bluuebunny/crossref_metadata_2025 huggingface dataset:
EV-Checklist: A fast documentation tool for enhancing transparency and accessibility in extracellular vesicle research
Authors: Poupardin Rodolphe , Wolf Martin , Maeding Nicole , Fuhrmann Gregor , Strunk Dirk
Published: May 2024

Extracellular vesicle (EV) research has experienced rapid growth in the past decade. While community-driven efforts, such as the MISEV guidelines (2014, 2018, 2023), have aimed to standardize reporting and enhance experimental rigor, these have not yet been universally adopted in daily EV research practices. The complexity and time commitment required by existing reporting tools can deter researchers from fully embracing them, leading to incomplete or inconsistent documentation in EV studies. Therefore, we created EV-checklist, a complementary digital tool to streamline documentation during manuscript submission and increase transparency for the reviewers, editors and readers. Our tool guides researchers through a straightforward checklist covering nomenclature, source, isolation, characterization, and functional studies of EVs. It generates a concise two-page PDF formatted table for easy integration into manuscripts and provides editors, reviewers, and readers with a clear overview of the conducted studies. EV-Checklist intends to complement existing comprehensive registries as a ‘fast-and-easy’ tool to enhance clarity and accessibility of methodological and functional insights. It may also promote higher adherence to documentation standards and in EV studies. Our tool is available onhttps://ev-zone.org.
LncCE: Landscape of Cellular-Elevated LncRNAs in Single Cells Across Normal and Cancer Tissues
Authors: Xu Kang , Liu Yujie , Lv Chongwen , Luo Ya , Shi Jingyi , Zou Haozhe , Zhou Weiwei , Lv Dezhong , Yang Changbo , Li Yongsheng , Xu Juan
Published: May 2024

Long non-coding RNAs (lncRNAs) have emerged as significant players for maintaining the morphology and function of tissues or cells. The precise regulatory effectiveness of lncRNA is closely associated with the spatial expression patterns across tissues and cells. Here, we proposed the Cellular-Elevated LncRNA (LncCE) database to systematically explore cellular-elevated (CE) lncRNAs across normal and cancer tissues in single cells. LncCE encompasses 87,946 CE lncRNAs of 149 cell types by analyzing 181 single-cell RNA sequencing (scRNA-seq) datasets, involved in 20 fetal normal tissues, 59 adult normal tissues, as well as 32 adult and 5 pediatric cancer tissues.Two main search options were provided via a given lncRNA name or a cell type. The output results emphasize both qualitative and quantitative expression features of lncRNAs across different cell types, co-expression with protein-coding genes as well as their involved in biological functions. For cancers, LncCE particularly provided quantitative figures for exhibiting their expression changes compared to control samples and clinical associations with patient overall survivals. Together, LncCE offers an extensive, quantitative and user-friendly interface to investigate cellular-elevated expression atlas for lncRNAs across normal and cancers tissues at single-cell level. The LncCE database is available athttp://bio-bigdata.hrbmu.edu.cn/LncCE.
The role of ADAR editing and nonsense-mediated decay in Parkinson’s Disease
Authors: Mercer Heather Milliken , Nair Aiswarya Mukundan , Tariq Ayesha , Piontkivska Helen
Published: May 2024

Parkinson’s Disease (PD) is a multifactorial disease with heterogenous phenotypes that vary across individuals, as well as by age and sex. Therefore, it is likely that multiple interacting factors, such as environmental influences and aging, as well as genetic factors, including dynamic RNA (ADAR, Adenosine Deaminases Acting on RNA) editing, may play a role in PD pathology. In this analysis of 317 transcriptomes of healthy controls, PD and prodromal patients aged 65 years or older, from Parkinson’s Project Markers Initiative dataset, we observe differences in ADAR expression, number of putative ADAR edits, editing index, and the number of high and moderate impact edits between control groups and diseased samples, particularly when ADAR editing is associated with nonsense-mediated decay (NMD). Likewise, differentially expressed genes between comparison groups were linked to NMD-related pathways. NMD is an important process in detecting deleterious nonsense sequences in mRNA transcripts and eliminating them from the cell. Thus, NMD regulation serves an important role in neurodevelopment, neural differentiation, and neural maturation. RNA misprocessing, which includes dysregulation of NMD, is known to play an important role in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and fronto-temporal dementia. Our results suggest that NMD may also be an important factor in PD physiology.
Emergence of potato virus Y outbreaks in tomatoes in Brazil, the disease and spread
Authors: Lucena Vivian S. , Nakasu Erich Y. T. , Pereira José L. , Rêgo-Machado Camila M. , Rodrigues Cristiano S. , Ueno Bernardo , Morais Ivair J. , Inoue-Nagata Alice K.
Published: May 2024

The emergence of ’Mexican Fire’ disease in Brazilian tomato fields, attributed to potato virus Y (PVY), has raised concerns. Characterized by severe necrosis on median leaves, the definitive etiological agent of this disease remained unverified despite PVY detection in symptomatic plants. Our study aimed to elucidate the causal agent, occurrence, spread, and symptomatology of Mexican Fire. Deep sequencing of tomato leaves with typical necrotic symptoms confirmed the association with PVY, reinforcing its role as the causal agent. Serological tests with a PVY-specific polyclonal antibody consistently correlated symptoms with virus presence in a fresh market tomato field, with higher PVY incidence near older tomato and maize plants. Necrotic leaf distribution analysis revealed a predominant occurrence in median leaves, progressing upwards. Deep sequencing of symptomatic field samples exclusively detected PVY, reaffirming its role in symptom induction. Importantly, PVY inoculation under field and greenhouse conditions fulfilled Koch’s Postulates, triggering leaf necrosis. Our findings unequivocally establish PVY as the causal agent of Mexican Fire disease, shedding light on its etiology, incidence, spread, and symptom expression, crucial for effective disease management strategies.
Synthetic organelles enable protein purification in a single operation
Authors: Guo Haotian , Chen Jionglin
Published: May 2024

Protein purification is crucial for various applications in life sciences research and biotechnological industry, yet traditional chromatography methods are often complex and suffer from limited scalability and sustainability. Here we report a biological approach utilizing synthetic organelles for protein purification, termed Program Architecture of Nano-Device Actions for Purification (PandaPure). PandaPure includes a biomimetic mechanism of sorting proteins of interest into organelles through signal sequence domains during expression, and subsequently releasing said proteins via cleavage of organelle-locating signals in a single operation. In the commonly-used expression host Escherichia coli, PandaPure enables high-yield, high-purity production of target proteins, and significantly reducing environmental impact. We demonstrate its scalability and versatility with different designs, scales, and across diverse proteins, promising wide-ranging benefits in research and industrial settings.
De novo pyrimidine biosynthesis inhibition synergizes with BCL-XLtargeting in pancreatic cancer
Authors: Zhang Huan , Yu Qijia , Santana-Codina Naiara , Poupault Clara , Campos Claudia , Qin Xingping , Padhye Aparna , Sindoni Nicole , Kuljanin Miljan , Wang Junning , Dorman Matthew J. , Aguirre Andrew J. , Dougan Stephanie K. , Sarosiek Kristopher A. , Mancias Joseph D.
Published: May 2024

Oncogenic KRAS, the genetic driver of 90% of pancreatic adenocarcinoma (PDAC), induces a metabolic rewiring characterized, in part, by dependency on de novo pyrimidine biosynthesis. Pharmacologic inhibition of dihydroorotate dehydrogenase (DHODH), an enzyme in the de novo pyrimidine synthesis pathway, delays pancreatic tumor growth in vivo; however, limited monotherapy efficacy suggests compensatory pathways and that combinatorial strategies are required for enhanced efficacy. Here, we use an integrated metabolomic, quantitative temporal proteomic and in vitro and in vivo DHODH inhibitor anchored CRISPR/Cas9 genetic screening approach to identify compensatory pathways to DHODH inhibition (DHODHi) and targets for combination strategies. We demonstrate that DHODHi alters the apoptotic regulatory proteome thereby enhancing sensitivity to inhibitors of the anti-apoptotic BCL2L1 (BCL-XL) protein. Combinatorial regimens with DHODH and BCL-XL inhibition synergistically induce apoptosis in PDAC cell lines and patient-derived PDAC organoids. In vivo DHODH inhibition with Brequinar and BCL-XL degradation with DT2216, a proteolysis targeting chimera (PROTAC), significantly inhibits the growth of PDAC tumors. Our data defines mechanisms of adaptation to DHODH inhibition and identifies a combination therapy strategy in PDAC.
The dynamic climate adaptation of indicator warbler species complex in the Pacific Rainforest
Authors: Wang Silu
Published: May 2024

Understanding the functional responses of ecosystems to climate change is critical to predict and conserve global biodiversity. The evolution of indicator species provides an efficient avenue towards the functional biome response to climate change. Leveraging decades of citizen science data coupled with high-resolution spatiotemporal climate data, I dissected the climate change response of hybridizingSetophagawarblers, indicator species breeding in the mysterious canopy of the Pacific Rainforest. Breeding habitat temperature and precipitation powerfully predicted the breeding occupancy of the northern (S. townsendi) and the southern species (S. occidentalis). Both species showed positive climatic responses in the recent decade when the recent breeding occupancy was greater than the expected contingency. This implies the potential climate adaptation or life history plasticity in the breeding warbler populations. However, theS. occidentalisshowed a compromised climate response in 2000-10, when the predicted breeding occupancy was significantly lower than the expected contingency. The compromised response might be due to rampant decline of July precipitation in 2000-10, compared to previous decades. Although the July precipitation continued to decline in 2010-20,S. occidentalisshowed signs of recovery, after generations of climate adaptation. I further evaluated their breeding niche competition, reflected by the overlap of breeding occupancy between species. I found that the competition potential was the lowest in 2000-10 when both species were at the trough of breeding occupancy, which recovered in 2010-20. This study shed light on eco-evo feedback in the indicating species complex and illuminated the functional response of the rainforest ecosystem to climate change.
lme4breeding: enabling genetic evaluation in the era of genomic data
Authors: Covarrubias-Pazaran Giovanny
Published: May 2024

Mixed models are a cornerstone in quantitative genetics to study the genetics of complex traits. A standard quantitative genetic model assumes that the effects of some random effects (e.g., individuals) are correlated based on their identity by descent and state. In addition, other relationships arise in the genotype by environment interactions (i.e., covariance structures). Open-source mixed model routines are available but do not account for complex covariance structures and are able to fit big genomic models. The lme4breeding R package was developed as an extension of the lme4 package and allows correlated random effects and complex covariance structures to be fitted for Gaussian, binary, and count responses. The correlation between levels of the random effect (e.g., individuals) is accounted for by post-multiplying the incidence matrix of the levels of this random factor by the Cholesky factor of the corresponding (co)variance matrix (e.g., the genomic relationship matrix). To enable big genomic models the eigen decomposition of relationship matrices is enabled. Maximum likelihood and REML estimation are available in lme4breeding. This note describes the type of models that can be fitted using lme4breeding and presents some examples.
Cardioprotection by Poloxamer 188 is Mediated through Increased Endothelial Nitric Oxide Production
Authors: Chen Gaoxian , Douglas Hunter F. , Li Zhu , Cleveland William J. , Balzer Claudius , Yannopolous Demetris , Chen Ian Ying-Li , Obal Detlef , Riess Matthias L.
Published: May 2024

Ischemia/reperfusion (I/R) injury significantly contributes to the morbidity and mortality associated with cardiac events. Poloxamer 188 (P188), a nonionic triblock copolymer, has been proposed to mitigate I/R injury by stabilizing cell membranes. However, the underlying mechanisms remain incompletely understood, particularly concerning endothelial cell function and nitric oxide (NO) production.

We employed human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs) and endothelial cells (ECs) to elucidate the effects of P188 on cellular survival, function, and NO secretion under simulated I/R conditions. iPSC-CMs contractility and iPSC-ECs’ NO production were assessed following exposure to P188. Further, an isolated heart model using Brown Norway rats subjected to I/R injury was utilized to evaluate the ex-vivo cardioprotective effects of P188, examining cardiac function and NO production, with and without the administration of a NO inhibitor.

In iPSC-derived models, P188 significantly preserved CM contractile function and enhanced cell viability after hypoxia/reoxygenation. Remarkably, P188 treatment led to a pronounced increase in NO secretion in iPSC-ECs, a novel finding demonstrating endothelial protective effects beyond membrane stabilization. In the rat isolated heart model, administration of P188 during reperfusion notably improved cardiac function and reduced I/R injury markers. This cardioprotective effect was abrogated by NO inhibition, underscoring the pivotal role of NO. Additionally, a dose-dependent increase in NO production was observed in non-ischemic rat hearts treated with P188, further establishing the critical function of NO in P188 induced cardioprotection.

In conclusion, our comprehensive study unveils a novel role of NO in mediating the protective effects of P188 against I/R injury. This mechanism is evident in both cellular models and intact rat hearts, highlighting the potential of P188 as a therapeutic agent against I/R injury. Our findings pave the way for further investigation into P188’s therapeutic mechanisms and its potential application in clinical settings to mitigate I/R-related cardiac dysfunction.
Developmental stage and level of submersion in water impact the viability of lone star and winter tick eggs during flooding
Authors: Alhawsawi Maher Ramadan I. , Lewis David A. , Frigard Ronja A. , Smith Ellen M. , Sivakumar Jaishna , Sharma Ajay M. , Nantz Adalynn R. , Sabile Chloe , Kennedy Jasmine , Loni Rashi , LeFefevre Gabrielle , Vaka Akshita , Leanza Quinn , Kelley Melissa , Stacey Crystal L. , Santosh Richa A. , Catlett Nathan , Cady Tabitha L. , Rizvi Raaidh S. , Wagner Zach , Olafson Pia U. , Benoit Joshua B.
Published: May 2024

Female ticks deposit large egg clusters that range in size from hundreds to thousands. These clusters are restricted to a deposition site, usually under leaf litter and other debris. These sites can be exposed to periodic flooding, where the cluster of tick eggs can float to the surface or remain underneath organic debris entirely underwater. Here, we examined the viability of egg clusters from winter ticks,Dermacentor albipictus, and lone star ticks,Amblyomma americanum, when partially submerged or fully submerged in water in relation to the developmental stages of the eggs. In general, egg clusters that were older and partially submerged had a higher viability than fully submerged, young eggs in water. A. americanum was much more resistant to water exposure between the two species. These studies highlight that egg clusters for specific tick species can remain viable when exposed to water for at least two weeks, where eggs float on the surface. These studies also suggest that water-based distribution of egg clusters could occur for some species, and flooding will differentially impact tick egg survival based on the specific developmental stage of exposure and species.
WEDAP: A Python Package for Streamlined Plotting of Molecular Simulation Data
Authors: Yang Darian T. , Chong Lillian T.
Published: May 2024

Given the growing interest in path sampling methods for extending the timescales of molecular dynamics (MD) simulations, there has been great interest in software tools that streamline the generation of plots for monitoring the progress of large-scale simulations. Here, we present the WEDAP Python package for simplifying the analysis of data generated from either conventional MD simulations or the weighted ensemble (WE) path sampling method, as implemented in the widely used WESTPA software package. WEDAP facilitates (i) the parsing of WE simulation data stored in highly compressed, hierarchical HDF5 files, and (ii) incorporates trajectory weights from WE simulations into all generated plots. Our Python package consists of multiple user-friendly interfaces: a command-line interface, a graphical user interface, and a Python application programming interface. We demonstrate the plotting features of WEDAP through a series of examples using data from WE and conventional MD simulations that focus on the HIV-1 capsid protein C-terminal domain dimer as a showcase system. The source code for WEDAP is freely available on GitHub athttps://github.com/chonglab-pitt/wedap.
Sex-specific expression of the human NAFLD-NASH transcriptional signatures in the liver of medaka with a history of ancestral bisphenol A exposure
Authors: Chakraborty Sourav , Anand Santosh , Bhandari Ramji Kumar
Published: May 2024

The progression of fatty liver disease to non-alcoholic steatohepatitis (NASH) is a leading cause of death in humans. Lifestyles and environmental chemical exposures can increase the susceptibility of humans to NASH. In humans, the presence of bisphenol A (BPA) in urine is associated with fatty liver disease, but whether ancestral BPA exposure leads to the activation of human NAFLD-NASH-associated genes in the unexposed descendants is unclear. In this study, using medaka fish as an animal model for human NAFLD, we investigated the transcriptional signatures of human NAFLD-NASH and their associated roles in the pathogenesis of the liver of fish who were not directly exposed but their ancestors were exposed to BPA during embryonic and perinatal development three generations prior. Comparison of bulk RNA-Seq data of the liver in BPA lineage male and female medaka with publicly available human NAFLD-NASH patient data revealed transgenerational alterations in the transcriptional signature of human NAFLD-NASH in medaka liver. Twenty percent of differentially expressed genes (DEGs) were upregulated in both human NAFLD patients and medaka. Specifically in females, among the total shared DEGs in the liver of BPA lineage fish and NAFLD patient groups, 27.69% DEGs were downregulated and 20% DEGs were upregulated. Off all DEGs, 52.31% DEGs were found in ancestral BPA-lineage females, suggesting that NAFLD in females shared majority of human NAFLD gene networks. Pathway analysis revealed beta-oxidation, lipoprotein metabolism, and HDL/LDL-mediated transport processes linked to downregulated DEGs in BPA lineage males and females. In contrast, the expression of genes encoding lipogenesis-related proteins was significantly elevated in the liver of BPA lineage females only. BPA lineage females exhibiting activationof myc, atf4, xbp1,stat4, and cancerous pathways, as well as inactivation ofigf1, suggest their possible association with an advanced NAFLD phenotype. The present results suggest that gene networks involved in the progression of human NAFLD and the transgenerational NAFLD in medaka are conserved and that medaka can be an excellent animal model to understand the development and progression of liver disease and environmental influences in the liver.
Vesicle-Mediated Regulation of Pollen tube Growth inTorenia fournieri
Authors: Jin Xingyue , Mizukami Akane G. , Okuda Satohiro , Higashiyama Tetsuya
Published: May 2024

In flowering plants, the growth of pollen tubes carrying sperm cells to the female gametophyte is necessary for double fertilization and seed development. The rate of growth of the pollen tube tip determines the fertility of many artificially cultivated plants and depends on the vesicle-mediated transport of cell membrane and cell wall components. However, the investigation of vesicle transport is hampered by the lack of transgenic methods for economically important plants. Here, we developed a method to transiently inhibit vesicle activity using brefeldin A (BFA) and antisense oligodeoxynucleotides (AS-ODNs) targeting key genes in the wishbone flower (Torenia fournieri), which has been used in studies of sexual reproduction in plants. BFA disrupted vesicle gradient homeostasis, thereby altering cell wall deposition and pollen tube morphology. The AS-ODN targetingTorenia fournieri ANXUR(TfANX), which is implicated in maintaining pollen tube growth and is used as a marker to assess inhibition by AS-ODNs, penetrated cell membranes and inhibited the expression of target genes. The treatment of pollen tubes with AS-ODN againstTfRABA4D, which is specifically expressed in pollen and involved in regulating vesicle targeting, resulted in pollen tubes with a bulging phenotype and disrupted pectin deposition, effects similar to those of BFA. In summary, vesicle-mediated mechanisms regulate the patterning of the pollen tube cell wall inT.fournieriand our findings could facilitate the genetic manipulation of horticultural crops.
PowerCHORD: constructing optimal experimental designs for biological rhythm discovery
Authors: Silverthorne Turner , Carlucci Matthew , Petronis Arturas , Stinchcombe Adam R
Published: May 2024

Equally spaced temporal sampling is the standard protocol for the study of biological rhythms. These equispaced designs perform well when calibrated to an oscillator’s period yet can have systematic detection biases when applied to rhythms of unknown periodicity. Here, we present a broadly-applicable set of computational methods for seeking optimal measurement schedules for rhythm detection. Our PowerCHORD methods generate experimental designs by maximizing a closed-form expression for the statistical power of the cosinor model using a black-box optimization method (differential evolution), a brute-force search, or mixed-integer conic programming. Application of these three methods showed numerically that they improve upon equispaced designs under many experimental contexts. Our numerical results also revealed an intuitive approach for achieving optimal power for simultaneous investigation of circadian, circalunar, and circannual rhythms. Our findings suggest that timing optimization is an effective yet under-explored tool for improving biological rhythm discovery.
A Human iPSC-derived Organoid System to Model Myelin Destruction and Repair
Authors: Ramesan Shwathy , Mason Joel , Swanson Molly E.V. , Mills Samuel , Turner Bradley J. , Rutar Matthew , Gonsalvez David G. , Barton Samantha K.
Published: May 2024

We engineered a novel human iPSC-derived organoid system enriched with mature, myelinating oligodendrocytes and functionally reactive microglia. This model enables the study of human CNS remyelination following a demyelinating insult, encapsulating: myelin fragmentation, microglial clearance of myelin debris and oligodendrocyte genesis, differentiation, and axon ensheathment. This system provides a powerful and unparalleled capacity to interrogate complex human cell behaviour, relevant to those studying glial biology and neurological disease mechanisms.
The human and mouse dendritic cell receptor DCIR binds to LRP1 N-glycans containing terminal galactoses, including the α-Gal antigen
Authors: Raymond Benjamin B. A. , Sneperger Tamara , Rousset Stella , Gilles Nelly , Bally Aurélie , Stella Alexandre , Hipgrave Ederveen Agnes L. , Marcoux Julien , Trimaglio Giulia , Thépaut Michel , Lagarrigue Frederic , Burlet-Schiltz Odile , Wuhrer Manfred , Fieschi Franck , Demange Pascal , Neyrolles Olivier , Rombouts Yoann
Published: May 2024

The dendritic cell immunoreceptor (DCIR) is a C-type lectin receptor expressed by myeloid cells that plays a key immunoregulatory role in a wide range of diseases, from inflammation to cancer. However, the ligand(s) of DCIR remain(s) unknown, hampering our understanding of the exact function of this immune receptor. Here, we found that both human DCIR and mouse DCIR1 bind specifically to the low-density lipoprotein receptor-related protein 1 (LRP1), a heavily glycosylated receptor mediating the clearance of various molecules from the extracellular matrix and apoptotic cells. This interaction is mediated by galactose-terminated biantennary complex-type N-glycans, including those carrying the immunogenic α-Gal epitope. Our study provides a deeper understanding of the role of DCIR in immune regulation and its potential impact on a range of immune disorders, highlighting its specificity in ligand recognition which is crucial for developing therapeutic strategies.
Rtf1 HMD domain facilitates global histone H2B monoubiquitination and regulates morphogenesis and virulence in the meningitis-causing pathogenCryptococcus neoformans
Authors: Jiang Yixuan , Liang Ying , Zhao Fujie , Lu Zhenguo , Wang Siyu , Meng Yao , Liu Zhanxiang , Zhang Jing , Zhao Youbao
Published: May 2024

Rtf1 is generally considered to be a subunit of the Paf1 complex (Paf1C), which is a multifunctional protein complex involved in histone modification and RNA biosynthesis at multiple stages. Rtf1 is stably associated with the Paf1C inSaccharomyces cerevisiae, but not in other species including humans. Little is known about its function in human fungal pathogens. Here, we show that Rtf1 is required for facilitating H2B monoubiquitination (H2Bub1), and regulates fungal morphogenesis and pathogenicity in the meningitis-causing fungal pathogenCryptococcus neoformans. Rtf1 is not tightly associated with the Paf1C, and its histone modification domain (HMD) is sufficient to promote H2Bub1 and the expression of genes related to fungal mating and filamentation. Moreover, Rtf1 HMD fully restores fungal morphogenesis and pathogenicity; however, it fails to restore defects of thermal tolerance and melanin production in thertf1Δ strain background. The present study establishes a role for cryptococcal Rtf1 as a Paf1C-independent regulator in regulating fungal morphogenesis and pathogenicity, and highlights the function of HMD in facilitating global H2Bub1 inC. neoformans.
The Peptonizer2000: bringing confidence to metaproteomics
Authors: Holstein Tanja , Verschaffelt Pieter , Van Den Bossche Tim , Van de Vyver Simon , Martens Lennart , Mesuere Bart , Muth Thilo
Published: May 2024

Metaproteomics, the large-scale study of proteins from microbial communities, faces challenges in identifying species due to similarities in protein sequences across different organisms. Current methods often rely on simple counting of matches between proteins and taxa, which can lead to low accuracy. We introduce the Peptonizer2000, a new tool that uses advanced modeling to provide more precise taxonomic identifications along with confidence scores. It combines peptide scores from any proteomic search engine with peptide-to-taxon links from the Unipept database. By applying statistical models, the Peptonizer2000 improves taxonomic resolution and delivers more reliable results. We validate its performance using publicly available datasets, demonstrating its ability to produce high-confidence identifications. Our results suggest that the Peptonizer2000 improves the specificity and confidence of taxonomic assignments in metaproteomics, providing a valuable resource for the study of complex microbial communities.
The Observed T cell receptor Space database enables paired-chain repertoire mining, coherence analysis and language modelling
Authors: Raybould Matthew I. J. , Greenshields-Watson Alexander , Agarwal Parth , Aguilar-Sanjuan Broncio , Olsen Tobias H. , Turnbull Oliver M. , Quast Nele P. , Deane Charlotte M.
Published: May 2024

T cell activation is governed through T cell receptors (TCRs), heterodimers of two sequence-variable chains (often an alpha [α] and beta [β] chain) that recognise linear antigen fragments presented on the cell surfaces. Early sequencing technologies limited the study of immune repertoire TCRs to unpaired transcripts, leading to extensive analysis ofβ-chain data alone as its greater sequence diversity suggested it should dominate antigen recognition. Over time, structural data has revealed that bothαandβchains contribute to binding most antigens and highthroughput single-cell handling technologies have been increasingly applied to obtain samples of complete TCR variable region sequences from repertoires. Despite this, there is currently no repository dedicated to the curation of publicly available paired TCR sequence data. We have addressed this gap by creating the Observed T cell receptor Space (OTS) database, a source of consistently processed and annotated, full-length, paired-chain TCR sequencing data from 50 studies and at least 75 individuals. Currently, OTS contains 5.35M redundant (1.63M nonredundant) predominantly human TCR sequences and, based on recent data availability trends, will grow rapidly. We perform an initial analysis of OTS, leading to the identification of pairing biases, public TCRs, and distinct chain coherence patterns relative to antibodies. We also harness the data to build a publicly available paired-chain TCR language model, providing paired embedding representations and a method for residue in-filling that is conditional on the partner chain. OTS will be updated and maintained as a central community resource and is freely downloadable and available as a web application athttps://opig.stats.ox.ac.uk/webapps/ots.
A PARP2-specific active site α-helix melts to permit DNA damage-induced enzymatic activation
Authors: Smith-Pillet Emily S. , Billur Ramya , Langelier Marie-France , Talele Tanaji T. , Pascal John M. , Black Ben E.
Published: May 2024

PARP1 and PARP2 recognize DNA breaks immediately upon their formation, generate a burst of local PARylation to signal their location, and are co-targeted by all current FDA-approved forms of PARP inhibitors (PARPi) used in the cancer clinic. Recent evidence indicates that the same PARPi molecules impact PARP2 differently from PARP1, raising the possibility that allosteric activation may also differ. We find that unlike for PARP1, destabilization of the autoinhibitory domain of PARP2 is insufficient for DNA damage-induced catalytic activation. Rather, PARP2 activation requires further unfolding of an active site α-helix absent in PARP1. Only one clinical PARPi, Olaparib, stabilizes the PARP2 active site α-helix, representing a structural feature with the potential to discriminate small molecule inhibitors. Collectively, our findings reveal unanticipated differences in local structure and changes in activation-coupled backbone dynamics between PARP1 and PARP2.
Identification of Small Molecule Inhibitors of PPM1D Using a Novel Drug Discovery Platform
Authors: Jiang Wei , Shaw Subrata , Rush Jason , Dumont Nancy , Kim John , Singh Ritu , Skepner Adam , Khodier Carol , Raffier Cerise , Yan Ni , Schluter Cameron , Yu Xiao , Szuchnicki Mateusz , Sathappa Murugappan , Kahn Josephine , Sperling Adam S. , McKinney David C. , Gould Alexandra E. , Garvie Colin W. , Miller Peter G.
Published: May 2024

Protein phosphatase, Mg2+/Mn2+dependent 1D (PPM1D), is a serine/threonine phosphatase that is recurrently activated in cancer, regulates the DNA damage response (DDR), and suppresses the activation of p53. Consistent with its oncogenic properties, genetic loss or pharmacologic inhibition of PPM1D impairs tumor growth and sensitizes cancer cells to cytotoxic therapies in a wide range of preclinical models. Given the therapeutic potential of targeting PPM1D specifically and the DDR and p53 pathway more generally, we sought to deepen our biological understanding of PPM1D as a drug target and determine how PPM1D inhibition differs from other therapeutic approaches to activate the DDR. We performed a high throughput screen to identify new allosteric inhibitors of PPM1D, then generated and optimized a suite of enzymatic, cell-based, andin vivopharmacokinetic and pharmacodynamic assays to drive medicinal chemistry efforts and to further interrogate the biology of PPM1D. Importantly, this drug discovery platform can be readily adapted to broadly study the DDR and p53. We identified compounds distinct from previously reported allosteric inhibitors and showedin vivoon-target activity. Our data suggest that the biological effects of inhibiting PPM1D are distinct from inhibitors of the MDM2-p53 interaction and standard cytotoxic chemotherapies. These differences also highlight the potential therapeutic contexts in which targeting PPM1D would be most valuable. Therefore, our studies have identified a series of new PPM1D inhibitors, generated a suite ofin vitroandin vivoassays that can be broadly used to interrogate the DDR, and provided important new insights into PPM1D as a drug target.
Synthetic Genitourinary Image Synthesis via Generative Adversarial Networks: Enhancing AI Diagnostic Precision
Authors: Van Booven Derek J , Chen Cheng-Bang , Malpani Sheetal , Mirzabeigi Yasamin , Mohammadi Maral , Wang Yujie , Arora Himanshu
Published: May 2024

In the realm of computational pathology, the scarcity and restricted diversity of genitourinary (GU) tissue datasets pose significant challenges for training robust diagnostic models. This study explores the potential of Generative Adversarial Networks (GANs) to mitigate these limitations by generating high-quality synthetic images of rare or underrepresented GU tissues. We hypothesized that augmenting the training data of computational pathology models with these GAN-generated images, validated through pathologist evaluation and quantitative similarity measures, would significantly enhance model performance in tasks such as tissue classification, segmentation, and disease detection. To test this hypothesis, we employed a GAN model to produce synthetic images of eight different GU tissues. The quality of these images was rigorously assessed using a Relative Inception Score (RIS) of 17.2 ± 0.15 and a Fréchet Inception Distance (FID) that stabilized at 120, metrics that reflect the visual and statistical fidelity of the generated images to real histopathological images. Additionally, the synthetic images received an 80% approval rating from board-certified pathologists, further validating their realism and diagnostic utility. We used an alternative Spatial Heterogeneous Recurrence Quantification Analysis (SHRQA) to assess quality in prostate tissue. This allowed us to make a comparison between original and synthetic data in the context of features, which were further validated by the pathologist’s evaluation. Future work will focus on implementing a deep learning model to evaluate the performance of the augmented datasets in tasks such as tissue classification, segmentation, and disease detection. This will provide a more comprehensive understanding of the utility of GAN-generated synthetic images in enhancing computational pathology workflows. This study not only confirms the feasibility of using GANs for data augmentation in medical image analysis but also highlights the critical role of synthetic data in addressing the challenges of dataset scarcity and imbalance. Future work will focus on refining the generative models to produce even more diverse and complex tissue representations, potentially transforming the landscape of medical diagnostics with AI-driven solutions.

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Loss of glia-neuronal interactions and age-dependent cell death in aDrosophilamodel of adult neurodegeneration
Authors: Jhuti Unmila P. , Blumenthal Edward M.
Published: May 2024

While glial dysfunction has been implicated in the development of multiple neurodegenerative diseases, the role of glial cell morphology in neurodegeneration is underexplored. In the fruit flyDrosophila melanogaster, mutants of the genedrop-dead(drd) exhibit adult neurodegeneration and extremely short lifespans. The morphology of one class of glia, the cortex glia (CG), is abnormal indrdmutants. In controls, the CGs form a continuous network that wraps around all neuronal cell bodies, but indrdmutants, individual CGs are stunted and the CG network is disrupted. These phenotypes are present on the first day of adulthood. Apoptosis is the central mechanism of cell death indrdmutants; widespread cell death is observed on the first day of adulthood and increases with age and is primarily neuronal. Apoptotic cells are found both within and outside of the remaining CG network, with significant variation in the distribution among individual brains. The degree of cell death and CG network breakdown in young adults could explain whydrdmutant flies die within the first week of adulthood. TheDrosophila drdmutant is a unique model of adult neurodegeneration that provides new insight into the breakdown in interaction between glia and neuronal cell bodies.
L-Dopa incorporation into tubulin alters microtubule dynamics and reduces dendritic spine invasion and synapse maintenance
Authors: Zorgniotti Agustina , Sharma Aditi , Ramirez-Rios Sacnicte , Sanyal Chadni , Aleman Martina , Ditamo Yanina , Moutin Marie-Jo , Bisig C. Gastón , Peris Leticia
Published: May 2024

Previous studies have shown that L-Dopa, a tyrosine analog used in Parkinson’s disease treatment, can be incorporated into α-tubulin C-terminal tail via the tubulin tyrosine ligase (TTL) and polymerize into microtubules. In this work, we demonstrated that mature wild type hippocampal neurons treated with L-Dopa exhibited reduced dendritic spine density, primarily affecting mature dendritic spines. In these neurons, L-Dopa treatment significantly reduced tyrosinated α-tubulin levels without altering detyrosinated or Δ2 α-tubulin levels, suggesting the formation of a new tubulin pool, likely composed of L-Dopa-α-tubulin.

In vitro analysis of the activity of the purified VASH1-SVBP complex, the most abundant tubulin carboxypeptidase in brain, revealed that L-Dopa incorporation into α-tubulin modified the binding of the complex to microtubules and reduced its carboxypeptidase activity. These results suggest that L-Dopa incorporation into tubulin alters the properties of microtubules and affects their ability to interact with the enzyme.

To confirm the implication of L-Dopa-microtubules in dendritic spine alterations observed in wild type neurons, we analyzed the effect of L-Dopa treatment in neurons lacking the enzymes of the α-tubulin detyrosination/tyrosination cycle. In these cells, L-Dopa cannot be incorporated into α-tubulin due to the absence of the ligase (in TTL KO neurons) or the reduction of detyrosinated α-tubulin levels (in SVBP KO neurons). L-Dopa treatment did not modify dendritic spine density in TLL KO or SVBP KO neurons, clearly demonstrating that the alterations in dendritic spines seen in WT neurons are due to the incorporation of L-Dopa into tubulin.

Further analysis revealed that L-Dopa treatment decreased the percentage of spines containing excitatory synapses in wild type neurons, but not in TTL KO or SVBP KO neurons, suggesting a cumulative synaptic defect due to L-Dopa incorporation into microtubules. Additionally, L-Dopa altered microtubule dynamics by increasing catastrophe frequency and reducing comet lifetime, which led to fewer microtubules entering dendritic spines and decreased spine resistance to pruning.

Taken together, our results demonstrate that L-Dopa incorporation into α-tubulin drastically affects synaptic homeostasis, reaffirming the importance of balanced detyrosination/tyrosination of tubulin within the synaptic compartment. The abnormal dynamics of L-Dopa-microtubules and the reduction of dendritic spines and excitatory synapses highlight a novel mechanism of L-Dopa-induced synaptotoxicity.
A Bioluminescent Assay for Quantification of Cellular Glycogen Levels
Authors: Leippe Donna , Choy Rebeca , Vidugiris Gediminis , Merritt Hanne , Mellem Kevin T , Beattie David T , Ullman Julie C , Vidugiriene Jolanta
Published: May 2024

Glycogen is a large polymer of glucose that functions as an important means of storing energy and maintaining glucose homeostasis. Glycogen synthesis and degradation pathways are highly regulated and their dysregulation can contribute to disease. Glycogen storage diseases are a set of disorders that arise from improper glycogen metabolism. Glycogen storage disease II, known as Pompe disease, is caused by a genetic mutation that leads to increased glycogen storage in cells and tissues, resulting in progressive muscle atrophy and respiratory decline for patients. One approach for treating Pompe disease is to reduce glycogen levels by interfering with the glycogen synthesis pathway through glycogen synthase inhibitors. To facilitate the study of glycogen synthase inhibitors in biological samples, such as cultured cells, a high-throughput approach for measuring cellular glycogen was developed. A bioluminescent glycogen detection assay was automated and used to measure glycogen content in cells grown in 384-well plates. The assay successfully quantified reduced glycogen stores in cells treated with a series of glycogen synthase 1 inhibitors, verifying inhibitor activity in biological systems and validating the utility of the assay for glycogen metabolism studies.
Acarbose Impairs GutBacteroidesGrowth by Targeting Intracellular GH97 Enzymes
Authors: Brown Haley A. , Morris Adeline L. , Pudlo Nicholas A. , Hopkins Ashley E. , Martens Eric C. , Golob Jonathan L. , Koropatkin Nicole M.
Published: May 2024

Acarbose is a type-2 diabetes medicine that inhibits dietary starch breakdown into glucose by inhibiting host amylase and glucosidase enzymes. Numerous gut species in theBacteroidesgenus enzymatically break down starch and change in relative abundance within the gut microbiome in acarbose-treated individuals. To mechanistically explain this observation, we used two model starch-degradingBacteroides,Bacteroides ovatus(Bo) andBacteroides thetaiotaomicron(Bt). Bt growth is severely impaired by acarbose whereas Bo growth is not. TheBacteroidesuse a starch utilization system (Sus) to grow on starch. We hypothesized that Bo and Bt Sus enzymes are differentially inhibited by acarbose. Instead, we discovered that although acarbose primarily targets the Sus periplasmic GH97 enzymes in both organisms, the drug affects starch processing at multiple other points. Acarbose competes for transport through the Sus beta-barrel proteins and binds to the Sus transcriptional regulators. Further, Bo expresses a non-Sus GH97 (BoGH97D) when grown in starch with acarbose. The Bt homolog, BtGH97H, is not expressed in the same conditions, nor can overexpression of BoGH97D complement the Bt growth inhibition in the presence of acarbose. This work informs us about unexpected complexities of Sus function and regulation inBacteroides, including variation between related species. Further, this indicates that the gut microbiome may be a source of variable response to acarbose treatment for diabetes.

Importance

Acarbose is a type 2 diabetes medication that works primarily by stopping starch breakdown into glucose in the small intestine. This is accomplished by inhibition of host enzymes, leading to better blood sugar control via reduced ability to derive glucose from dietary starches. The drug and undigested starch travel to the large intestine where acarbose interferes with the ability of some bacteria to grow on starch. However, little is known about how gut bacteria interact with acarbose, including microbes that can use starch as a carbon source. Here, we show that two gut species,Bacteroides ovatus(Bo) andBacteroides thetaiotaomicron(Bt), respond differently to acarbose: Bt growth is inhibited by acarbose while Bo growth is not. We reveal a complex set of mechanisms involving differences in starch import and sensing behind the different Bo and Bt responses. This indicates the gut microbiome may be a source of variable response to acarbose treatment for diabetes via complex mechanisms in common gut microbes.
Asymmetry of both repressor and operator is important for the transcriptional regulation of theP. putidaXre-RES system
Authors: Henriksen Frederik Oskar G. , Brodersen Ditlev E. , Skjerning Ragnhild B.
Published: May 2024

Transcriptional regulation is a fundamental mechanism in bacteria and is often mediated by repressor proteins. In the type II toxin-antitoxin (TA) system.xre-res, the Xre antitoxin contains a putative helix-turn-helix (HTH) DNA-binding domain and could thus potentially bind to and repress transcription from thexre-respromoter. The structure of thePseudomonas putidaXre-RES TA complex revealed an unusual 4:2 stoichiometry with two potential DNA-binding sites, suggesting a non-canonical mechanism of transcriptional autoregulation. Here, we show that the activity of thexre-respromoter requires both an intact σ70element and the transcriptional start site, and that the Xre–RES complex represses transcription via binding to an imperfect inverted repeat region downstream of the σ70element. We furthermore confirm the presence of the unusual 4:2 TA complex in solution and show that it preferably binds the imperfect inverted repeat in a 1:1 ratio. In addition, we show that the isolated Xre antitoxin is an aggregation-prone monomerin vitroand a weak repressorin vivo.We find that the Xre dimer can dissociates within the 4:2 complex and result in a 2:2 TA complex that still neutralizes the RES toxin but cannot bind DNA. Together, our data suggests that the asymmetry of the promoter is important for both transcription and repression, and we propose a model in which the Xre-RES complex regulates transcription through a dynamic and concentration-dependent equilibrium between a non-binding (2:2) and a DNA-binding (4:2) form.

IMPORTANCE

Transcriptional regulation is fundamental for bacteria to survive in a constantly changing environment and requires the RNA polymerase, which recognizes and binds to a DNA element (the promoter) located upstream of the gene and initiates transcription. Transcriptional repressors can bind DNA and inhibit transcription by the RNA polymerase, but the exact recognition of DNA binding sites remains unclear. The Xre antitoxin from thePseudomonas putidatoxin-antitoxin system Xre-RES contains a putative DNA-binding domain, and the unusual ratio of antitoxin to toxin within the protein complex suggests two potential DNA-binding sites. In this work, we show that Xre-RES auto-regulates its own transcription through an asymmetrical DNA binding-site, optimized to bind both the RNA polymerase and the Xre-RES complex, as well as a concentration-dependent ratio of toxin to antitoxin within the complex. Our work provides insight on how promoters can evolve to fine-tune the regulation of bacterial transcription.