bluuebunny/crossref_metadata_2025 huggingface dataset:
Culture method surveillance ofVibrio choleraein a non-sewered sanitation refugee camp setting: Dzaleka Camp, Malawi
Authors: Shackelford Brandie Banner , Chigwechokha Petros , Ziba Lucious , Misomali Christopher , Kanjiru Mphatso , Buleya Patrick , Nyirenda Ruth Lusungu , Wolfe Marlene K. , Holm Rochelle H.
Published: February 2025

Refugees living in camps are particularly vulnerable to infectious disease outbreaks because of overcrowding, inadequate preventative health care, and limited water, sanitation, and hygiene services. As a result of the high disease prevalence, surveillance of cholera in Malawi provides a strong rationale for site and pathogen selection for refugee camp wastewater and environmental surveillance feasibility studies. We conducted a study in Dzaleka camp, Malawi, during a nationwide cholera outbreak. Incentivized refugee volunteers took samples for 19 weeks from seven high-use public pit latrines and a vacuum pump truck used for desludging fecal sludge. The National Microbiological Reference Laboratory of the Public Health Institute of Malawi used culture methods with confirmation via VITEK®MS or Analytical Profile Index to detectVibrio cholerae. Academic partners provided technical input, training, and quality assurance through the study. The results were reviewed weekly at partner-coordination meetings. NoV. choleraewas detected in samples, but one quality assurance sample tested positive during the study period. Here, we discuss the unique challenges of conducting wastewater and environmental surveillance in refugee camp settings, including our operational framework. This study provides a framework for up-scaling wastewater and environmental surveillance efforts in other humanitarian contexts through robust partnerships, processes and in-country tools.

Graphical Abstract

Highlights

A refugee camp in Malawi was investigated during a nationwide cholera outbreak

Samples were collected for 19 weeks from seven pit latrines and a pump truck

Vibrio choleraewas largely absent using culture methods

Surveillance of non-sewered sanitation in a camp setting is achievable through partnerships
Differences between Scaly-sided Merganser (Mergus squamatus) and Common Merganser (M. merganser) feather microstructure
Authors: Li Donghong , Zhang Shiyu , Tian Ran , Zhao Yongbin , Yi Guodong
Published: February 2025

The microstructures of breast feathers, flank feathers, rectrices, and down feathers of both Scaly-sided Merganser (Mergus squamatus) and Common Merganser (M. merganser) were meticulously examined using scanning electron microscope. The observed data were subjected to the Mann-WhitneyUtest, followed by hierarchical clustering and Fisher’s discriminant analysis of indicators with significant differences in morphological structure. The findings revealed statistically significant differences between Scaly-sided Merganser and Common Merganser in terms of the length of the base handle from distal barbules in the breast feathers, the length of the base handle, the number of hooked barbules, the number of cilia from distal barbules, and the number of ventral teeth from proximal barbules in rectrices. Additionally, variations in the distance between nodules, the nodular diameter, and the length of the base nodular of the down feathers were noted. Hierarchical clustering successfully categorized Scaly-sided Merganser and Common Merganser into distinct groups, achieving a 100% accuracy rate. Moreover, Fisher’s discriminant analysis demonstrated a 100% correctness rate when the back-substitution method was used to test the discriminant equation. Consequently, the length of the base handle of distal barbules in breast feathers, along with all selected indicators from rectrices and down feathers, may provide references to identify Scaly-sided Merganser and Common Merganser.
Polygenic scores for disease risk are not associated with clinical outcomes in Parkinson’s disease
Authors: Tan Manuela MX , Iwaki Hirotaka , Bandres-Ciga Sara , Sosero Yuri , Shoai Maryam , Brockmann Kathrin , Williams Nigel M , Alcalay Roy N , Maple-Grødem Jodi , Alves Guido , Tysnes Ole-Bjørn , Auinger Peggy , Eberly Shirley , Heutink Peter , Simon David K. , Kieburtz Karl , Hardy John , Williams-Gray Caroline H , Grosset Donald G , Corvol Jean-Christophe , Gan-Or Ziv , Toft Mathias , Pihlstrøm Lasse
Published: February 2025

Polygenic risk scores (PRS) in Parkinson’s disease (PD) are associated with disease risk. Recently, pathway-specific PRS have been created to take advantage of annotations inking variants to biological pathways or cell types. Here, we investigated 8 biological pathways or regions of open chromatin using pathway-specific PRS: alpha-synuclein pathway, adaptive immunity, innate immunity, lysosomal pathway1, endocytic membrane-trafficking pathway, mitochondrial pathway, microglial open chromatin single nucleotide polymorphisms (SNPs), and monocyte open chromatin SNPs. We analysed 7,402 PD patients across 18 ‘in-person’ PD cohorts, and 6,717 patients from the online Fox Insight study. We did not find any significant associations between the 8 pathway-specific PRSs and 8 clinical outcomes in PD. Though this may be due to a lack of statistical power and limited sample size, it may also suggest that the genetic architecture of sporadic PD risk is different from the genetics of PD progression and clinical outcomes.
An immunocompetent mouse model of liposarcoma
Authors: Shafer Amanda M. , Kenna Emma , Golden Lexi-Ann F. , Elhossiny Ahmed M. , Perry Kyle D. , Wilkowski Jodi , Yan Wei , Kaczkofsky Brynn , McGue J , Bresler Scott C. , Courtney Adam H. , Dalman Jessie M. , Galban Craig J. , Jiang Wei , Espinoza Carlos E. , Chugh Rashmi , Iyer Matthew K. , Frankel Timothy L. , Pasca di Magliano Marina , Dlugosz Andrzej A. , Angeles Christina V.
Published: February 2025

Liposarcoma (LPS) is the most prevalent soft tissue sarcoma. The most common biological subtypes are well-differentiated (WDLPS), a low-grade disease that can evolve to high-grade dedifferentiated LPS (DDLPS), with increased rates of recurrence and metastasis and low response rates to chemotherapy and targeted therapies. Preclinical testing of immunotherapeutics for LPS has been held back by the lack of an immunocompetent mouse model. Here, we present a spontaneous immunocompetent LPS mouse model, ACPP, with targeted deletion of Trp53 and Pten in adipocytes to mimic signaling alterations observed in human LPS. Similar to human LPS, tumors arising in ACPP mice produce WDLPS and DDLPS, along with tumors that exhibit both WD and DD components. Murine and human DDLPS tumors possess transcriptional similarities, including increased expression of oncogenes Cdk4 and Hmga2 and reduced expression of the tumor suppressor Cebpa; further, both mouse and human DDLPS exhibit either high or low T cell infiltration. Syngeneic cell lines derived from spontaneous ACPP DDLPS reliably produce tumors following orthotopic injection, each with distinct growth patterns, aggressiveness and tumor infiltrating lymphocyte profiles. These models provide much needed tools to understand the complex immunobiology of LPS and greatly accelerate the pace of preclinical studies to uncover new therapies for patients with this aggressive malignancy.
Discovery of Epigenetically Silenced Tumour Suppressor Genes in Aggressive Breast Cancer Through a Computational Approach
Authors: Vitte Anne-Laure , Chuffart Florent , Jacquet Emmanuelle , Nika Eleni , Mousseau Mireille , Jung Ina , Tabone-Eglinger Séverine , Bachelot Thomas , Rousseaux Sophie , Khochbin Saadi , Bourova-Flin Ekaterina
Published: February 2025

Breast cancer is characterized by genetic and epigenetic deregulations, leading to aberrant expression of tissue-specific genes that are normally silent in healthy breast tissue. Our previous work identified the embryonic stem cell-specific geneDNMT3B, ade novoDNA methyltransferase, as aberrantly activated in breast cancer, correlating with aggressive tumour behaviour and high relapse risk, regardless of molecular subtype. Through integrative bioinformatic analyses of DNA methylation and transcriptomic data, we identified 154 genes downregulated viaDNMT3B-driven promoter hypermethylation, many of which are associated with high relapse risk. Notably, the tumour suppressor geneGATA3emerged as a primary target of functional inactivation through either loss-of-function mutations orDNMT3B-controlled hypermethylation, in a mutually exclusive manner.

Both mechanisms ofGATA3inactivation were associated with similar molecular signatures linked to tumour progression, increased malignancy, and poorer prognosis. However, distinct differences were observed, with immune- and inflammation-related genes enriched inGATA3hypermethylation cases but depleted in mutation-driven silencing. Additionally, our analysis uncovered other potential tumour suppressor genes epigenetically repressed in aggressive breast cancers. These findings underscore a broader role ofGATA3inactivation beyond genetic alterations and suggest therapeutic opportunities to target epigenetically silenced tumour suppressors in aggressive breast tumours.
The WIPI homolog Atg18 binds to and tethers membranes containing phosphatidylinositol-3,4,5-triphosphate
Authors: Noguera-Cañas Fernando L. , Díaz-Agustín Aitor , Gavira Jose A. , Paredes Jose M. , Salto Rafael , Orte Angel , Conejero-Lara Francisco , Martín-Martínez Mercedes , Pérez-Lara Ángel
Published: February 2025

Atg18 —for Autophagy-related gene 18— is a member of the PROPPIN (β-propellers that bindpolyphosphoinositides) family, which is known for its binding to phosphorylated phosphoinositides through two conserved binding sites. Although Atg18 binding to polyphosphoinositides is crucial for its roles in both autophagic and non-autophagic functions within cells, the precise molecular mechanism by which Atg18 selectively binds to specific phosphatidylinositols remains unresolved. Here, we combined molecular dynamic simulations and biophysical methods —including isothermal titration calorimetry (ITC), cosedimentation, fluorescence resonance energy transfer (FRET), and dynamic light scattering (DLS)— to characterize the interaction between Atg18 and polyphosphoinositides. In contrast to previous findings, we demonstrate that Atg18 binds to and clusters liposomes containing phosphatidylinositol-3,4,5-triphosphate (PtdIns(3,4,5)P3), suggesting that Atg18 oligomerizes and tethers opposing membranes containing this physiological phosphatidylinositol. Hence, our results provide new insights into how Atg18 and its mammalian homologs, WIPI —for WD-repeat domain phosphoinositide-interacting— proteins, may regulate organelle membrane organization and vesicle trafficking required for both autophagic and non-autophagic functions.
Branched chain amino acid metabolism and microbiome in adolescents with obesity during weight loss therapy
Authors: McCann Jessica R. , Yang Chengxin , Bihlmeyer Nathan , Tang Runshi , Truong Tracy , An Jie , Jawahar Jayanth , Ilkayeva Olga , Muehlbauer Michael , Hu Zheng Zheng , Dressman Holly , Poppe Lisa , Granek Joshua , David Lawrence A. , Shi Pixu , Balikcioglu Pinar Gumus , Shah Svati , Armstrong Sarah C. , Newgard Christopher B. , Seed Patrick C. , Rawls John F.
Published: February 2025

BACKGROUND

Towards improving outcomes for adolescents with obesity, we aimed to define metabolic and microbiome phenotypes at baseline and post-weight loss intervention.

METHODS

The Pediatric Obesity Microbiome and Metabolism Study enrolled 220 adolescents aged 10-18 with severe obesity (OB) and 67 healthy weight controls (HWC). Blood, stool, and clinical measures were collected at baseline and after a 6-month intervention for the OB group. Serum metabolomic and fecal microbiome data were analyzed for associations with BMI, insulin resistance, and inflammation. Fecal microbiome transplants were performed on germ-free mice using samples from both groups to assess weight gain and metabolomic changes.

RESULTS

Adolescents with OB exhibited elevated serum branched-chain amino acids (BCAA) but reduced ketoacid metabolites (BCKA) compared to HWC. This pattern was sex- and age-dependent, unlike adults with OB, who showed elevated levels of both. The fecal microbiomes of adolescents with OB and HWC had similar diversity but differed in membership and functional potential. FMT from OB and HWC donors had similar effects on mouse body weight, with specific taxa linked to weight gain in FMT recipients. Longitudinal analysis identified metabolic and microbial features correlated with changes in health measures during the intervention.

CONCLUSION

Adolescents with OB have unique metabolomic adaptations and microbiome signatures compared to their HWC counterparts and adults with OB.

TRIAL REGISTRATION

ClinicalTrials.govIdentifier:NCT03139877(Observational Study) andNCT02959034(Repository)

FUNDING SOURCES

American Heart Association Grants: 17SFRN33670990, 20PRE35180195

National Institute of Diabetes and Digestive and Kidney Diseases Grant: R24-DK110492
Large language models improve transferability of electronic health record-based predictions across countries and coding systems
Authors: Kirchler Matthias , Ferro Matteo , Lorenzini Veronica ,   FinnGen, Lippert Christoph , Ganna Andrea
Published: February 2025

Variation in medical practices and reporting standards across healthcare systems limits the transferability of prediction models based on structured electronic health record (EHR) data. We introduce GRASP, a novel transformer-based architecture that enhances the generalizability of EHR-based prediction by embedding medical codes into a unified semantic space using a large language model. We applied GRASP to predict the onset of 21 diseases and all-cause mortality in over one million individuals from UK Biobank (UK), FinnGen (Finland) and Mount Sinai (USA), all harmonized to OMOP common data model. Trained on the UK Biobank and evaluated in FinnGen and Mount Sinai, GRASP achieved an average ΔC-index that was 83% and 35% higher than language-unaware models, respectively. GRASP also showed significantly higher correlations with polygenic risk scores for 62% of diseases. Notably, GRASP mantained robust performance even when datasets were not harmonized to the same data model, accurately predicting disease risk from ICD-10-CM codes without direct mappings to OMOP. GRASP enables accurate and transferable disease predictions across heterogeneous healthcare systems with minimal resource requirements.
Monocyte-derived macrophage recruitment mediated by TRPV1 is required for eardrum wound healing
Authors: Zhang Yunpei , Wang Pingting , Neng Lingling , Sharma Kushal , Kachelmeier Allan , Shi Xiaorui
Published: February 2025

The tympanic membrane (TM), or eardrum, is a thin, sensitive tissue critical for hearing by vibrating and transmitting sound waves to the inner ear. TM perforation and development of otitis media and conductive hearing loss are commonly seen in the clinic. In this study, we demonstrate the role of TRPV1 signaling mediated macrophage recruitment and angiogenesis in TM repair. By creating a wounded TM mouse model with a perforation in the anteroinferior region of the pars tensa — a region in humans often damaged in traumatic injury, we observed a massive accumulation of macrophages in the vicinity of the acutely wounded TM. Using 5-Ethynyl-2’-deoxyuridine pause labeling and a chimeric bone marrow transplant model, we found that most of the recruited macrophages did not originate from local tissue-resident macrophages but rather from blood-circulating monocytes. Parallel to macrophage recruitment, angiogenesis was observed near the wound on day 3 after perforation and further progressed by day 7. The angiogenic process was strongly associated with the recruited macrophages, as macrophage depletion resulted in a notable reduction in angiogenesis. At the transcriptional level, we found that macrophages facilitate angiogenesis through several signaling pathways. Additionally, we identified direct intercellular communication between macrophages and endothelial cells mediated by phosphoprotein 1 signaling. Furthermore, Gene Ontology analysis of bulk RNA sequencing data from TMs revealed that the macrophage recruitment is associated with neuroinflammatory responses. Using a fluorescence reporter mouse driven by TRPV1, we discovered that the TM contains rich sensory nerve fibers expressing TRPV1. A genetic mutation in theTrpv1gene resulted in a marked decrease in the expression of neuroinflammatory genes, such asTac1. This decrease subsequently resulted in reduced macrophage recruitment, impaired angiogenesis, and delayed wound healing. Together, these findings highlight the crucial role of TRPV1 signaling in monocyte migration and macrophage-related angiogenesis, both of which are crucial for facilitating healing of the TM. These results also open new opportunities for clinical interventions. Targeting TRPV1 signaling could enhance TM immunity, improve blood circulation, promote the repair of damaged TM, and ultimately prevent middle ear infections.
A novel deep optimal transport framework reveals prostate cancer risk heterogeneity, Alzheimer’s disease risk heterogeneity, and myeloma cells associated with a short-term sham bortezomib response and progressive disease
Authors: Liu Ziyu , Li Sihong , Couetil Justin , Zhang Jie , Huang Kun , Johnson Travis S.
Published: February 2025

Leveraging single-cell gene expression profiles can significantly enhance our understanding of diseases by associating single cells with traits such as disease subtypes, prognosis, and drug response. Although previous efforts have linked single cell clusters and groups with these attributes, they have primarily focused on changes in cell proportions while overlooking transcriptional changes at the single cell level. To further unravel cell heterogeneity with clusters and reveal the nuanced behaviors of cellular subtypes, it is essential to assess the disease associations of individual cells. Previous methods often fail to capture complex patterns that are only discernible through summarizing non-linear relationships across multiple genes. The Diagnostic Evidence GAuge of Single-cells/Spatial-transcriptomics (DEGAS) framework advances these efforts by aligning single cells and/or spatial transcriptomics regions with patients through a unified latent space using non-linear transformations learned from deep neural networks (DNNs). DEGAS achieves superior performance in analyzing single cell and spatial transcriptomics datasets, including Alzheimer’s disease (AD), multiple myeloma (MM), and prostate cancer (PDAC). Here we present DEGAS version 2, which has been updated with optimial transport based transfer learning and improved time-to-event loss functions, more advanced model architecture, and improved model baseline evaluations. DEGASv2 outperformed other methods in both single cell and spatial transcriptomic baseline comparisons. On the multiple myeloma discovery dataset, DEGASv2 enabled us to discover cell types that exhibited distinct drug response patterns over various time frames and were validated with time series single cell multiomic data that we generated, demonstrating a dangerous subtype of cell and novel therapeutic target.
Duration discrimination in the bumblebeeBombus terrestris
Authors: Davidson Alexander , Nanda Ishani , Ong Lay Mun Anita , Chittka Lars , Versace Elisabetta
Published: February 2025

The ability to process temporal information is crucial for animal activities like foraging, mating, and predator avoidance. While circadian rhythms have been extensively studied, there is limited knowledge regarding how insects process durations in the range of seconds and sub-seconds. This study aimed to assess bumblebees’ (Bombus terrestris) ability to differentiate the durations of flashing lights, and use this information in a free-foraging task. Bees were trained to associate either the long or short-duration stimulus with a sugar reward versus an unpalatable solution until reaching a criterion, and then tested without sucrose solution with the same stimuli. In Experiment 1, we tested the ability to discriminate between a long stimulus (2.5 or 5 seconds) vs a short stimulus (0.5 or 1 second). The bees learned to discriminate between the two stimuli. To check whether bees solve the task without using the absolute difference in proximal stimulation as a cue, we ran a second experiment. In Experiment 2, the flashing stimuli were presented for the same total amount of time in a cycle. Bees could discriminate between durations even when the overall amount of stimulation in each presentation cycle was the same. This shows general learning abilities in bumblebees, that can discriminate seconds/subseconds intervals in visual flashing stimuli. This reveals an insect’s ability to use non-naturalistic stimuli and temporal cues in free foraging.
Endoderm differentiates into a transient epidermis in the mouse perineum
Authors: Larkins Christine E. , Grunberg Daniel M. , Daniels Gabriel M. , Feldtmann Erik J. , Cohn Martin J.
Published: February 2025

In eutherian mammals, the embryonic cloaca is partitioned into genitourinary and anorectal canals by the urorectal septum. At the caudal end of the mouse embryo, the urorectal septum contributes to the perineum, which separates the anus from the external genitalia. Growth of the urorectal septum displaces cloacal endoderm to the surface of the perineum, where it is incorporated into epidermis, an enigmatic fate for endodermal cells. Here we show that endodermal cells differentiate into true epidermis in the perineum, expressing basal, spinous, and granular cell markers. Endodermal epidermis is lost through terminal differentiation and desquamation postnatally, when it is replaced by ectoderm. Live imaging and single-cell tracking reveal that ectodermal cells move at a faster velocity in a lateral-to-medial direction, converging towards the narrow band of endoderm between the anus and external genitalia. Although the perineum is sexually dimorphic, similar spatiotemporal patterns of cell movement were observed in males and females. These results demonstrate that cloacal endoderm differentiates into a non-renewing, transient epidermis at the midline of the perineum. Differential movement of endodermal and ectodermal cells suggests that perineum epidermis develops by convergent extension. These findings provide a foundation for further studies of perineum development and of sex-specific epidermal phenotypes.
Cross-Species Communication via Fungal Extracellular Vesicles
Authors: Piraine Renan E. A. , Oliveira Henrique T. , Santos Patrick W. , Froldi Julia L. , Oliveira Bianca T. M. , Rezende Caroline P. , Trentin Gabriel E. S. , Nogueira Lucas F. B. , Colombo Arnaldo L. , Casadevall Arturo , Rodrigues Marcio L. , Almeida Fausto
Published: February 2025

Extracellular vesicles (EVs) play crucial roles in fungal communication and host immune modulation, representing potential therapeutic targets for fungal infections. This study investigated the role of fungal EVs in both intra- and interspecies communication, focusing on their effects on virulence and immune responses. Co-incubation experiments were performed using EVs derived fromCandida albicansandCandida auristo assess interactions withC. albicansplanktonic cells and biofilms, as well asCryptococcus neoformansandCryptococcus gattiiEVs interacting withC. neoformanscultures. EVs were observed associating with recipient cell surfaces, suggesting subsequent internalization. Functional assays revealed that EV exposure led to increased expression ofCap59, Lac1, Ure1,andErg11genes, correlating with reduced antifungal susceptibility in both planktonic and biofilm forms. Additionally, EVs facilitated cross-species communication, enhancing biofilm adhesion and dispersion, which underscores their role in phenotypic modulation. Macrophages stimulated with fungal EVs exhibited receptor-specific gene expression changes, notably the upregulation ofgalectin-3, along with a pro-inflammatory phenotype marked by increasediNOSexpression and elevated cytokine levels (IL-1β, IL-6, and IL-8). Collectively, these findings underscore a critical role for fungal EVs in interspecies communication, biofilm regulation, and immune modulation, offering valuable insights into fungal pathogenicity mechanisms.

Importance

Currently, no vaccines exist to prevent fungal infections, underscoring the need for new therapies. As fungal diseases increase globally, understanding fungal biology is essential to identifying treatment targets. Fungi use EVs to communicate and evade immune responses. EVs mediate cell-cell communication, transporting proteins, polysaccharides, lipids, and nucleic acids – serving as “messages” exchanged within a fungal network. Understanding how these vesicles facilitate communication not only within a single species but also across different fungal species can shed light on their contribution to infection persistence and cross-species adaptability. Moreover, EVs may have a broader role in inter-kingdom communication, influencing how fungi interact with host immune cells. The impact of fungal EVs on human innate immune responses remains a largely underexplored area, with significant gaps in our knowledge. This study aims to examine how fungal EVs affect immune responses and whether their signaling varies across species, potentially revealing new therapeutic targets.
Manipulating PARK7/DJ-1 Levels by Genotoxic Stress Alters Noncoding RNAs and Cellular Homeostasis
Authors: Zohar Keren , Zoubi Haya Obied , Goldberg Michal , Eliyahu Tsiona , Linial Michal
Published: February 2025

DJ-1/PARK7 is a multifunctional protein that plays a vital role in sensing oxidative stress and maintaining redox homeostasis. As an oncogene, DJ-1 influences p53-mediated stress responses and contributes to cancer progression. This study investigates the impact of X-ray-induced DNA breaks on cellular responses post-irradiation under varying DJ-1 expression levels. Using siRNA knockdown and overexpression to modulate DJ-1 levels, transcriptional changes were analyzed through RNA-seq. Naïve cells exhibited only a moderate transcriptional response to X-ray exposure, including suppression of the cell cycle and activation of stress pathways. Overexpression of DJ-1 led to pronounced gene expression suppression, particularly suppression of most ribosomal and mitochondrial genes. Conversely, DJ-1 knockdown caused extensive, non-specific transcriptional changes, indicating disrupted cellular homeostasis. Notably, around 25% of non-coding RNAs were differentially expressed under these conditions. DJ-1 overexpression suppressed lncRNA of the host genes for snoRNAs, thus altering the miRNA sponging capacity after X-ray exposure. These findings underscore DJ-1’s critical role in modulating cellular responses to genotoxic stress, reshaping transcriptional landscapes, and regulating non-coding RNA profiles. DJ-1 emerges as essential for maintaining genomic stability.
Proteasome Caspase-Like Activity Regulates Stress Granules and Proteasome Condensates
Authors: Steinberger Shirel , Adler Julia , Myers Nadav , Shaul Yosef
Published: February 2025

The 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

Desmoplakin (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.
Distinct filament morphology and membrane tethering features of the dual FtsZs in Odinarchaeota
Authors: Kumari Jayanti , Uthaman Akhilesh , Kundu Ananya , Dhar Anubhav , Sharma Vaibhav , Bose Sucharita , Dutta Soumyajit , Roy Srijita , Srinivasan Ramanujam , Pande Samay , Vinothkumar Kutti R. , Gayathri Pananghat , Palani Saravanan
Published: February 2025

The deep-branching Asgard archaea have emerged as a model to study Eukaryogenesis as they harbor homologs of many of the major eukaryotic protein families. In this study, we use Candidatus Odinarchaeota FtsZs as representatives to understand the probable origin, evolution, and assembly of the FtsZ-Tubulin protein superfamily in Asgard archaea. We have performed a comparative analysis of the biochemical properties and cytoskeletal assembly of FtsZ1 and FtsZ2, the two FtsZ isoforms in the Odinarchaeota metagenome. Our electron microscopy analysis reveals that OdinFtsZ1 assembles into curved single protofilaments, while OdinFtsZ2 forms stacked spiral ring-like structures. Upon sequence analysis, we identified an N-terminal amphipathic helix in OdinFtsZ1, which mediates direct membrane tethering. In contrast, OdinFtsZ2 is recruited to the membrane by the membrane-anchor OdinSepF via OdinFtsZ2’s C-terminal tail. Overall, we report the presence of two distant evolutionary paralogs of FtsZ in Odinarchaeota, with distinct filament assemblies and differing modes of membrane targeting mechanisms, namely direct vs SepF-mediated membrane binding. Our findings highlight the diversity of FtsZ proteins in the transitional archaeal phylum Odinarchaeota, providing valuable insights into the evolution and differentiation of tubulin family proteins.
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

Structural 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

Maintaining 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

Centromere 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

In 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

Abstract

For 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%.

Importance

Taxonomic 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

Retinal 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

Bacteriophages, 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.