency is expected relatively low, even where all cells in a single layer contain a specific mutation. To address this challenge, we employed a read assignment strategy, selecting the appropriate reference sequence (pseudo-haploid or diploid) based on alignment score and mapping quality. Ultimately, we identified 198 high-confidence somatic mutations, compared with 17 somatic mutations identified before with the same set of short reads. Our approach thus increased the total estimated annual mutation rate by a factor of five.
Depolarizing afterpotentials in rodent mEC layer II neurons vary gradually across all principal cell types
Authors: Kümpfbeck Franziska S. , Nagele Johannes , Felmy Felix , Herz Andreas V.M.
Published: February 2025

Principal cells in Layer II (LII) of the rodent medial entorhinal cortex (mEC) often discharge in short burst episodes, whose biophysical mechanism and biological function are not fully understood. As shown byin vitrorecordings in rats andin vivorecordings in mice, action potentials of stellate cells (SCs) and pyramidal cells (PCs) in mEC LII can trigger depolarizing afterpotentials (DAPs) that facilitate spike doublets and burst firing.In vitrorecordings in mice confirm these findings for SCs and two additional cell types intermediate between SCs and PCs but suggest that mouse PCs generate minute DAPs only. To better understand the diversity and physiology of the intermediate LII mEC cell types, we performed slice experiments in Long Evans rats of both sexes and analyzed the data using clustering techniques. The analysis suggests four distinct cell clusters, reminiscent of previous observations in mice. On purpose, DAP properties were not used for the cluster analysis so that they could be studied post-hoc in an unbiased manner. Our results show that characteristic features of DAPs and other physiological markers are broadly distributed within each cell class and vary gradually from cluster to cluster, forming a continuum of biophysical properties.
Ist2, a protein involved in phosphatidylserine transport, is an ER lipid scramblase
Authors: Sebinelli Heitor Gobbi , Syska Camille , Razmazma Hafez , Albanese Veronique , Araujo Ana Rita Dias , Hilpert Cecile , Montigny Cedric , Jaxel Christine , Tchamba Manuella , Belingar Karolina , D'Ambrosio Juan Martin , Monticelli Luca , Lenoir Guillaume , Copic Alenka
Published: February 2025

Lipid scramblases allow passive flip-flop of phospholipids between bilayer leaflets, thereby promoting membrane symmetry. At the endoplasmic reticulum (ER), where phospholipid synthesis is restricted to one of the two leaflets, scramblase activity should be essential for equilibrated membrane growth. However, phospholipid scramblases at the ER are poorly understood. The yeast protein Ist2 contains an ER domain and a cytosolic tail that binds the plasma membrane (PM) and participates in the transfer of phosphatidylserine (PS). Here, we show both in vitro and in silico that the ER- domain of Ist2, which bears homology to the TMEM16 proteins, possesses a lipid scramblase activity. Ist2 activity is not regulated by Ca2+, in contrast to TMEM16 proteins, but is affected by the lipid composition of the bilayer used in simulations. In cells, we do not find a strong impact of the scramblase domain of Ist2 in on PS distribution; however, its over-expression or deletion affects processes at the ER such as vesicular transport, lipid droplet biogenesis and general phospholipid transport, with a specific contribution of residues important for lipid scrambling. Our study therefore identifies the first dedicated phospholipid scramblase in yeast and demonstrates that membrane asymmetry can impact diverse membrane-remodeling processes at the ER.
Dynamic and Biphasic Regulation of Cell Migration by Ras
Authors: Lin Yiyan , Parajón Eleana , Yuan Qinling , Ye Siyu , Qin Guanghui , Deng Yu , Borleis Jane , Koyfman Ariel , Iglesias Pablo A. , Konstantopoulos Konstantinos , Robinson Douglas N. , Devreotes Peter N.
Published: February 2025

Ras has traditionally been regarded as a positive regulator and therapeutic target due to its role in cell proliferation, but recent findings indicate a more nuanced role in cell migration, where suppressed Ras activity can unexpectedly promote migration. To clarify this complexity, we systematically modulate Ras activity using various RasGEF and RasGAP proteins and assess their effects on migration dynamics. Leveraging optogenetics, we assess the immediate, non-transcriptional effects of Ras signaling on migration. Local RasGEF recruitment to the plasma membrane induces protrusions and new fronts to effectively guide migration, even in the absence of GPCR/G-protein signaling whereas global recruitment causes immediate cell spreading halting cell migration. Local RasGAP recruitment suppresses protrusions, generates new backs, and repels cells whereas global relocation either eliminates all protrusions to inhibit migration or preserves a single protrusion to maintain polarity. Consistent local and global increases or decreases in signal transduction and cytoskeletal activities accompany these morphological changes. Additionally, we performed cortical tension measurements and found that RasGEFs generally increase cortical tension while RasGAPs decrease it. Our results reveal a biphasic relationship between Ras activity and cellular dynamics, reinforcing our previous findings that optimal Ras activity and cortical tension are critical for efficient migration.

Significance

This study challenges the traditional view of Ras as solely a positive regulator of cell functions by controlling of gene expression. Using optogenetics to rapidly modulate Ras activity inDictyostelium, we demonstrate a biphasic relationship between Ras activity and migration: both excessive and insufficient Ras activity impair cell movement. Importantly, these effects occur rapidly, independent of transcriptional changes, revealing the mechanism by which Ras controls cell migration. The findings suggest that optimal Ras activity and cortical tension are crucial for efficient migration, and that targeting Ras in cancer therapy should consider the cell’s initial state, aiming to push Ras activity outside the optimal range for migration. This nuanced understanding of the role of Ras in migration has significant implications for developing more effective cancer treatments, as simply inhibiting Ras might inadvertently promote metastasis in certain contexts.
Mrx6 binds the Lon protease Pim1 N-terminal domain to confer selective substrate specificity and regulate mtDNA copy number
Authors: Schrott Simon , Gerle Charlotte , Bibinger Sebastian , Bertgen Lea , Marino Giada , Schwenkert Serena , Rathberger Romina , Herrmann Johannes M. , Osman Christof
Published: February 2025

Mitochondrial DNA (mtDNA) copy number regulation remains incompletely understood, despite its importance in cellular function. InSaccharomyces cerevisiae, Mrx6 belongs to the Pet20-domain-containing protein family, consisting of Mrx6, Pet20, and Sue1. Notably, absence of Mrx6 leads to increased mtDNA copy number. Here, we identify the C-terminus of Mrx6 as essential for its stability and interaction with the mitochondrial matrix protein Mam33. Deletion of Mam33 mimics the effect of Mrx6 loss, resulting in elevated mtDNA copy number. Bioinformatics, mutational analyses, and immunoprecipitation studies reveal that a subcomplex of Mam33 and Mrx6 trimers interacts with the substrate recognition domain of the conserved mitochondrial Lon protease Pim1 through a bipartite motif in the Pet20 domain of Mrx6. Loss of Mrx6, its paralog Pet20, Mam33, or mutations disrupting the interaction between Mrx6 and Pim1 stabilize key proteins required for mtDNA maintenance, the RNA polymerase Rpo41 and the HMG-box-containing protein Cim1. We propose that Mrx6, alongside Pet20 and Mam33, regulates mtDNA copy number by modulating substrate degradation through Pim1. Additionally, Mrx6 loss alters Cim1’s function, preventing the detrimental effect on mtDNA maintenance observed upon Cim1 overexpression. The presence of three Pet20-domain proteins in yeast implies broader roles of Lon protease substrate recognition beyond mtDNA regulation.
Predicting the topography of fitness landscapes from the structure of genotype-phenotype maps
Authors: Srivastava Malvika , Louis Ard A. , Martin Nora S.
Published: February 2025

Ruggedness, the prevalence of fitness peaks, and navigability, the existence of fitness-increasing paths to a target, are key factors affecting evolution on fitness landscapes. We analytically predict landscape ruggedness for genotype-phenotype maps with randomly assigned fitness, using only the sizes of neutral components – mutationally connected genotype sets sharing the same phenotype– and their evolvabilities, the number of neighbouring phenotypes. Further, more evolvable peaks tend to have higher fitness and a minimal evolvability is required for navigability.
Balancing Act: Groundwater microbiome’s resilience and vulnerability to hydroclimatic extremes
Authors: Wang He , Herrmann Martina , Schroeter Simon A. , Zerfaß Christian , Lehmann Robert , Lehmann Katharina , Ivanova Arina , Pohnert Georg , Gleixner Gerd , Trumbore Susan E. , Totsche Kai Uwe , Küsel Kirsten
Published: February 2025

Groundwater health is increasingly threatened by climate change, which alters precipitation patterns, leading to groundwater recharge shifts. These shifts impact subsurface microbial communities, crucial for maintaining ecosystem functions. In this decade-long study of carbonate aquifers, we analyzed 815 bacterial 16S rRNA gene datasets, 226 dissolved organic matter (DOM) profiles, 387 metabolomic datasets, and 174 seepage microbiome sequences. Our findings reveal distinct short- and long-term temporal patterns of groundwater microbiomes driven by environmental fluctuations. Microbiomes of hydrologically connected aquifers exhibit lower temporal stability due to stochastic processes and greater susceptibility to surface disturbances, yet they demonstrate remarkable resilience. Conversely, isolated aquifer microbiomes show resistance to short-term changes, governed by deterministic processes, but exhibit reduced stability under prolonged stress. Variability in seepage-associated microorganisms, DOM, and metabolic diversity further drive microbiome dynamics. These findings highlight the dual vulnerability of groundwater systems to acute and chronic pressures, emphasizing the critical need for sustainable management strategies to mitigate the impacts of hydroclimatic extremes.
Forward genetic screen in zebrafish identifies new fungal regulators that limit host-protectiveCandida-innate immune interaction
Authors: Blair Bailey A. , Bragdon Emma , Dhillon Gursimran , Baker Nnamdi , Stasiak Lena , Muthig Mya , Miramon Pedro , Lorenz Michael C. , Wheeler Robert T.
Published: February 2025

Candidais one of the most frequent causes of bloodstream infections, and our first line of defense against these invasive infections is the innate immune system. The early immune response is critical in controllingC. albicansinfection, butC. albicanshas several strategies to evade host immune attack. Phagocytosis ofC. albicansblocks hyphal growth, limiting host damage and virulence, but howC. albicanslimits early recruitment and phagocytosis in vertebrate infection is poorly understood. To study innate immune evasion by intravital imaging, we utilized the transparent larval zebrafish infection model to screen 131C. albicansmutants for altered virulence and phagocyte response. Infections with each of seven hypovirulent mutants led to altered phagocyte recruitment and/or phagocytosis, falling into four categories. Of particular interest among these isNMD5, a predicted β-importin and newly-identified virulence factor. Thenmd5Δ/Δ mutant fails to limit phagocytosis and its virulence defects are eliminated when phagocyte activity is compromised, suggesting that its role in virulence is limited to immune evasion. These quantitative intravital imaging experiments are the first to document alteredCandida-phagocyte interactions for several additional mutants, and clearly distinguish recruitment from phagocytic uptake, suggesting thatCandidamodulates both events. This initial large-scale screen of individualC. albicansmutants in a vertebrate, coupled with high-resolution imaging ofCandida-phagocyte interactions, provides a more nuanced view of how diverse mutations can lead to more effective phagocytosis, a key immune process which blocks germination and drives anti-fungal immunity.

Importance

Candida albicansis part of the human microbial community and is a dangerous opportunistic pathogen, able to prevent its elimination by the host immune system. AlthoughCandidaavoids immune attack through several strategies, we still understand little about how it regulates when immune phagocytes get recruited to the infection site and when they engulf fungal cells. We tested over 130 selectedCandidamutants for their ability to cause lethal infection and found several avirulent mutants which provoked altered innate immune responses, resulting in lower overall inflammation and greater host survival. Of particular interest isNMD5, which acts to limit fungal phagocytosis and is predicted to regulate the activity of stress-associated transcription factors. Our high-content screening was enabled by modelingCandidainfection in transparent vertebrate zebrafish larva. Our findings help us understand howCandidasurvives immune attack during commensal and pathogenic growth, and may eventually inform new strategies for controlling disease.
Analysis of crosslinking sites suggestsC. elegnasPIWI Argonaute exhibits flexible conformations for target recognition
Authors: Wu Wei-Sheng , Lee Dong-En , Chung Chi-Jung , Lu Shang-Yi , Brown Jordan S. , Zhang Donglei , Lee Heng-Chi
Published: February 2025

Small RNAs play critical roles in gene regulation in diverse processes across organisms. Crosslinking, ligation, and analyses of sequence hybrid (CLASH) experiments have shown PIWI and Argonaute proteins bind to diverse mRNA targets, raising questions about their functional relevance and the degree of flexibility in target recognition.

As crosslinking-induced mutations (CIMs) provides nucleotide-resolution of RNA binding sites, we developed MUTACLASH to systematically analyze CIMs in piRNA and miRNA CLASH data inC. elegans. We found CIMs are enriched at the nucleotide positions of mRNA corresponding to the center of targeting piRNAs and miRNAs. Notably, CIMs are also enriched at nucleotides with local pairing mismatches to piRNA. In addition, distinct patterns of CIMs are observed between canonical and non-canonical base pairing interactions, suggesting that the worm PIWI Argonaute PRG-1 adopts distinct conformations for canonical vs. non-canonical interactions. Critically, non-canonical miRNA or piRNA binding sites with CIMs exhibit more regulatory effects than those without CIMs, demonstrating CIM analysis as a valuable approach in assessing functional significance of small RNA targeting sites in CLASH data. Together, our analyses reveal the landscapes of Argonaute crosslinking sites on mRNAs and highlight MUTACLASH as an advanced tool in analyzing CLASH data.
Divergent modulation of dopaminergic neurons by hypocretin/orexin receptors-1 and-2 shapes dopaminergic cell activity and socio-emotional behavior
Authors: Tzanoulinou Stamatina , Astori Simone , Grandi Laura Clara , Gullo Francesca , Kalusivikako Richie , Rai Simran , Tafti Mehdi , Becchetti Andrea , Vassalli Anne
Published: February 2025

Many neuropsychiatric disorders involve dysregulation of the dopaminergic (DA) input to the forebrain. Of particular relevance are DA projections originating from the midbrain ventral tegmental area (VTA). A key neuromodulatory influence onto DAVTAneurons arises from the dense axonal projections emanating from lateral hypothalamic area hypocretin/orexin (OX) neurons. Despite being a major input, the differential action of orexin peptides A and B (OXA and OXB) on orexin receptors 1 and 2 (OX1R and OX2R) in DA cells is poorly characterized. We thus genetically engineered mice whose DA neurons are selectively unresponsive to OX input via OX1R (DAOx1R-KOmice) or OX2R (DAOx2R-KOmice) and compared their behavior and DA cell electrophysiology to genetic controls. We previously showed a profound functional divergence between OX1R-and OX2R-mediated modulation of DA neurons in controlling vigilance states, brain oscillations and cognitive behavior. Inactivation of OX2R, but not OX1R, in DA neurons dramatically increased time spent in EEG theta-rich wakefulness, improved reward-driven learning and attentional skills, while it impaired inhibitory control. Here, we interrogate DAOx1R-KOand DAOx2R-KOmice in further behavioral domains. We show that mice with DA-specific OX1R loss exhibit hyperactivity, or anxiety-like responding, in context-dependent manners. OX2R loss in contrast decreases sociability and aversion-driven learning. We next investigate the underlying electrophysiological substrates and uncover previously unrecognized effects of OX peptides on DAVTAcell responses. In WT and control mice, we show that while OXA enhances, OXB diminishes DAVTAneuronal excitability. OX1R-deficient DA cells lose OXA responding and OX2R-deficient DA cells lose OXB responding. We altogether evidence strikingly distinct functions of OX1vs OX2R signaling in modulating the intrinsic excitability of DAVTAneurons and influencing DA-related behaviors. Our data position OX→DA neurotransmission via OX1or OX2R as relevant to endophenotypes observed in the context of disorders such as obsessive-compulsive, attention-deficit/hyperactivity, and autism spectrum disorders.
Peroxisomal Dysfunction Drives Loss of Dynamic Nutrient Responses to Initiate Metabolic Inflexibility During Aging
Authors: Sharma Arpit , Mistry Meeta , Yao Pallas , Liang Yanshan , Hui Sheng , Mair William B.
Published: February 2025

Aging results in a loss of metabolic flexibility during fasting, characterized by the inability to acutely switch between metabolic substrates and reduced lipid mobilization. However, the drivers of these effects intracellularly remain unclear. Here, inCaenorhabditis elegans, we show that loss of coordinated inter-organelle dynamics causally initiates metabolic inflexibility with age. In young animals, peroxisomes emerge as the priming orchestrators of the fasting response, simultaneously governing lipid droplets (LDs) utilization and mitochondrial bioenergetics. With age, peroxisomal priming is lost, leading to mitochondrial fragmentation and impaired dynamic nutrient responses during fasting. Notably, dietary restriction (DR) exerts a rejuvenating effect on peroxisomal function, thereby preserving mitochondrial integrity and promoting longevity. Our study uncovers the expansive network of organelles enabling lipid mobilization during youth, providing critical context to the poorly understood role of peroxisomes in actively maintaining organelle homeodynamics and metabolic flexibility throughout the aging process.
Transformation ofGardnerella vaginaliswith aBifidobacterium-Escherichia colishuttle vector plasmid
Authors: Kularatne B. M. D. N. , Hill Janet E.
Published: February 2025

Gardnerellaspp. significantly influence female reproductive health, and are indicators of bacterial vaginosis, a common gynecological disorder. Lack of genetic tools forGardnerellaspp. is a hindrance in fully understanding their role in the vaginal microbiome and no naturally occurring plasmids have yet been identified in these organisms. The aim of this study was to transformGardnerella vaginalisand characterize transformants carryingBifidobacterium-E. colishuttle vector pKO403-lacZ′-Sp.G. vaginalisATCC 49145 was selected for protocol development based on its high growth rate, lack of restriction activity and susceptibility to spectinomycin. Low efficiency (∼102cfu/µg of plasmid DNA) but reproducible transformation was achieved. The expression of the spectinomycin resistance gene and the β-galactosidase gene of pKO403-lacZ′-Sp inG. vaginalisATCC 49145 resulted in an increase in spectinomycin tolerance from 2 µg/ml (MIC) to >512 µg/ml, and an appreciable increase in β-galactosidase activity compared to the wild type. Plasmid copy number was determined to be ∼3 per genome copy. Plasmid was lost rapidly in the absence of spectinomycin selection, with only ∼13% of colony forming units retaining the resistant phenotype after 24 h of growth without selection. These results demonstrate thatG. vaginaliscan be transformed by electroporation and that pKO403-lacZ′-Sp can be maintained and its genes expressed in this host, offering a starting point for the development of genetic tools for mechanistic studies of this important member of the vaginal microbiome.

Importance

The healthy human vaginal microbiome is mainly dominated byLactobacillusspp. An imbalance or shift in this population can lead to a gynecological disorder known as bacterial vaginosis (BV). In BV, there is a reduction inLactobacillusspp. and overgrowth of mixed anaerobes and facultative bacteria includingGardnerellaspp. The reason for this increase in theGardnerellapopulation and associated changes in the vaginal microbiota composition is yet not understood, and a lack of genetic tools is one of the major barriers to performing mechanistic research to study the biology of these clinically significant organisms. A first step in developing genetic tools is introducing foreign DNA. In this study, we have developed a protocol for transformation and identified a plasmid that can be maintained inG. vaginalis.