bluuebunny/crossref_metadata_2025_split huggingface dataset:
Tuft cell-derived acetylcholine regulates epithelial fluid secretion
Authors: Billipp Tyler E. , Fung Connie , Webeck Lily M. , Sargent Derek B. , Gologorsky Matthew B. , McDaniel Margaret M. , Kasal Darshan N. , McGinty John W. , Barrow Kaitlyn A. , Rich Lucille M. , Barilli Alessio , Sabat Mark , Debley Jason S. , Myers Richard , Howitt Michael R. , von Moltke Jakob 
Published: March 2023

AbstractTuft cells are solitary chemosensory epithelial cells that can sense lumenal stimuli at mucosal barriers and secrete effector molecules to regulate the physiology and immune state of their surrounding tissue. In the small intestine, tuft cells detect parasitic worms (helminths) and microbe-derived succinate, and signal to immune cells to trigger a Type 2 immune response that leads to extensive epithelial remodeling spanning several days. Acetylcholine (ACh) from airway tuft cells has been shown to stimulate acute changes in breathing and mucocilliary clearance, but its function in the intestine is unknown. Here we show that tuft cell chemosensing in the intestine leads to release of ACh, but that this does not contribute to immune cell activation or associated tissue remodeling. Instead, tuft cell-derived ACh triggers immediate fluid secretion from neighboring epithelial cells into the intestinal lumen. This tuft cell-regulated fluid secretion is amplified during Type 2 inflammation, and helminth clearance is delayed in mice lacking tuft cell ACh. The coupling of the chemosensory function of tuft cells with fluid secretion creates an epithelium-intrinsic response unit that effects a physiological change within seconds of activation. This response mechanism is shared by tuft cells across tissues, and serves to regulate the epithelial secretion that is both a hallmark of Type 2 immunity and an essential component of homeostatic maintenance at mucosal barriers.
The intensities of canonical senescence biomarkers integrate the duration of cell-cycle withdrawal
Authors: Ashraf Humza M. , Fernandez Brianna , Spencer Sabrina L. 
Published: March 2023

AbstractSenescence, a state of permanent cell-cycle withdrawal, is difficult to distinguish from quiescence, a transient state of cell-cycle withdrawal. This difficulty arises because quiescent and senescent cells are defined by overlapping biomarkers, raising the question of whether quiescence and senescence are truly distinct states. To address this, we used single-cell time-lapse imaging to distinguish slow-cycling quiescent cells frombona fidesenescent cells after chemotherapy treatment, followed immediately by staining for various senescence biomarkers. We found that the staining intensity of multiple senescence biomarkers is graded rather than binary and primarily reflects the duration of cell-cycle withdrawal, rather than senescence per se. Together, our data suggest that quiescence and senescence are not distinct cellular states but rather fall on a continuum of cell-cycle withdrawal, where the intensities of canonical senescence biomarkers reflect the likelihood of cell-cycle re-entry.
CMS Final Rule for Organ Donation: Unintended Consequences of the Pancreas Research Loophole
Authors: Goldberg David , Chyou Darius , Wulf Rachael , Wadsworth Matthew 
Published: March 2023

AbstractCMS updated the ‘Final Rule’ for OPO Conditions for Coverage on 11/20/2020 to include new OPO metrics, tiers of classification, and a new definition of a donor that included an individual with: a) ≥1 organ transplanted; or b) pancreas procured for research or islet cell transplantation. We conducted a retrospective cohort study using data from the OPTN/ UNOS. The number of pancreata procured for research increased four-fold from 2020 to 2022, despite stable numbers of other organs procured for research. Of the 57 OPOs, 8 (14.0%) procured >100 pancreata for research in 2022, accounting for 1,548 (58.2%) pancreata research procurements. Of those 8 OPOs, seven would be at risk for possible (Tier 2; n=3) or definite (Tier 3; n=4) decertification based on CMS’s 2022 interim report. Based on these data, two Tier 3 OPOs would be reclassified as Tier 2 and one as Tier 1; three Tier 2 OPOs would be reclassified as Tier 1; and three Tier 3s would be reclassified as borderline Tier 2. The pancreas research carveout may detrimentally impact the system of organ donation by failing to accurately measure OPO performance and flag (and decertify) underpeforming OPOs unless CMS revises its final rule.
Usefulness of Current sgRNA Design Guidelines andin vitroCleavage Assays for Plant CRISPR/Cas Genome Editing: A Case in Eggplant (Solanum melongenaL.)
Authors: Sagarbarria Mark Gabriel S. , Caraan John Albert M. 
Published: March 2023

ABSTRACTThe advent of genome editing platforms such as the CRISPR/Cas9 system ushers an unprecedented speed on the development of new crop varieties that can withstand agricultural challenges of the 21stcentury. The CRISPR/Cas9 system depends on the specificity of engineered single guide RNAs (sgRNAs). However, sgRNA design in plants can be challenging due to a multitude of design tools to choose from, many of which use guidelines that are based on animal experiments yet allow the use of plant genomes. Upon choosing sgRNAs, it is also unclear whether anin vitroassay is needed to validate the targeting efficiency of a particular sgRNA prior toin vivodelivery of the CRISPR/Cas9 system. Here, we demonstrate thein vitroandin vivoactivity of four different sgRNAs that we selected based on their ability to target multiple members of the eggplant polyphenol oxidase gene family. Some sgRNAs that have highin vitrocleavage activity did not produce editsin vivo, suggesting that anin vitroassay may not be a reliable basis to predict sgRNAs with highly efficientin vivocleavage activity. Further analysis of our sgRNAs using other design algorithms suggest that plant-validated criteria such as the presence of necessary secondary structures and appropriate base-pairing may be the reason for the discrepancy between our observedin vitroandin vivocleavage efficiencies. However, recent reports and our data suggests that there is no guaranteed way to ensurein vivocleavage of chosen sgRNAs.Key Messagein vitrocleavage assay of sgRNAs was able to identify low activity sgRNAs but did not 13 reliably predictin vivomutagenesis.Using multiple sgRNAs that meet the plant-validated parameters and have high activityin vitroin plant genome editing is critical to ensure success.
Ras-dependent activation of BMAL2 regulates hypoxic metabolism in pancreatic cancer
Authors: Maurer H. Carlo , Garcia-Curiel Alvaro , Holmstrom Sam R. , Castillo Cristina , Palermo Carmine F. , Sastra Steven A. , Andren Anthony , Zhang Li , Le Large Tessa Y.S. , Sagalovskiy Irina , Ross Daniel R. , Wong Winston , Shaw Kaitlin , Genkinger Jeanine , Manji Gulam A. , Iuga Alina C. , Schmid Roland M. , Johnson Kristen , Badgley Michael A. , Lyssiotis Costas A. , Shah Yatrik , Califano Andrea , Olive Kenneth P. 
Published: March 2023

SummaryTo identify drivers of malignancy in human pancreatic ductal adenocarcinoma (PDAC), we performed regulatory network analysis on a large collection of expression profiles from laser capture microdissected samples of PDAC and benign precursors. We discovered that BMAL2 plays a role in the initiation, progression, post resection survival, and KRAS activity in PDAC. Functional analysis of BMAL2 target genes led us to hypothe-size that it plays a role in regulating the response to hypoxia, a critical but poorly understood feature of PDAC physiology. Knockout of BMAL2 in multiple human PDAC cell lines revealed effects on viability and invasion, particularly under hypoxic conditions. Loss of BMAL2 also affected glycolysis and other metabolic processes. We found that BMAL2 directly regulates hypoxia-responsive target genes. We also found that BMAL2 is necessary for the stabilization of HIF1A upon exposure to hypoxia, but destabilizes HIF2A under hypoxia. These data demonstrate that BMAL2 is a master transcriptional regulator of hypoxia responses in PDAC and may serve as a long-sought molecular switch that distinguishes HIF1A- and HIF2A-dependent modes of hypoxic metabolism.Statement of significanceWe provide a comprehensive portrait of the molecular drivers behind pancreatic cancer initiation, progression and outcome. We discover a new genetic regulator of metabolic responses to low oxygen environments, a key feature of pancreatic cancer biology. This protein, BMAL2, is a new driver of pancreatic cancer malignancy.
The Sde Phosphoribosyl-Linked Ubiquitin Transferases protect theLegionella pneumophilavacuole from degradation by the host
Authors: Kim Seongok , Isberg Ralph R. 
Published: March 2023

AbstractLegionella pneumophilagrows intracellularly within a host membrane-bound vacuole that is formed in response to a bacterial type IV secretion system (T4SS). T4SS translocated Sde proteins promote phosphoribosyl-linked ubiquitination of endoplasmic reticulum protein Rtn4, but the role played by this modification is obscure due to lack of clear growth defects of mutants. To identify the steps in vacuole biogenesis promoted by these proteins, mutations were identified that unmasked growth defects in Δsdestrains. Mutations in thesdhA,ridLandlegA3genes aggravated the Δsdefitness defect, resulting in disruption of theLegionella-containing vacuole (LCV) membrane within 2 hrs of bacterial contact with host cells. Depletion of Rab5B and sorting nexin 1 partially bypassed loss of Sde proteins, consistent with Sde blocking early endosome and retrograde trafficking, similar to roles previously demonstrated for SdhA and RidL proteins. Sde protein protection of LCV lysis was only observed shortly after infection, presumably because Sde proteins are inactivated by the metaeffector SidJ during the course of infection. Deletion of SidJ extended the time that Sde proteins could prevent vacuole disruption, indicating that Sde proteins are negatively regulated at the posttranslational level and are limited to protecting membrane integrity at the earliest stages of replication. Transcriptional analysis was consistent with this timing model for an early point of execution of Sde protein. Therefore, Sde proteins act as temporally-regulated vacuole guards during establishment of the replication niche, possibly by constructing a physical barrier that blocks access of disruptive host compartments early during biogenesis of the LCV.Significance statementMaintaining replication compartment integrity is critical for growth of intravacuolar pathogens within host cells. By identifying genetically redundant pathways,Legionella pneumophilaSde proteins that promote phosphoribosyl-linked ubiquitination of target eukaryotic proteins are shown to be temporally-regulated vacuole guards, preventing replication vacuole dissolution during early stages of infection. As targeting of reticulon 4 by these proteins leads to tubular endoplasmic reticulum aggregation, Sde proteins are likely to construct a barrier that blocks access of disruptive early endosomal compartments to the replication vacuole. Our study provides a new framework for how vacuole guards function to support biogenesis of theL. pneumophilareplicative niche.
Mutant Prion Protein Endoggresomes are Hubs for Local Axonal Organelle-Cytoskeletal Remodeling
Authors: Chaiamarit Tai , Verhelle Adriaan , Chassefeyre Romain , Shukla Nandini , Novak Sammy Weiser , Andrade Leonardo R. , Manor Uri , Encalada Sandra E. 
Published: March 2023

AbstractDystrophic axons comprising misfolded mutant prion protein (PrP) aggregates are a characteristic pathological feature in the prionopathies. These aggregates form inside endolysosomes -called endoggresomes-, within swellings that line up the length of axons of degenerating neurons. The pathways impaired by endoggresomes that result in failed axonal and consequently neuronal health, remain undefined. Here, we dissect the local subcellular impairments that occur within individual mutant PrP endoggresome swelling sites in axons. Quantitative high-resolution light and electron microscopy revealed the selective impairment of the acetylated vs tyrosinated microtubule cytoskeleton, while micro-domain image analysis of live organelle dynamics within swelling sites revealed deficits uniquely to the MT-based active transport system that translocates mitochondria and endosomes toward the synapse. Cytoskeletal and defective transport results in the retention of mitochondria, endosomes, and molecular motors at swelling sites, enhancing mitochondria-Rab7 late endosome contacts that induce mitochondrial fission via the activity of Rab7, and render mitochondria dysfunctional. Our findings point to mutant Pr Pendoggresome swelling sites as selective hubs of cytoskeletal deficits and organelle retention that drive the remodeling of organelles along axons. We propose that the dysfunction imparted locally within these axonal micro-domains spreads throughout the axon over time, leading to axonal dysfunction in prionopathies.
Expansion microscopy with trypsin digestion and tyramide signal amplification (TT-ExM) for protein and lipid staining
Authors: Wang Ueh-Ting Tim , Tian Xuejiao , Liou Yae-Huei , Lee Sue-Ping , Lu Chieh-Han , Chen Peilin , Cheb Bi-Chang 
Published: March 2023

AbstractExpansion microscopy, whereby the relative positions of biomolecules are physically increased via hydrogel expansion, can be used to reveal ultrafine structures of cells under a conventional microscope. Despite its utility for achieving super-resolution imaging, expansion microscopy suffers two major drawbacks, namely proteolysis and swelling effects that, respectively, induce protein loss and dilute fluorescence signals. Here, we report two improvements to expansion microscopy that overcome these two challenges, i.e., deploying trypsin digestion to reduce protein loss and tyramide signal amplification to enhance fluorescence signal. We name our new methodology TT-ExM to indicate dual trypsin and tyramide treatments. TT-ExM may be applied for both antibody and lipid staining. Notably, we demonstrate better protein retention for endoplasmic reticulum and mitochondrial markers in COS-7 cell cultures following 2-h trypsin treatment. Subsequent lipid staining revealed the complex 3D membrane structures in entire cells. Through combined lipid and DNA staining, our TT-ExM methodology highlighted mitochondria by revealing their DNA and membrane structures in cytoplasm, as well as the lipid-rich structures formed via phase separation in nuclei at interphase. We also observed lipid-rich chromosome matrices in the mitotic cells. Thus, TT-ExM significantly enhances fluorescent signals and generates high-quality and ultrafine-resolution images under confocal microscopy.
Pathologic polyglutamine aggregation begins with a self-poisoning polymer crystal
Authors: Kandola Tej , Venkatesan Shriram , Zhang Jiahui , Lerbakken Brooklyn , Schulze Alex Von , Blanck Jillian F , Wu Jianzheng , Unruh Jay , Berry Paula , Lange Jeffrey J. , Box Andrew , Cook Malcolm , Sagui Celeste , Halfmann Randal 
Published: March 2023

AbstractA long-standing goal of amyloid research has been to characterize the structural basis of the rate-determining nucleating event. However, the ephemeral nature of nucleation has made this goal unachievable with existing biochemistry, structural biology, and computational approaches. Here, we addressed that limitation for polyglutamine (polyQ), a polypeptide sequence that causes Huntington’s and other amyloid-associated neurodegenerative diseases when its length exceeds a characteristic threshold. To identify essential features of the polyQ amyloid nucleus, we used a direct intracellular reporter of self-association to quantify frequencies of amyloid appearance as a function of concentration, conformational templates, and rational polyQ sequence permutations. We found that nucleation of pathologically expanded polyQ involves segments of three glutamine (Q) residues at every other position. We demonstrate using molecular simulations that this pattern encodes a four-stranded steric zipper with interdigitated Q side chains. Once formed, the zipper poisoned its own growth by engaging naive polypeptides on orthogonal faces, in a fashion characteristic of polymer crystals with intramolecular nuclei. We further show that self-poisoning can be exploited to block amyloid formation, by genetically oligomerizing polyQ prior to nucleation. By uncovering the physical nature of the rate-limiting event for polyQ aggregation in cells, our findings elucidate the molecular etiology of polyQ diseases.
Evo-devo dynamics of hominin brain size
Authors: González-Forero Mauricio 
Published: March 2023

AbstractBrain size tripled in the human lineage over four million years, but why this occurred remains uncertain. To advance our understanding of what caused hominin-brain expansion, I mechanistically replicate it in-silico by modelling the evolutionary and developmental (evo-devo) dynamics of hominin-brain size. I show that, starting from australopithecine brain and body sizes, the model recovers the evolution of brain and body sizes of seven hominin species, the evolution of the hominin brain-body allometry, and major patterns of human development and evolution. Analysis shows that in this model the brain expands because it is “socio-genetically” correlated with developmentally late preovulatory ovarian follicles, not because brain size is directly selected for. The socio-genetic correlation causing the recovered hominin brain expansion is generated over development by ecology and possibly culture. Thus, in this model, direct selection that does not favour brain expansion provides a force that developmental constraints divert causing hominin-brain expansion.
Contingency, Repeatability and Predictability in the Evolution of a Prokaryotic Pangenome
Authors: Beavan Alan , Domingo-Sananes Maria Rosa , McInerney James O. 
Published: March 2023

AbstractPangenomes exhibit remarkable variability in many prokaryotic species. This variation is maintained through the processes of horizontal gene transfer and gene loss. Repeated acquisitions of near-identical homologs can easily be observed across pangenomes, leading to the question of whether these parallel events potentiate similar evolutionary trajectories, or whether the remarkably different genetic background of the recipients mean that post-acquisition evolutionary trajectories end up being quite different. In this study, we present a machine learning method that predicts the presence or absence of genes in theEscherichia colipangenome based on the presence of other accessory genes within the genome. We are, in effect, asking whether gene acquisitions potentiate similar evolutionary trajectories or not. Our analysis leverages the repeated transfer of genes through theE. colipangenome to observe patterns of repeated evolution following similar events. The presence or absence of a substantial set of genes is highly predictable, from other genes alone, indicating that selection potentiates and maintains gene-gene co-occurrence and avoidance relationships deterministically over long-term bacterial evolution despite differences in host evolutionary history. We propose that the pangenome can be understood as a set of genes with relationships that govern their likely cohabitants, analogous to an ecosystem’s set of interacting organisms. Our findings highlight intra-genomic gene fitness effects as key drivers of prokaryotic evolution, with ensuing pangenome-wide emergence of repeated patterns of community structure.
Microglial IL-1β plays a protective role in epilepsy
Authors: Wei Li , Wei Zhang , Ming Chen , Yang Liu , Si-Yan Li 
Published: March 2023

AbstractEpilepsy is a neurological disorder characterized by recurrent seizures that affect about 50 million people worldwide. Although the exact mechanisms underlying epilepsy remain elusive, it is known that neuroinflammation contributes to the pathogenesis of the disease. Microglia, the resident immune cells of the central nervous system, play a key role in neuroinflammation and are activated in response to seizures. Interleukin-1β (IL-1β) is a pro-inflammatory cytokine produced by microglia and other immune cells. While IL-1β has been implicated in the pathogenesis of various neurological disorders, recent studies have revealed a protective role for microglial IL-1β in epilepsy. This paper aims to review the current knowledge about microglial IL-1β and its potential therapeutic implications for epilepsy.
Two sequence- and two structure-based ML models have learned different aspects of protein biochemistry
Authors: Kulikova Anastasiya V. , Diaz Daniel J. , Chen Tianlong , Cole T. Jeffrey , Ellington Andrew D. , Wilke Claus O. 
Published: March 2023

ABSTRACTDeep learning models are seeing increased use as methods to predict mutational effects or allowed mutations in proteins. The models commonly used for these purposes include large language models (LLMs) and 3D Convolutional Neural Networks (CNNs). These two model types have very different architectures and are commonly trained on different representations of proteins. LLMs make use of the transformer architecture and are trained purely on protein sequences whereas 3D CNNs are trained on voxelized representations of local protein structure. While comparable overall prediction accuracies have been reported for both types of models, it is not known to what extent these models make comparable specific predictions and/or generalize protein biochemistry in similar ways. Here, we perform a systematic comparison of two LLMs and two structure-based models (CNNs) and show that the different model types have distinct strengths and weaknesses. The overall prediction accuracies are largely uncorrelated between the sequence- and structure-based models. Overall, the two structure-based models are better at predicting buried aliphatic and hydrophobic residues whereas the two LLMs are better at predicting solvent-exposed polar and charged amino acids. Finally, we find that a combined model that takes the individual model predictions as input can leverage these individual model strengths and results in significantly improved overall prediction accuracy.
Gain without pain: Adaptation and increased virulence of Zika virus in vertebrate host without fitness cost in mosquito vector
Authors: Jaeger Anna S. , Marano Jeffrey , Riemersma Kasen , Castañeda David , Pritchard Elise , Pritchard Julia , Bohm Ellie K. , Baczenas John J. , O’Connor Shelby L. , Weger-Lucarelli James , Friedrich Thomas C. , Aliota Matthew T. 
Published: March 2023

AbstractZika virus (ZIKV) is now in a post-pandemic period, for which the potential for re-emergence and future spread is unknown. Adding to this uncertainty is the unique capacity of ZIKV to directly transmit between humans via sexual transmission. Recently, we demonstrated that direct transmission of ZIKV between vertebrate hosts leads to rapid adaptation resulting in enhanced virulence in mice and the emergence of three amino acid substitutions (NS2A-A117V, NS2A- A117T, and NS4A-E19G) shared among all vertebrate-passaged lineages. Here, we further characterized these host-adapted viruses and found that vertebrate-passaged viruses also have enhanced transmission potential in mosquitoes. To understand the contribution of genetic changes to the enhanced virulence and transmission phenotype, we engineered these amino acid substitutions, singly and in combination, into a ZIKV infectious clone. We found that NS4A- E19G contributed to the enhanced virulence and mortality phenotype in mice. Further analyses revealed that NS4A-E19G results in increased neurotropism and distinct innate immune signaling patterns in the brain. None of the substitutions contributed to changes in transmission potential in mosquitoes. Together, these findings suggest that direct transmission chains could enable the emergence of more virulent ZIKV strains without compromising mosquito transmission capacity, although the underlying genetics of these adaptations are complex.