bluuebunny/crossref_metadata_2025_split huggingface dataset:
Proteasome Caspase-Like Activity Regulates Stress Granules and Proteasome Condensates
Authors: Steinberger Shirel , Adler Julia , Myers Nadav , Shaul Yosef 
Published: February 2025

AbstractThe 20S proteasome maintains cellular protein homeostasis, particularly during stress responses. In a previous study, we identified numerous 20S proteasome substrates through mass spectrometry analysis of peptides generated from cellular extracts degraded by purified 20S proteasome. Many substrates were found to be components of liquid-phase separation, such as stress granules (SGs). Here, we demonstrate the degradation products predominantly arise from the caspase-like (CL) proteasomal activity. To investigate the functional implications of CL activity, we generated cell lines devoid of CL function by introducing the PSMB6 T35A mutation. These mutant cells exhibited slower growth rates, heightened sensitivity to stress, and activation of the unfolded protein response (UPR), as indicated by elevated levels of spliced XBP1 (sXBP1) and stress markers. Cells were subjected to arsenite and osmotic stress to assess their responses. Our findings reveal that CL activity is crucial for efficient SG assembly but does not significantly affect SG disassembly. Interestingly, in these mutant cells, proteasomes were more cytoplasmic under normal conditions but formed nuclear condensates/granules (PGs) upon NaCl osmotic stress. However, the PGs were unstable and rapidly disassembled. These findings underscore the important role of the proteasome’s CL activity in managing stress-induced dynamics of liquid-liquid phase, highlighting its importance in cellular adaptation to proteotoxic and genotoxic stress conditions.
Desmoplakin tail domain position in the desmosomal plaque is isoform dependent
Authors: Ainslie Collin M. , Patel Krishna , Tran Yen T. B. , Bharathan Navaneetha Krishnan , Spindler Volker , Mattheyses Alexa L. 
Published: February 2025

AbstractDesmoplakin (DP) is the anchoring subunit of desmosomes, macromolecular junctions that provide mechanical integrity to the skin and heart. DP has three isoforms, DPI, DPIa, and DPII that arise from alternative splicing. The isoforms are structurally identical excluding the length of their central rod domain. As desmosomes are macromolecular complexes, the precise arrangement of their component proteins, or architecture, is essential to maintain physiological function. Alterations of the tissue-specific expression of DP isoforms underlies rare human diseases impacting the skin and heart. Overall DP is oriented with its head domain closest to the plasma membrane and tail domain extending into the cytosol. However, the differences in the architecture of the DP isoforms within the desmosomal plaque remains unknown. Here, we sought to define the architectural arrangement of each DP isoform. To address this, we utilized direct stochastic optical reconstruction microscopy (dSTORM) and analysis of DP KO HaCaT cells stably expressing DPI, DPIa, or DPII with a C-terminal mEGFP tag. Our results show the DP head domain position in the desmosomal plaque is isoform independent and the DP tail domain position correlates with rod length. The tail domain of DPI, the isoform with the longest rod, is furthest from the plasma membrane and that of DPII, the isoform with the shortest rod, is closest. We propose a variable tail location model to describe the architectural arrangement of each isoform. In this model, the DP isoforms are arranged with their rod domains parallel at an angle between 21° to 25° from the plasma membrane. These results provide valuable insight into the role of DP isoforms in desmosomal architecture and function.
Structural Characterization of Native RNA Polymerase II Transcription Complexes and Nucleosomes inDrosophila melanogaster
Authors: Venette-Smith Natalie L. , Vishwakarma Rishi K. , Venkatakrishnan Varun , Dollinger Roberta , Schultz Josie , Babitzke Paul , Anand Ganesh , Gilmour David S. , Armache Jean-Paul , Murakami Katsuhiko S. 
Published: February 2025

AbstractStructural studies of eukaryotic RNA polymerase II (Pol II) transcription complexes often depend onin vitroassembly by mixing purified Pol II with synthetic DNA/RNA scaffolds, recombinant transcription factors, and/or histones, followed by stalling transcription at defined positions by adding selected nucleotide triphosphate substrates. These studies have yielded remarkable results for understanding nucleosome transcription by Pol II with elongation factors but may fail to represent transcription in native conditions. To investigate Pol II transcription within metazoan cells, we developed an approach to isolate the native transcription complexes fromDrosophila melanogasterembryos. Utilizing one-step FLAG-tag affinity purification and mild chromatin treatment with Micrococcal Nuclease (MNase), we preserved the native transcription complex for cryo-EM and proteomics studies.In silicopurification through the cryo-EM classifications determined structures of multiple forms of native transcription complex, nucleosome and other macromolecules. Remarkably, we determined the structures of metazoan Rpb4/Rpb7 stalk-less elongation complex as well as hexameric nucleosome lacking an H2A/H2B dimer, revealing that diverse elongation complexes and nucleosomes are involved in active transcriptionin vivo. Nucleosome is positioned only downstream of Pol II in the nucleosome elongation complex, underscoring it as a major energy barrier and a time-consuming step during Pol II progression through nucleosomal DNA. Proteomics identified co-purified factors responsible for initiation and elongation stages of transcription, as well as RNA modification factors. This study lays the groundwork for structural study of native transcription in eukaryotes, with future work focused on studies of transient and minor populations of transcription complexes.
Deep Learning-Powered Colloidal Digital SERS for Precise Monitoring of Cell Culture Media
Authors: Zheng Peng , Wu Lintong , Lee Michael Ka Ho , Nelson Andy , Betenbaugh Michael , Barman Ishan 
Published: February 2025

AbstractMaintaining consistent quality in biopharmaceutical manufacturing is essential for producing high-quality complex biologics. Yet, current process analytical technologies (PAT) struggle to achieve rapid and highly accurate monitoring of small molecule critical process parameters and critical quality attributes. While Raman spectroscopy holds great promise as a highly sensitive and specific bioanalytical tool for PAT applications, its conventional implementation, surface-enhanced Raman spectroscopy (SERS), is constrained by considerable temporal and spatial intensity fluctuations, limiting the achievable reproducibility and reliability. Herein, we introduce a deep learning-powered colloidal digital SERS platform to address these limitations. Rather than addressing the intensity fluctuations, the approach leverages their very stochastic nature, arising from highly dynamic analyte-nanoparticle interactions. By converting the temporally fluctuating SERS intensities into digital binary “ON/OFF” signals using a predefined intensity threshold by analyzing the characteristic SERS peak, this approach enables digital visualization of single-molecule events and significantly reduces false positives and background interferences. By further integrating colloidal digital SERS with deep learning, the applicability of this platform is significantly expanded and enables detection of a broad range of analytes, unlimited by the lack of characteristic SERS peaks for certain analytes. We further implement this approach for studying AMBIC 1.1, a chemically-defined, serum-free complete media for mammalian cell culture. The obtained highly accurate and reproducible results demonstrate the unique capabilities of this platform for rapid and precise cell culture media monitoring, paving the way for its widespread adoption and scaling up as a new PAT tool in biopharmaceutical manufacturing and biomedical diagnostics.
Epigenetically dynamic human centromeres are maintained within a stable DNA methylation signature
Authors: Mahlke Megan A. , Lumerman Lior , Nath Poulomi , Chittenden Cy , Hoyt Savannah , Koeppel Jonas , Xu Yuan , Raphael Rebecca , Zaffina Kylie , Hook Paul W. , Timp Winston , Miga Karen H. , Campbell Peter J. , O’Neill Rachel , Altemose Nicolas , Nechemia-Arbely Yael 
Published: February 2025

AbstractCentromere location is specified by CENP-A, a centromere-specific histone that epigenetically propagates centromere identity. How CENP-A is epigenetically maintained at one location in rapidly evolving centromeric DNA is unknown. Using single cell-derived clones of human cell lines, we demonstrate heterogeneity in CENP-A position within cell populations at neocentromeres and a native centromere. CENP-A heterogeneity is accompanied by heterogenous DNA methylation and H3K9me3 patterns, with DNA methylation shifting according to CENP-A position. We further reveal temporary precise CENP-A maintenance but evolution of CENP-A, DNA methylation and H3K9me3 position over prolonged proliferation, with neocentromeres exhibiting DNA methylation instability, H3K9me3 gain, boundary loss and increased epigenetic heterogeneity. Lastly, prolonged CENP-A and HJURP overexpression leads to neocentromere expansion, gradual CENP-A depletion, neocentromere destabilization and re-localization. This study reveals the evolving epigenetic plasticity of human centromeres and neocentromeres and highlights the importance of repressive chromatin boundaries in maintaining centromere stability across cellular proliferation.
Ancient DNA indicates 3,000 years of genetic continuity in the Northern Iranian Plateau, from the Copper Age to the Sassanid Empire
Authors: Ala Amjadi Motahareh , Can Özdemir Yusuf , Ramezani Maryam , Jakab Kristóf , Megyes Melinda , Bibak Arezoo , Salehi Zeinab , Hayatmehar Zahra , Taheri Mohammad Hossein , Moradi Hossein , Zargari Peyman , Hasanpour Ata , Jahani Vali , Sharifi Abdol Motalleb , Egyed Balázs , Mende Balázs Gusztáv , Tavallaie Mahmood , Szécsényi-Nagy Anna 
Published: February 2025

AbstractIn this study, we present new ancient DNA data from prehistoric and historic populations of the Iranian Plateau. By analysing 50 samples from nine archaeological sites across Iran, we report 23 newly sequenced mitogenomes and 13 nuclear genomes, spanning 4700 BCE to 1300 CE. We integrate an extensive reference sample set of previously published ancient DNA datasets from Western and South-Central Asia, enhancing our understanding of genetic continuity and diversity within ancient Iranian populations. A new Early Chalcolithic sample, predating all other Chalcolithic genomes from Iran, demonstrates mostly Early Neolithic Iranian genetic ancestry. This finding reflects long-term cultural and biological continuity in and around the Zagros area, alongside evidence of some western genetic influence. Our sample selection prioritizes northern Iran, with a particular focus on the Achaemenid, Parthian, and Sassanid periods (355 BCE–460 CE). The genetic profiles of historical samples from this region position them as intermediates on an east-west genetic cline across the Persian Plateau. They also exhibit strong connections to local and South-Central Asian Bronze Age populations, underscoring enduring genetic connections across these regions. Diachronic analyses of uniparental lineages on the Iranian Plateau further highlight population stability from prehistoric to modern times.
Predicting characterization of microbiome taxonomy from imaging using machine learning approaches
Authors: Niese Brandon , Sweet Philip , Dinh Bryan , Scholes Amanda N. , LeSassier Danielle S. , Ternus Krista L. , Contreras Lydia , Gordon Vernita 
Published: February 2025

AbstractFor this study, a total of 47 mock human skin microbiome communities were created using microorganisms collected from human donors and grownin vitrofor between eight and 32 days. Each mock community sample was split. Ten mL of each sample was used to determine the taxonomy of the community, using metatranscriptomics and Kraken2 to provide population-level taxonomic information; five mL of each sample was used for imaging. The resulting micrographs served as the basis for establishing a new analysis pipeline that sequentially used two different methods for machine learning and one statistical technique: (1) confocal microscopy images were segmented into individual cells using the generalist, deep learning, publicly available machine learning model Cellpose; (2) continuous probability density functions describing the joint distribution of the cell area and eccentricity were found using algorithms expressing the statistical technique of kernel density estimation; (3) these probability density functions were used as input for convolutional neural networks, that were trained to predict both the taxonomic diversity and the most common bacterial class, independently of metatranscriptomics. Specifically, models were made to predict the Shannon index (a quantitative measure of taxonomic diversity) and to predict the most common bacterial class, for each micrograph. Measured Shannon indices (based on metatranscriptomics) ranged from nearly 0 to 1.4. The model predictions of Shannon indices had a mean squared error of 0.0321 +/- 0.0035. The model predictions of the most common taxonomic class of bacteria had an accuracy of 94.0% +/- 0.7%.ImportanceTaxonomic diversity is a useful metric for describing microbial communities and can be used as a measure of ecosystems’ health, resilience, and biological interactions. Characterization of microbial community diversity also has diagnostic applications. For the human skin microbiome in particular, microbial diversity directly impacts skin health, including resilience against pathogens and regulation of immune responses. Currently, microbial diversity can be determined either using traditional staining methods that are limited to pure cultures or using sequencing methods that require high investment in cost, time, and expertise. In this study, we demonstrate an innovative method that employs microscopy images of bacterial communities and machine learning to predict taxonomic diversity and the dominant bacterial classes of bacterial communities. The underlying framework of the pipeline for taxonomy prediction has the potential to be adapted and extended to other organisms and microbiomes and to make taxonomic analyses less expensive and more feasible in low-resource settings.
Active DNA demethylation is upstream of rod-photoreceptor fate determination and required for retinal development
Authors: Hernández-Núñez Ismael , Urman Alaina , Zhang Xiaodong , Jacobs William , Hoffman Christy , Rebba Sohini , Harding Ellen G , Li Qiang , Mao Fengbiao , Cani Andi K , Chen Shiming , Dawlaty Meelad M , Rao Rajesh C , Ruzycki Philip A , Edwards John R , Clark Brian S 
Published: February 2025

ABSTRACTRetinal cell fate specification from multipotent retinal progenitors is governed by dynamic changes in chromatin structure and gene expression. Methylation at cytosines in DNA (5mC) is actively regulated for proper control of gene expression and chromatin architecture. Numerous genes display active DNA demethylation across retinal development; a process that requires oxidation of 5mC to 5-hydroxymethylcytosine (5hmC) and is controlled by the ten-eleven translocation methylcytosine dioxygenase (TET) enzymes. Using an allelic series of conditional TET enzyme mutants, we determine that DNA demethylation is required upstream of NRL and NR2E3 expression for the establishment of rod-photoreceptor fate. Using histological, behavioral, transcriptomic, and base-pair resolution DNA methylation analyses, we establish that inhibition of active DNA demethylation results in global changes in gene expression and methylation patterns that prevent photoreceptor precursors from adopting a rod-photoreceptor fate, instead producing a retina in which all photoreceptors specify as cones. Our results establish the TET enzymes and DNA demethylation as critical regulators of retinal development and cell fate specification, elucidating a novel mechanism required for the specification of rod-photoreceptors.
Bacteriophage purification using CIMmultus monolithic OH-column chromatography for therapeutic purposes
Authors: Echterhof Arne , Dharmaraj Tejas , Blankenberg Patrick , Hajfathalian Maryam , Blankenberg Francis , Sacher Jessica , Bollyky Paul L. 
Published: February 2025

AbstractBacteriophages, viruses that selectively infect bacteria, are increasingly explored as potential therapies to combat antibiotic-resistant infections. Effective phage purification is vital for therapeutic use, particularly to reduce endotoxin (lipopolysaccharide, LPS) and host protein contamination from production processes in gram-negative bacterial lysates. This study investigates the purification efficiency of CIMmultus OH-monolithic chromatography for seven similarly sized tailed phages (Myoviridae and Siphoviridae families) infectingPseudomonas aeruginosa. Using preferential exclusion chromatography with a potassium phosphate gradient, we achieved significant reductions in host proteins and endotoxins, yielding therapeutically compliant samples suitable for human intravenous injection. We observed recovery rates of 85-95% for smaller phages (e.g., PAML-31-1, LPS5), with lower recoveries for larger “giant” phages (e.g., OMKO1, PhiKZ). All purified samples met endotoxin limits, with average reductions of 98.5% for proteins and up to 6.30 log reductions for endotoxins. Despite increased DNA content post-purification in most samples, the approach shows promise for clinical-grade phage purification. This chromatographic strategy is scalable, reproducible, and free from hazardous chemicals, making it suitable for industrial phage production.
Huntingtin inclusion bodies have distinct immunophenotypes and ubiquitination profiles in the Huntington’s disease human cerebral cortex
Authors: Swanson Molly E. V. , Tan Adelie Y. S. , Tippett Lynette J. , Turner Clinton P. , Curtis Maurice A. , Scotter Emma L. , Lashuel Hilal A. , Dragunow Mike , Faull Richard L. M. , Murray Helen C. , Singh-Bains Malvindar K. 
Published: February 2025

AbstractHuntington’s disease (HD) is a hereditary neurodegenerative condition caused by a CAG repeat expansion mutation in the gene encoding the huntingtin (Htt) protein. The accumulation of Htt inclusion bodies is a pathological hallmark of HD and a common target for therapeutic strategies. However, the limited efficacy of treatments targeting the Htt protein highlights the need for a better understanding of the role of Htt inclusion bodies in HD pathogenesis. This study examined the heterogeneity of Htt inclusion body composition by co-labelling with three Htt epitope-specific antibodies to characterize Htt inclusion body ‘immunophenotype’. We then characterized the size and sub-cellular location of Htt inclusions with distinct immunophenotypes. Using multiplex immunohistochemistry, we also examined the ubiquitination profile of each immunophenotype. Our findings demonstrate that Htt inclusions have a range of immunophenotypes, with some labelled by only one of the three antibodies and others exhibiting co-labelling by several antibodies, thus demonstrating the heterogeneity in inclusion composition and structure. We outline evidence that inclusion bodies exclusively labelled with the EM48 antibody are small, non-nuclear, and more abundant in HD cases with increased CAG repeat length, higher Vonsattel grade, and earlier age of onset. We also find that Htt inclusion bodies labelled by multiple antibodies are more likely to be ubiquitinated, predominantly by K63-rather than K48-linked ubiquitin, suggesting preferential degradation by autophagy. Lastly, we show that ubiquitinated Htt inclusion bodies are more highly immunoreactive for ubiquilin 2 than p62. Our findings highlight the need for multiple antibodies to capture the full spectrum of Htt pathology in HD and imply that future studies should consider the diversity of inclusion body composition and structure when correlating pathology formation to neurodegeneration, clinical symptoms, or disease severity.
TLR4 competence and mouse models of leptospirosis
Authors: Abil Olifan Zewdie , Kundu Suman , Midon Leonardo Moura , Gomes-Solecki Maria 
Published: February 2025

AbstractMice are slowly being accepted as alternative models for investigation of leptospiral infection. The strain most used to analyse sublethal disease (C3H-HeJ) expresses atlr4gene in its immune cells that is hyporesponsive to leptospiral LPS and thus the model is deemed immunocompromised. To help resolve this valid scientific concern we did a study in which we compared infection withLeptospira interrogansserovar Copenhageni Fiocruz in mice expressing fully competenttlr4(C3H-HeN, C57BL6) versustlr4hyporesponsive mice (C3H- HeJ) over a period of two weeks. We found that the C3H-HeJ mouse was the only strain that sustained lethal infection, thus confirming the association betweentlr4function and acute disease. We found that the two mouse strains with a fully functionaltlr4gene (C3H-HeN and C57BL/6) also developed clinical and molecular signs of sublethal leptospirosis nearly on par with C3H-HeJ, as quantified by weight loss, survival curves, presence ofLeptospira16S rRNA in blood and urine, and burden of viable spirochetes in kidney. Regarding kidney markers of inflammation and fibrosis, C3H-HeN and C57BL/6 produced less IL-1 beta, iNOS and ColA1 than C3H-HeJ, which is consistent with increased resilience to infection expected of thetlr4competent strains. Regarding Leptospira-specific antibody, all mouse strains produced IgM and T-cell independent IgG3. Interestingly, in contrast to C57BL/6, both C3H strains produced circulating IgG1 but only C3H-HeN produced IgG2a toL. interroganstwo weeks post infection. Thus, TLR4-competent C3H-HeN and C57BL/6 may be more appropriate mouse models of sublethal leptospirosis, whereas C3H-HeJ can be used to further establish a mouse model of lethal leptospirosis. Our data strongly suggests that TLR4 function is important but not sufficient to cause susceptibility to leptospirosis.Author summaryPrevious studies by different laboratories have shown C3H-HeJ to be a susceptible mouse strain to sublethal and lethal leptospirosis. The goal of our study was to evaluate if lowtlr4responsiveness to leptospiral LPS in C3H-HeJ mice is the factor that drives susceptibility to infection with pathogenicLeptospira. We did a comparative study using mouse strains immunocompetent and hyporesponsive totlr4. The data shows that althoughtlr4does affect the virulence of leptospirosis (only C3H-HeJ succumbed to infection), thetlr4competent strains (C3H-HeN and C57BL/6) also developed measurable sublethal leptospirosis. Thus, competent recognition of leptospiral-LPS by murine TLR4 plays a role but it is not sufficient to determine susceptibility to leptospirosis. Furthermore, the data suggests that T-cell dependent responses could be involved in susceptibility to leptospirosis in the C3H models of infection.