bluuebunny/crossref_metadata_2025_split huggingface dataset:
Efficient delivery of small RNAs to podocytesin vitroby direct exosome transfection
Authors: Lange Tim , Maron Luzia , Weber Claudia , Schlüter Rabea , Endlich Nicole 
Published: October 2024

SummaryPodocytes are a crucial component of the glomerular filtration barrier and changes in their 3D structure contribute to over 80% of chronic kidney disease (CKD) cases. Exosomal small RNAs play a key role in cell-cell communication in CKD and may serve as nanocarriers for delivering small RNAs into podocytes. However, the uptake of exosomal cargo by podocytes remains poorly understood. This study explores the use of isolated exosomes, directly transfected with fluorescently labeled small RNAs, for tracking and delivering small RNAs to cultured podocytes.Exosomes were isolated from immortalized murine podocytes and transfected with Cy3-labeled siRNA and miRNA controls using the ExoFect siRNA/miRNA Transfection Kit. We characterized the transfected exosomes via transmission electron microscopy (TEM) and Western blot for exosomal markers CD9 and TSG101. Subsequently, we co-cultured these exosomes with podocytes and used confocal laser-scanning microscopy (cLSM) and structured illumination microscopy (SIM) to visualize cargo uptake. The isolated exosomes were also transfected with pre-miR-21 and filamin A (FlnA)-siRNAs before being co-cultured with podocytes. We confirmed the efficiency of transfection and knockdown using RT-qPCR, Western blotting, and immunofluorescence staining.TEM revealed that the exosomes maintained a consistent shape and size of approximately 20 nm post-transfection and exhibited a stable expression of CD9 and TSG101. Our imaging showed that podocytes take up Cy3-labeled exosomal miRNAs and siRNAs, utilizing various mechanisms, including encapsulation within vesicular structures, endocytosis and free distribution within the cells. Transfection of exosomes with FlnA-siRNAs resulted in a significant 2.8-fold reduction of FlnA expression in co-cultured podocytes, while pre-miR-21-transfected exosomes led to a remarkable 338-fold increase in mature miR-21 levels.These findings demonstrate that direct exosome transfection with fluorescently labeled small RNAs is an effective tool for exosomal cargo tracking in podocytes. This study is the first to show that directly transfected exosomes can deliver small RNAs to podocytes in vitro, suggesting their potential as RNA carriers for therapeutic strategies in more complex settings.
Protective effects of PDGF-AB/BB against cellular senescence in human intervertebral disc
Authors: Zhang Changli , Diaz-Hernandez Martha Elena , Fukunaga Takanori , Sreekala Shenoy , Yoon Sangwook Tim , Haglund Lisbet , Drissi Hicham 
Published: October 2024

AbstractCellular senescence, characterized by a permanent state of cell cycle arrest and a secretory phenotype contributing to inflammation and tissue deterioration, has emerged as a target for age-related interventions. Accumulation of senescent cells is closely linked with intervertebral disc (IVD) degeneration, a prevalent age-dependent chronic disorder causing low back pain. Previous studies have highlighted that platelet-derived growth factor (PDGF) mitigated IVD degeneration through anti-apoptosis, anti-inflammation, and pro-anabolism. However, its impact on IVD cell senescence remains elusive. In this study, human NP and AF cells derived from aged, degenerated IVDs were treated with recombinant human (rh) PDGF-AB/BB for 5 days and changes of transcriptome profiling were examined through mRNA sequencing. NP and AF cells demonstrated similar but distinct responses to the treatment. However, the effects of PDGF-AB and BB on human IVD cells were comparable. Specifically, PDGF-AB/BB treatment resulted in downregulation of gene clusters related to neurogenesis and response to mechanical stimulus in AF cells while the downregulated genes in NP cells were mainly associated with metabolic pathways. In both NP and AF cells, PDGF-AB and BB treatment upregulated the expression of genes involved in cell cycle regulation, mesenchymal cell differentiation, and response to reduced oxygen levels, while downregulating the expression of genes related to senescence associated phenotype, including oxidative stress, reactive oxygen species (ROS), and mitochondria dysfunction. Network analysis revealed that PDGFRA and IL6 were the top hub genes in treated NP cells. Furthermore, in irradiation-induced senescent NP cells, PDGFRA gene expression was significantly reduced compared to non-irradiated cells. However, rhPDGF-AB/BB treatment increased PDGFRA expression and mitigated the senescence progression through increased cell population in the S phase, reduced SA-β-Gal activity, and decreased expression of senescence related regulators including P21, P16, IL6, and NF-κB. Our findings reveal a novel anti-senescence role of PDGF in the IVD, demonstrating its ability to alleviate the senescent phenotype and protect against the progression of senescence. This makes it a promising candidate for preventing or treating IVD degeneration by targeting cellular senescence.Abstract Figure
Mitochondrial Dysfunction in Alzheimer’s Disease Is Driven By Excess ER-Calcium Release in Patient-Derived Neurons
Authors: Mustaly-Kalimi Sarah , Gallegos Wacey , Marr Robert A. , Peterson Daniel A. , Sekler Israel , Stutzmann Grace E. 
Published: October 2024

AbstractTight regulation of mitochondrial Ca2+is essential for neuronal bioenergetics and cellular metabolism. Ca2+transfer from ER-localized ryanodine receptors (RyR) and inositol triphosphate receptors (IP3R) to the mitochondria maintains a steady Ca2+source that fuels oxidative phosphorylation and ATP production. In Alzheimer’s disease (AD), RyR-evoked Ca2+release is markedly increased, contributing to synaptic deficits, protein mishandling, and memory impairment. Here, we demonstrate that dysregulated RyR-Ca2+release directly compromises mitochondrial activity and is an early contributor to AD cellular pathology. We measured an array of mitochondrial functions using fluorescent biosensors and optical imaging in RyR2-expressing HEK cells and iPSC-derived neurons from familial AD and nonAD patients. In neurons from AD patients, resting mitochondrial Ca2+levels were elevated alongside increased free radical production and higher caspase-3 activity relative to nonAD neurons. RyR-evoked Ca2+release further potentiated pathogenic mitochondrial responses in AD neurons, with increased Ca2+uptake and exaggerated membrane depolarization. Additionally, clearance of damaged mitochondria was impaired in AD neurons, demonstrating consequences from dysfunctional lysosomes. Notably, impairments to mitochondria in AD neurons were largely prevented with the RyR negative allosteric modulator, Ryanodex. These findings highlight how excess RyR-Ca2+release broadly contributes to early cellular pathology in AD which includes a cascade of ER, lysosomal, and mitochondrial deficits culminating in neuronal decline and degeneration. Additionally, pharmacological suppression of RyR-Ca2+release preserves mitochondrial, ER and lysosomal function, thus providing a novel and effective therapeutic.Significance StatementMitochondrial dysfunction plays a central role in the cellular pathogenesis of Alzheimer’s disease, yet the upstream mechanisms driving these deficits remain largely unknown. Here, in human neurons derived from Alzheimer’s patients, we reveal how early Ca2+mishandling through the ER-localized ryanodine receptor (RyR) is sensed by the mitochondria and triggers a pathogenic metabolic cascade. Pharmacologically restoring Ca2+homeostasis reversed these deficits, including the increased free radicals and defective mitophagy, and restored the nonAD phenotype. The findings reveal the early neuronal signaling mechanisms that affect ER Ca2+handling, proteolysis, and mitochondrial activity, highlighting the potential role of the dysregulated RyR as a therapeutic target for AD using clinically relevant model systems.
Allele-specific silencing of a dominantSETXmutation in familial amyotrophic lateral sclerosis type 4
Authors: Winkelsas Audrey , Apfel Athena , Johnson Brian , Harmison George , Li Dongjun , Cheung Vivian , Grunseich Christopher 
Published: October 2024

ABSTRACTAmyotrophic lateral sclerosis 4 (ALS4) is an autosomal dominant motor neuron disease that is molecularly characterized by reduced R-loop levels and caused by pathogenic variants insenataxin(SETX).SETXencodes an RNA/DNA helicase that resolves three-stranded nucleic acid structures called R-loops. Currently, there are no disease-modifying therapies available for ALS4. Given thatSETXis haplosufficient, removing the product of the mutated allele presents a potential therapeutic strategy. We designed a series of siRNAs to selectively target the RNA transcript from the ALS4 allele containing the c.1166T>C mutation (p.Leu389Ser). Transfection of HEK293 cells with siRNA and plasmids encoding either wild-type or mutant (Leu389Ser) epitope tagged SETX revealed that three siRNAs specifically reduced mutant SETX protein levels without affecting the wild-type SETX protein. In ALS4 primary fibroblasts, siRNA treatment silenced the endogenous mutantSETXallele, while sparing the wild-type allele, and restored R-loop levels in patient cells. Our findings demonstrate that mutantSETX, differing from wild-type by a single nucleotide, can be effectively and specifically silenced by RNA interference, highlighting the potential of allele-specific siRNA as a therapeutic approach for ALS4.
Characterization of neural infection by Oropouche orthobunyavirus
Authors: Connors Kaleigh A. , Pedlow Maris R. , Frey Zachary D. , McGaughey Jackson J. , Amarasinghe Gaya K. , Duprex W. Paul , D’Aiuto Leonardo , Wills Zachary P. , Hartman Amy L. 
Published: October 2024

AbstractOropouche fever is a re-emerging global viral threat caused by infection with Oropouche orthobunyavirus (OROV). While disease is generally self-limiting, historical and recent reports of neurologic involvement highlight the importance of understanding the neuropathogenesis of OROV. In this study, we characterize viral replication kinetics in neurons and microglia derived from immortalized, primary, and induced pluripotent stem cell-derived cells, which are all permissive to infection. We demonstrate that ex vivo rat brain slice cultures can be infected by OROV and produce antiviral cytokines and chemokines, including IL-6, TNF-α and IFN-β, which introduces an additional model to study viral kinetics in the central nervous system. These findings provide additional insight into OROV neuropathogenesis and in vitro modeling strategies for a newly re-emerging arbovirus.
Reshaping the tumor microenvironment by degrading glycoimmune checkpoints Siglec-7 and -9
Authors: Wang Chao , Hou Yingqin , Zak Jaroslav , Zheng Qinheng , McCord Kelli A. , Wu Mengyao , Zhang Ding , Chung Shereen , Shi Yujie , Ye Jinfeng , Zhao Yunlong , Hajjar Stephanie , Wilson Ian A. , Paulson James C. , Teijaro John R. , Zhou Xu , Barry Sharpless K. , Macauley Matthew S. , Wu Peng 
Published: October 2024

AbstractCancer treatment has been rapidly transformed by the development of immune checkpoint inhibitors targeting CTLA-4 and PD-1/PD-L1. However, many patients fail to respond, especially those with an immunosuppressive tumor microenvironment (TME), suggesting the existence of additional immune checkpoints that act through orthogonal mechanisms. Sialic acid-binding immunoglobulin-like lectin (Siglec)-7 and -9 are newly designated glycoimmune checkpoints that are abundantly expressed by tumor-infiltrating myeloid cells. We discovered that T cells express only basal levels of Siglec transcripts; instead, they acquire Siglec-7 and -9 from interacting myeloid cells in the TME via trogocytosis, which impairs their activation and effector function. Mechanistically, Siglec-7 and -9 suppress T cell activity by dephosphorylating T cell receptor (TCR)-related signaling cascades. Using sulfur fluoride exchange (SuFEx) click chemistry, we developed a ligand that binds to Siglec-7 and -9 with high-affinity and exclusive specificity. Using this ligand, we constructed a Siglec-7/9 degrader that targets membrane Siglec-7 and -9 to the lysosome for degradation. Administration of this degrader induced efficient Siglec degradation in both T cells and myeloid cells in the TME. We found that Siglec-7/9 degradation has a negligible effect on macrophage phagocytosis, but significantly enhances T cell anti-tumor immunity. The degrader, particularly when combined with anti-CTLA-4, enhanced macrophage antigen presentation, reshaped the TME, and resulted in long-lasting T cell memory and excellent tumor control in multiple murine tumor models. These findings underscore the need to consider exogenous checkpoints acquired by T cells in the TME when selecting specific checkpoint blockade therapy to enhance T cell immunity.
Identification of functional rare coding variants in IGF-1 gene in humans with exceptional longevity
Authors: Ali Amanat , Zhang Zhengdong , Gao Tina , Aleksic Sandra , Gavathiotis Evripidis , Barzilai Nir , Milman Sofiya 
Published: October 2024

AbstractDiminished signaling via insulin/insulin-like growth factor-1 (IGF-1) axis is associated with longevity in different model organisms. IGF-1 gene is highly conserved across species, with only few evolutionary changes identified in it. Despite its potential role in regulating life span, no coding variants in IGF-1 have been reported in human longevity cohorts to date. This study investigated the whole exome sequencing data from 2,487 individuals in a cohort of Ashkenazi Jewish centenarians, their offspring, and controls without familial longevity to identify functional IGF-1 coding variants. We identified two likely functional coding variantsIGF-1:p.Ile91Leu andIGF-1:p.Ala118Thr in our longevity cohort. Notably, a centenarian specific novel variantIGF-1:p.Ile91Leu was located at the binding interface of IGF-1 – IGF-1R, whereasIGF-1:p.Ala118Thr was significantly associated with lower circulating levels of IGF-1. We performed extended all-atom molecular dynamics simulations to evaluate the impact of Ile91Leu on stability, binding dynamics and energetics of IGF-1 bound to IGF-1R. TheIGF-1:p.Ile91Leu formed less stable interactions with IGF-1R’s critical binding pocket residues and demonstrated lower binding affinity at the extracellular binding site compared to wild-type IGF-1. Our findings suggest thatIGF-1:p.Ile91Leu andIGF-1:p.Ala118Thr variants attenuate IGF-1R activity by impairing IGF-1 binding and diminishing the circulatory levels of IGF-1, respectively. Consequently, diminished IGF-1 signaling resulting from these variants may contribute to exceptional longevity in humans.
Pre-fusion AAA+ remodeling of target-SNARE protein complexes enables synaptic transmission
Authors: White K. Ian , Khan Yousuf A. , Qiu Kangqiang , Balaji Ashwin , Couoh-Cardel Sergio , Esquivies Luis , Pfuetzner Richard A. , Diao Jiajie , Brunger Axel T. 
Published: October 2024

SummaryMembrane fusion is driven by SNARE complex formation across cellular contexts, including vesicle fusion during synaptic transmission. Multiple proteins organize trans-SNARE complex assembly and priming, leading to fusion. One target membrane SNARE, syntaxin, forms nanodomains at the active zone, and another, SNAP-25, enters non-fusogenic complexes with it. Here, we show that the AAA+ protein NSF (N-ethylmaleimide sensitive factor) and SNAP (soluble NSF attachment protein) must act prior to fusion. We show that syntaxin clusters are conserved, that NSF colocalizes with them, and characterize SNARE populations within and near these clusters using cryo-EM. Supercomplexes of NSF, α-SNAP, and either a syntaxin tetramer or two binary complexes of syntaxin—SNAP-25 reveal atomic details of SNARE processing and show how sequential ATP hydrolysis drives disassembly. These results suggest a functional role for syntaxin clusters as reservoirs and a corresponding role for NSF in syntaxin liberation and SNARE protein quality control preceding fusion.
Amplification of olfactory transduction currents implements sparse stimulus encoding
Authors: Belonio Kai Clane , Haile Eyerusalem S. , Fyke Zach , Vivona Lindsay , Konanur Vaibhav , Zak Joseph D. 
Published: October 2024

AbstractSensory systems must perform the dual and opposing tasks of being sensitive to weak stimuli while also maintaining information content in dense and variable sensory landscapes. This occurs in the olfactory system, where OSNs are highly sensitive to low concentrations of odors and maintain discriminability in complex odor environments. How olfactory sensory neurons (OSNs) maintain both sensitivity and sparsity is not well understood. Here, we investigated whether the calcium-activated chloride channel, TMEM16B, may support these dual roles in OSNs. We used multiphoton microscopy to image the stimulus-response density of OSNs in the olfactory epithelium. In TMEM16B knockout mice, we found that sensory representations were denser, and the magnitude of OSN responses was increased. Behaviorally, these changes in sensory representations were associated with an increased aversion to the odorant trimethylamine, which switches perceptual valence as its concentration increases, and a decreased efficiency of olfactory-guided navigation. Together, our results indicate that the calcium-activated chloride channel TMEM16B sparsens sensory representations in the peripheral olfactory system and contributes to efficient integrative olfactory-guided behaviors.
Complex responses of soil prokaryotes, fungi and protists to prairie restoration on retired agricultural lands
Authors: Tosi Micaela , MacColl Kevin , Obregón Dasiel , MacDougall Andrew S. , Maherali Hafiz , Dunfield Kari 
Published: October 2024

AbstractRestoring native ecosystems on marginal croplands has many benefits but the impacts on belowground biodiversity are less clear, in part because the limiting factors regulating soil biota are complex and poorly described. Here, we studied how grassland prairie restoration of marginal croplands affected the diversity and composition of soil microbiota on 5 conventional farms from Ontario, Canada. Soil samples (0-15 cm) were collected from annually cultivated fields and adjacent planted perennial grassland where cultivation and chemical inputs had ceased several years previously. Following DNA extraction, we estimated bacterial and fungal abundance using quantitative PCR, and microbial diversity of prokaryotes, fungi and protists using amplicon high-throughput sequencing. Under both land uses, prokaryotic communities were dominated by Proteobacteria, Actinobacteria and Acidobacteria, fungal communities by Ascomycota, and protist communities by Rhizaria (TSAR), Evosea (Amoebozoa) and Chlorophyta (Archaeplastida). Prairie restoration did not have a consistent effect on soil microbial abundance, richness or evenness, which responses varied across farms. Microbial genetic and taxonomic community composition (i.e., sequence variant and genus level) were affected by land use, farm and the interaction between these two factors. Generally, prairie soils had higher relative abundance of Latescibacterota, Desulfobacterota, Acidobacteriota and Glomeromycota, and lower of Deinococcota, Chytridiomycota and Amoebozoa_X. In terms of differentially abundant fungal genera, prairies promoted more fungal plant symbionts, less saprotrophs and no plant pathogens. Interkingdom networks revealed changes in potential microbe-microbe associations with prairie restoration, with only 8 associations in common between land uses. The relationship between soil microbial diversity and physicochemical properties varied across microbial groups, diversity metrics and land uses. Our results evidence the complexity associated with restoring soils from agricultural land to natural ecosystems, with unspecified farm-specific factors (e.g., soil type, prairie species, management history) strongly modulating the response of different microbial groups and variables.
Feature Selection Enhances Peptide Binding Predictions for TCR-Specific Interactions
Authors: Teimouri Hamid , Ghoreyshi Zahra S. , Kolomeisky Anatoly B. , George Jason T. 
Published: October 2024

AbstractT-cell receptors (TCRs) play a critical role in the immune response by recognizing specific ligand peptides presented by major histocompatibility complex (MHC) molecules. Accurate prediction of peptide binding to TCRs is essential for advancing immunotherapy, vaccine design, and understanding mechanisms of autoimmune disorders. This study presents a novel theoretical method that explores the impact of feature selection techniques on enhancing the predictive accuracy of peptide binding models tailored for specific TCRs. To evaluate the universality of our approach across different TCR systems, we utilized a dataset that includes peptide libraries tested against three distinct murine TCRs. A broad range of physicochemical properties, including amino acid composition, dipeptide composition, and tripeptide features, were integrated into the machine learning-based feature selection framework to identify key features contributing to binding affinity. Our analysis reveals that leveraging optimized feature subsets not only simplifies the model complexity but also enhances predictive performance, enabling more precise identification of TCR-peptide interactions. The results of our feature selection method are consistent with findings from hybrid approaches that utilize both sequence and structural data as input as well as experimental data. Our theoretical approach highlights the role of feature selection in peptide-TCR interactions, providing a powerful tool for uncovering the molecular mechanisms of the T-cell response and assisting in the design of more advanced targeted therapeutics.
Fluorescence lifetime-based FRET biosensors for monitoring N-terminal domain interactions of TDP-43 in living cells: A novel resource for ALS and FTD drug discovery
Authors: Nathan Kochen Noah , Murray Marguerite , Vunnam Nagamani , Liao Elly E. , Chen Lihsia , Braun Anthony R. , Sachs Jonathan N. 
Published: October 2024

AbstractTAR DNA-binding protein 43 (TDP-43) pathological aggregates are widely implicated in Alzheimer’s disease, frontotemporal dementia and amyotrophic lateral sclerosis. While therapeutic platforms targeting TDP-43 have predominantly targeted its aggregation, recent findings suggest that loss of functional TDP-43 dimers and multimers — essential for RNA processing — occur upstream of aggregation and is driven through disruption of N-terminal domain (NTD) interactions. Here, we demonstrate that these interactions are targetable via cellular fluorescence lifetime-based FRET biosensors which we used to screen the FDA-approved Selleck library. Our NTD-specific hit ketoconazole rescues sorbitol-induced TDP-43 mislocalization and aggregation, and ameliorates TDP-43 induced downregulation of SREBP2, a TDP-43 mRNA binding target with known implication in ALS. In addition, ketoconazole improves neurite outgrowth in a TDP-43 overexpressing neuron model and motor dysfunction in TDP-43 overexpressing C. elegans. Taken together, our platform represents a novel approach for targeting NTD-dependent TDP-43 interactions, and the identification of ketoconazole validates an exciting translational premise for TDP-43 drug discovery.