bluuebunny/crossref_metadata_2025_split huggingface dataset:
Clustering of Tandem Inverse Duplications in Centromeric and Subtelomeric Chromosomal Regions ofAspergillus flavus
Authors: Cary Jeffrey W. , Malik Amna , Smith Alexie , Flemington Erik , Lustig Arthur J 
Published: January 2025

ABSTRACTRearrangements associated with gene amplification are frequently initiated by the formation of inverted repeats. Among these rearrangements are breakage-fusion-bridge cycles and tandem inverted duplications (TIDs), both of which can be mediated through palindromic sequences. We have investigated spontaneous rearrangements within the genome of the filamentous fungusAspergillus flavusby high-coverage third-generation sequencing of vegetatively cultured strains. Analysis of sequence data identified a class of rare and transient rearrangements having structures typical of TIDs. These putative TIDs (pTIDs) form both unprocessed and processed species and have a variably sized deletion at the junction between direct and inverted sequences. Most intact junctions contain the predicted palindromic sequences, supporting the TID model. However, palindrome-free junctions are also infrequently observed. pTIDs are highly enriched in heterochromatic centromeric and subtelomeric A+T rich regions, suggesting that this process is a source of genetic instability in these domains. Consistent with this finding, A+T rich regions contain elevated levels of direct, inverted, and perfect palindromic repeats. We propose a replication-based model in which palindromes and short inverted repeats, under condition of replication stress, are responsible for pTID structures.TEASERThe dynamic formation of duplication/inversions clustered in fungal chromosomes reveals the genetic instability of centromeric and subtelomeric regions.
Growth but not corticosterone, oxidative stress or telomere length is negatively affected by microplastic exposure in a filter-feeding amphibian
Authors: Martin Colette , Ruthsatz Katharina , Gomez-Mestre Ivan , Burraco Pablo 
Published: January 2025

AbstractMicroplastics (MPs) are of increasing global concern for species inhabiting aquatic habitats. However, the mechanisms behind animal responses to MPs need comprehensive exploration. Amphibians are the most threatened vertebrate group with most species having a complex life cycle, commonly with an aquatic larval stage. Here, we investigated whether exposure to an environmentally-relevant concentration of MPs affects the growth of filter-feeding larvae of the African clawed frog (Xenopus laevis), and the consequences for their stress physiology (corticosterone (CORT) levels), or health and ageing physiology (oxidative stress and telomere length). We conducted a 3×2 experiment with three levels of fibre exposure (fibres absent -control-, and MP and cellulose fibre treatments), and two stress levels (CORT absent –control-, and CORT present simulating a stressful condition). We observed a negative impact of MP exposure on larval growth; however, this did not alter the CORT levels, oxidative stress or telomere length. Our study shows that realistic concentrations of MPs is not enough to induce major alterations on the stress or health and ageing physiology of a filter-feeding amphibian. Whether compensatory growth responses during the post-metamorphic stages could lead to detrimental effects later in life should be explored in amphibians and other organisms with complex-life cycles.
A Novel Optimized Silver Nitrate Staining Method for Visualizing the Osteocyte Lacuno-Canalicular System
Authors: Wu Jinlian , Xue Chunchun , Li Qiang , Wu Hongjin , Zhang Jie , Wang Chenglong , Dai Weiwei , Wang Libo 
Published: January 2025

AbstractA 50% silver nitrate solution is commonly used to stain and characterize the osteocyte lacuno-canalicular system (LCS) in bone biology research. However, variability in reagent concentrations and types, along with inconsistent staining procedures, have limited the broader application of this method in osteocyte research. In this study, we present a novel optimized silver nitrate staining technique aimed at addressing these limitations. This new method utilizes a 1 mol/L silver nitrate solution in combination with a type-B gelatin-formic acid solution at various concentrations (0.05%-0.5% gelatin and 0.05%-5% formic acid, or 1%-2% gelatin and 0.1%-2% formic acid) in volume ratios of 4:1, 2:1, or 1:1, or a 0.5 mol/L silver nitrate solution at a 4:1 ratio. The staining process is carried out for 1 hour under ultraviolet light or 90 minutes under regular room light (or dark), followed by washing with Milli-Q water to terminate the reaction. We applied this new method to stain the osteocyte LCS in bone samples from different species and pathological bone models. The technique consistently produced clear, distinct staining patterns across all samples. Moreover, our novel method revealed a greater number of LCS compared to the traditional 50% silver nitrate solution. This suggests that the commonly used 50% silver nitrate method may disrupt or inadequately reveal the LCS in certain species, potentially leading to an underestimation of LCS density and number. In conclusion, our novel silver nitrate staining method provides a simpler and more cost-effective alternative to the traditional technique. By offering a more accurate and comprehensive analysis of the LCS across species, this approach has the potential to advance research on osteocyte morphogenesis, as well as the functional and evolutionary adaptations of the osteocyte LCS across different taxa.
Contact mode high-speed AFM of phase separated lipid bilayers to study Amyloid-β interactions
Authors: Robinson Morgan , Payton Oliver , Picco Loren , Beazely Michael , Miles Mervyn , Leonenko Zoya 
Published: January 2025

AbstractThe lipid bilayer membrane is an essential feature of life, the composition and structure of which is important in governing cellular processes, some of which are mediated by the changes induced by proteins. Of particular interest is the role that the lipid membrane plays in amyloidogenic diseases such as Alzheimer’s disease (AD), including the role of lipid composition and cholesterol in mediating amyloid-β (Aβ) toxicity. Atomic force microscopy (AFM) is a key tool in studying the structures of lipid membranes as nanoscale structure is important for function, unfortunately many of these dynamic processes occur on time scales where traditional AFM cannot operate fast enough. Recent advances in contact mode high-speed AFM (HS-AFM) have made it possible to capture processes at video rate. We used a unique custom-built contact mode HS-AFM to image model lipid membranes in liquid and compare this to standard and HS-AFM images. We demonstrate that this technique can be used to capture the dynamics of interactions of Aβ(1-42) monomers and oligomers with phase separated lipid bilayers composed of three and five lipid components to elucidate the role of nanoscale domains in amyloid-membrane interactions. Overall, we demonstrate the potential of the HS-AFM to image biological processes on biologically relevant soft samples and discuss tip-sample interactions at high-speed operation in contact mode on lipid membrane models in liquid environment.
Anionic lipid catalyzes the generation of cytotoxic insulin oligomers
Authors: Saha Jhinuk , Wolszczak Audrey , Kaur Navneet , Dickwella Widanage Malitha C. , McCalpin Samuel D , Fu Riqiang , Ali Jamel , Ramamoorthy Ayyalusamy 
Published: January 2025

AbstractMisfolding and aggregation of proteins into amyloidogenic assemblies are key features of several metabolic and neurodegenerative diseases. Human insulin has long been known to form amyloid fibrils under various conditions, which affects its bioavailability and function. Clinically, insulin aggregation at recurrent injection sites poses a challenge for diabetic patients who rely on insulin therapy. Furthermore, decreased responsiveness to insulin in type 2 diabetic (T2D) patients may lead to its overproduction and accumulation as aggregates. Earlier reports have reported that various factors such as pH, temperature, agitation, and the presence of lipids or other proteins influence insulin aggregation. Our present study aims to elucidate the effects of non-micellar anionic DMPG (1,2-dimyristoyl-sn-glycero-3-phosphoglycerol) lipids on insulin aggregation. Distinct pathways of insulin aggregation and intermediate formation were observed in the presence of DMPG using a ThT fluorescence assay. The formation of soluble intermediates, alongside large insulin fibrils, was observed in insulin incubated with DMPG via TEM, DLS and NMR, as opposed to insulin aggregates generated without lipids.13C magic angle spinning solid-state NMR and FTIR experiments indicated that lipids do not alter the conformation of insulin fibrils but do alter the time scale of motion of aromatic and aliphatic sidechains. Furthermore, the soluble intermediates were found to be more cytotoxic as compared to fibrils generated with or without lipids. Overall, our study elucidates the importance of anionic lipids in dictating the pathways and intermediates associated with insulin aggregation. These findings could be useful in determining various approaches to avoid toxicity and enhance the effectiveness of insulin in therapeutic applications.
Altered relaxation and Mitochondria-Endoplasmic Reticulum Contacts Precede Major (Mal)adaptations in Aging Skeletal Muscle and are Prevented by Exercise
Authors: Allen Ryan J. , Kronemberger Ana , Shi Qian , Pope Marshall , Cuadra-Muñoz Elizabeth , Son Wangkuk , Song Long-Sheng , Anderson Ethan J. , Pereira Renata O. , Lira Vitor A. 
Published: January 2025

AbstractSarcopenia, or age-related muscle dysfunction, contributes to morbidity and mortality. Besides decreases in muscle force, sarcopenia is associated with atrophy and fast-to-slow fiber type switching, which is typically secondary to denervation in humans and rodents. However, very little is known about cellular changes preceding these important (mal)adaptations. To this matter, mitochondria and the sarcoplasmic reticulum are critical for tension generation in myofibers. They physically interact at the boundaries of sarcomeres forming subcellular hubs called mitochondria-endo/sarcoplasmic reticulum contacts (MERCs). Yet, whether changes at MERCs ultrastructure and proteome occur early in aging is unknown. Here, studying young adult and older mice we reveal that aging slows muscle relaxation leading to longer excitation-contraction-relaxation (ECR) cycles before maximal force decreases and fast-to-slow fiber switching takes place. We reveal that muscle MERC ultrastructure and mitochondria-associated ER membrane (MAM) protein composition are also affected early in aging and are closely associated with rate of muscle relaxation. Additionally, we demonstrate that regular exercise preserves muscle relaxation rate and MERC ultrastructure in early aging. Finally, we profile a set of muscle MAM proteins involved in energy metabolism, protein quality control, Ca2+homeostasis, cytoskeleton integrity and redox balance that are inversely regulated early in aging and by exercise. These may represent new targets to preserve muscle function in aging individuals.
Environmental effects on constructed wetland microbial diversity and function in the context of wastewater management
Authors: Grandmont-Lemire Sandrine , Gearheart Bob , Cuellar-Gempeler Catalina 
Published: January 2025

ABSTRACTConsidering temporal and spatial change in biodiversity-ecosystem function (BEF) relationships is critical to predict and manage ecosystem services, especially in human mediated and impacted ecosystems. We propose that species responses to seasonal change and spatial distributions can act as a laboratory to reveal diversity-function relationships with management implications. This study investigates the relationship between bacterial diversity and ammonia removal function in a wastewater secondary treatment constructed wetland system. We took 8 samples across a system of 6 interconnected ponds, from August 2019 to February 2020, at the Arcata Wastewater Treatment Facility (AWTF), in Coastal Humboldt County (California, USA). We used 16S rRNA gene amplicon sequencing to measure bacterial diversity and composition, and an ammonia electrode probe to measure NH4at the influx and efflux positions of each pond. We found a significant negative relationship between ASV richness and ammonia removal, suggesting that nitrifying and denitrifying bacteria are poor competitors, known a negative selection effect. Bacterial richness effect on ammonia removal was strongest, followed by direct effects of season on richness and location on function, based on structural equation modeling. We identify taxa associated with function that may influence management strategies, including Planktophila, Legionella, Sulfurimonadaceae and Sporichtyaceae that thrive in ponds located after chlorination steps. This result challenges the traditional wastewater treatment reactor paradigm to reveal negative BEF relationships that appear stronger than environmental influences. By expanding our views of BEF relationships, we can further unravel how community diversity and composition influence ecosystem processes in natural and humanized systems.IMPORTANCEThis study sheds new light on how biodiversity impacts ecosystem functions in human-made environments, specifically wastewater treatment systems. By examining bacterial diversity and ammonia removal efficiency across interconnected ponds, we challenge the conventional assumption that more species always lead to better ecosystem performance. The surprising finding that higher bacterial diversity can reduce ammonia removal efficiency (due to competition among key bacteria) offers fresh insights into how microbial communities work. This understanding is critical for improving wastewater treatment and designing systems that maximize efficiency. Moreover, identifying specific bacteria linked to ammonia removal provides practical information for better managing and enhancing treatment processes. By broadening how we think about the relationship between biodiversity and ecosystem function, this study offers valuable tools for both scientists and environmental managers working to balance human impact with ecosystem health.
Functionally diversified BiP orthologs control body growth, reproduction, stress resistance, aging, and ER-Phagy inCaenorhabditis elegans
Authors: Urban Nicholas D. , Lacy Shannon M. , Van Pelt Kate M. , Abdon Benedict , Mattiola Zachary , Klaiss Adam , Tabler Sarah , Truttmann Matthias C. 
Published: January 2025

AbstractCellular systems that govern protein folding rely on a delicate balance of functional redundancy and diversification to maintain protein homeostasis (proteostasis). Here, we useCaenorhabditis elegansto demonstrate how both overlapping and divergent activities of two homologous endoplasmic reticulum (ER)-resident HSP70 family chaperones, HSP-3 and HSP-4, orchestrate ER proteostasis and contribute to organismal physiology. We identify tissue-, age-, and stress-specific protein expression patterns and find both redundant and distinct functions for HSP-3 and HSP-4 in ER stress resistance, reproduction, and body size regulation. We show that only HSP-3 overexpression is sufficient to improve longevity and that loss of HSP-3 or HSP-4 during distinct stages of the worm cycle or specific tissues have opposing effects on worm lifespan. Furthermore, we find that loss of HSP-4, but not HSP-3, improves tolerance to protein aggregation induced-stress by activating ER-Phagy through the engagement of IRE-1 and the putative ER-Phagy receptor, C18E9.2. Mechanistically, we show that de-repression of IRE-1 via HSP-4 dissociation allows for direct inhibition of C18E9.2- mediated ER-Phagy and demonstrate that a conserved orthologous mechanism involving the respective human orthologs, BiP, Sec-62, and IRE-1, contributes to ER proteostasis regulation in human cells. Taken as a whole, our study demonstrates that functional diversification of orthologous proteins within a single organelle is an efficient mechanism to maximize stress resilience while also defining a novel link between ER- phagy and proteostasis regulation.
Sequence-based Drug-Target Complex Pre-training Enhances Protein-Ligand Binding Process Predictions Tackling Crypticity
Authors: Zhang Shuo , Xie Li , Tiourine Daniel , Xie Lei 
Published: January 2025

AbstractPredicting protein-ligand binding processes, such as affinity and kinetics, is critical for accelerating drug discovery. However, many existing computational methods face key limitations, including insufficient integration of comprehensive databases, inadequate representation of protein structural dynamics, and incomplete modeling of microscale protein-ligand interactions. To address these challenges, we introduce ProMoNet, a sequence-based pre-training and fine-tuning framework to enhance protein-ligand binding process prediction. ProMoNet connects protein and molecular foundation models to expand data coverage and enhance diversity, and it integrates large-scale binding site pre-training with efficient fine-tuning for affinity and kinetics prediction. During pre-training, it effectively models microscale protein-ligand interactions and captures the dynamic nature of proteins, including binding site crypticity, without relying on 3-dimensional structural inputs. Notably, ProMoNet’s pre-training module surpasses or matches state-of-the-art structure-based methods in identifying exposed and cryptic binding sites. In the fine-tuning stage, it transfers pre-trained knowledge, achieving superior performance in affinity and kinetics prediction tasks with high computational efficiency. The combination of ProMoNet’s powerful modeling capabilities and demonstrated success across multiple tasks highlight its potential for broad applications in drug discovery.
Use of cellular FAD autofluorescence as a label-free cellular attribute for the production of chimeric antigen receptor-T cells
Authors: Cheung Ka-Wai , Kairi Faris , Lin Teo Denise Bei , Sin Wei-Xiang , Lee Yie Hou , Birnbaum Michael E. 
Published: January 2025

AbstractChimeric antigen receptor T (CAR-T) cell therapy has become an attractive approach for treating hematological malignancies. However, the accessibility of this therapy is limited by factors such as complex manufacturing process, limited capacity of manufacturing facilities and the requirement of highly skilled workforce for the manual steps of CAR-T cell production. To minimize the manual processes, CAR-T cell manufacturing field is shifting towards closed and automated systems, including analytical tools that offer intermittent monitoring of cells in production. Therefore, label-free technologies for closely monitoring CAR-T cells in closed systems are needed. Here, we evaluate the use of a flow cytometer equipped with a 405nm violet laser for investigating the NADH and FAD autofluorescence in T cells. Our results revealed the increase of NADH and FAD autofluorescence were significantly correlated with the upregulation of T cell activation marker, CD25 and the increase of extracellular lactate in spent media in the first three days after T cell activation. We demonstrate the potential use of FAD for determining the endpoint of CAR-T cell manufacture by establishing a relationship between the rate of change in the mean fluorescence intensity (MFI) of FAD in CAR-T cells and the rate of change in T cell proliferation using a G-Rex bioreactor. Collectively, these findings suggest that autofluorescence, particularly FAD autofluorescence, can serve as a label-free biomarker (cellular attribute) for monitoring T cell activation and expansion during CAR-T cell production. The use of 405nm visible light to substitute the genotoxic UV wavelengths for assessing the NADH and FAD autofluorescence, paves the way to incorporate autofluorescence measurements into closed and automated systems for in-process monitoring of CAR-T cell manufacturing.
Leveraging AI to Explore Structural Contexts of Post-Translational Modifications in Drug Binding
Authors: Medvedev Kirill E. , Schaeffer R. Dustin , Grishin Nick V. 
Published: January 2025

AbstractPost-translational modifications (PTMs) play a crucial role in allowing cells to expand the functionality of their proteins and adaptively regulate their signaling pathways. Defects in PTMs have been linked to numerous developmental disorders and human diseases, including cancer, diabetes, heart, neurodegenerative and metabolic diseases. PTMs are important targets in drug discovery, as they can significantly influence various aspects of drug interactions including binding affinity. The structural consequences of PTMs, such as phosphorylation-induced conformational changes or their effects on ligand binding affinity, have historically been challenging to study on a large scale, primarily due to reliance on experimental methods. Recent advancements in computational power and artificial intelligence, particularly in deep learning algorithms and protein structure prediction tools like AlphaFold3, have opened new possibilities for exploring the structural context of interactions between PTMs and drugs. These AI-driven methods enable accurate modeling of protein structures including prediction of PTM-modified regions and simulation of ligand-binding dynamics on a large scale. In this work, we identified small molecule binding-associated PTMs that can influence drug binding across all human proteins listed as small molecule targets in the DrugDomain database, which we developed recently. 6,131 identified PTMs were mapped to structural domains from Evolutionary Classification of Protein Domains (ECOD) database. Using recent AI-based approaches for protein structure prediction (AlphaFold3, RoseTTAFold All-Atom, Chai-1), we generated 14,178 models of PTM-modified human proteins with docked ligands. Our results demonstrate that these methods can predict PTM effects on small molecule binding, but precise evaluation of their accuracy requires a much larger benchmarking set. We also found that phosphorylation of NADPH-Cytochrome P450 Reductase, observed in cervical and lung cancer, causes significant structural disruption in the binding pocket, potentially impairing protein function. All data and generated models are available from DrugDomain database v1.1 (http://prodata.swmed.edu/DrugDomain/) and GitHub (https://github.com/kirmedvedev/DrugDomain). This resource is the first to our knowledge in offering structural context for small molecule binding-associated PTMs on a large scale.
MetaLigand: A database for predicting non-peptide ligand mediated cell-cell communication
Authors: Xin Ying , Jin Yang , Qian Cheng , Blackshaw Seth , Qian Jiang 
Published: January 2025

ABSTRACTNon-peptide ligands (NPLs), including lipids, amino acids, carbohydrates, and non-peptide neurotransmitters and hormones, play a critical role in ligand-receptor-mediated cell-cell communication, driving diverse physiological and pathological processes. To facilitate the study of NPL-dependent intercellular interactions, we introduce MetaLigand, an R-based and web-accessible tool designed to infer NPL production and predict NPL-receptor interactions using transcriptomic data. MetaLigand compiles data for 233 NPLs, including their biosynthetic enzymes, transporter genes, and receptor genes, through a combination of automated pipelines and manual curation from comprehensive databases. The tool integrates both de novo and salvage synthesis pathways, incorporating multiple biosynthetic steps and transport mechanisms to improve prediction accuracy. Comparisons with existing tools demonstrate MetaLigand’s superior ability to account for complex biogenesis pathways and model NPL abundance across diverse tissues and cell types. Furthermore, analysis of single-nucleus RNA-seq datasets from age-related macular degeneration samples revealed that distinct retinal cell types exhibit unique NPL profiles and participate in specific NPL-mediated pathological cell-cell interactions. Finally, MetaLigand supports single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics data, enabling the visualization of predicted NPL production levels and heterogeneity at single-cell resolution.