bluuebunny/crossref_metadata_2025_split huggingface dataset:
Hippo pathway-mediated YAP1/TAZ inhibition is essential for proper pancreatic endocrine specification and differentiation
Authors: Wu Yifan , Qin Kunhua , Lopez Kevin , Liu Jun , Nipper Michael , Deng Janice J , Yin Xue , Ramjit Logan , Ye Zhengqing , Wang Pei 
Published: June 2022

AbstractThe Hippo pathway plays a central role in tissue development and homeostasis. However, the function of Hippo in pancreatic endocrine development remains obscure. Here, we examined the roles of Hippo pathway mediated YAP1/TAZ inhibition in the development stages of endocrine specification and differentiation. While YAP1 protein was localized to the nuclei in bipotent progenitor cells, Ngn3 expressing endocrine progenitors completely lost YAP1. Using genetic mouse models, we found that this inactivation of YAP1 requires both intact Hippo pathway and NGN3 protein. Deleting the Lats1 and 2 kinases (Lats1&2) in endocrine progenitor cells of developing mouse pancreas with Ngn3-Cre blocked endocrine progenitor cell differentiation and specification, resulting in reduced islets size and disorganized pancreas at birth. Loss of Lats1&2 in NGN3 expressing cells activated YAP1/TAZ transcriptional activity and recruited macrophages to the developing pancreas. These defects were rescued by deletion of YAP1/TAZ genes, suggesting that tight regulation of YAP1/TAZ by Hippo signaling is crucial for pancreatic endocrine specification. In contrast, deletion of Lats1&2 using beta-cell specific MIP1CreER resulted in a phenotypically normal pancreas, indicating that Lasts1&2 are dispensable for pancreatic beta-cell function. Our results demonstrate that YAP1/TAZ inhibition in the pancreatic endocrine compartment is not a passive consequence of endocrine specification. Rather, Hippo pathway-mediated YAP1/TAZ inhibition in endocrine progenitors is a prerequisite for endocrine specification and differentiation.
HSV-1 exploits host heterochromatin for egress
Authors: Lewis Hannah C , Kelnhofer-Millevolte Laurel E , Brinkley Mia R , Arbach Hannah E , Arnold Edward A , Ramachandran Srinivas , Avgousti Daphne C 
Published: June 2022

AbstractHerpes simplex virus (HSV-1) progeny form in the nucleus and must exit to successfully infect other cells. These newly formed viral capsids navigate the complex chromatin architecture of the nucleus to reach the inner nuclear membrane and egress. Here, we demonstrate by transmission electron microscopy (TEM) that HSV-1 capsids traverse dense heterochromatin in the nuclear periphery to reach the inner nuclear membrane. We found that this heterochromatin is dependent on the specific chromatin marks of trimethylation on histone H3 lysine 27 (H3K27me3) and the histone variant macroH2A1. Through chromatin profiling over the course of infection, we revealed massive host genomic regions bound by macroH2A1 and H3K27me3 that correlate with decreased host transcription in active compartments. This indicates the formation of new heterochromatin during infection. We found that loss of these markers resulted in significantly lower viral titers but did not impact viral genome or protein accumulation. Strikingly, we discovered by TEM that loss of macroH2A1 or H3K27me3 resulted in nuclear trapping of viral capsids. Thus, our work demonstrates that HSV-1 takes advantage of the dynamic nature of host heterochromatin formation during infection for efficient viral egress.
The conserved AAA-ATPase PCH-2 distributes its regulation of meiotic prophase events by remodeling multiple meiotic HORMADs inC. elegans
Authors: Russo Anna E. , Giacopazzi Stefani , Deshong Alison , Menon Malaika , Ortiz Valery , Ego Kaori M. , Corbett Kevin D. , Bhalla Needhi 
Published: June 2022

AbstractDuring meiotic prophase, the essential events of homolog pairing, synapsis, and recombination are coordinated with meiotic progression to promote fidelity and prevent aneuploidy. The conserved AAA+ ATPase PCH-2 coordinates these events to guarantee crossover assurance and accurate chromosome segregation. How PCH-2 accomplishes this coordination is poorly understood. Here, we provide evidence that PCH-2 decelerates pairing, synapsis and recombination inC. elegansby remodeling meiotic HORMADs. We propose that PCH-2 converts the closed versions of these proteins, which drive these meiotic prophase events, to unbuckled conformations, destabilizing interhomolog interactions and delaying meiotic progression. Further, we find that PCH-2 distributes this regulation among three essential meiotic HORMADs inC. elegans: PCH-2 acts through HTP-3 to regulate pairing and synapsis, HIM-3 to promote crossover assurance, and HTP-1 to control meiotic progression. In addition to identifying a molecular mechanism for how PCH-2 regulates interhomolog interactions, our results provide a possible explanation for the expansion of the meiotic HORMAD family as a conserved evolutionary feature of meiosis. Taken together, our work demonstrates that PCH-2’s remodeling of meiotic HORMADs has functional consequences for the rate and fidelity of homolog pairing, synapsis, recombination and meiotic progression, ensuring accurate meiotic chromosome segregation.
Mitochondrial function determines severity but not risk of amyotrophic lateral sclerosis
Authors: Harvey Calum , Weinreich Marcel , Zhang Sai , Hop Paul J , Zwamborn Ramona A J , van Eijk Kristel , Julian Thomas H , Moll Tobias , Iacoangeli Alfredo , Khleifat Ahmad Al , Quinn John P , Pfaff Abigail L , Koks Sulev , Poulton Joanna , Battle Stephanie L , Arking Dan E , Snyder Michael P ,   Project MinE ALS Sequencing Consortium, Veldink Jan , Kenna Kevin P , Shaw Pamela J , Cooper-Knock Johnathan 
Published: June 2022

AbstractAmyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease. Selective vulnerability of energy-intensive motor neurons (MNs) has fostered speculation that mitochondrial function is a determinant of ALS. Previously, the position of mitochondrial function in the pathogenic cascade leading to neurotoxicity has been unclear. We separated upstream genetic determinants of mitochondrial function, including genetic variation within the mitochondrial genome or autosomes; from downstream changeable factors including mitochondrial copy number (mtCN) and MN gene expression. We discovered that functionally validated mitochondrial haplotypes are a determinant of ALS survival but not ALS risk. Loss-of-function genetic variants within, and reduced MN expression of, ACADM and DNA2 lead to shorter ALS survival; both genes impact mitochondrial function. MtCN responds dynamically to the onset of ALS independent of mitochondrial haplotype, and is also significantly correlated with disease severity. We conclude that mitochondrial function impacts ALS progression but not risk; our findings have therapeutic implications.
Wolbachiainteracts with the microbiome to shape fitness-associated traits during seasonal adaptation inDrosophila melanogaster
Authors: Henry Lucas P. , Fernandez Michael , Wolf Scott , Ayroles Julien F. 
Published: June 2022

AbstractThe microbiome contributes to many different host traits, but its role in host adaptation remains enigmatic. The fitness benefits of the microbiome often depend on ecological conditions, but fluctuations in both the microbiome and environment modulate these fitness benefits. Moreover, vertically transmitted bacteria might constrain the ability of both the microbiome and host to respond to changing environments.Drosophila melanogasterprovides an excellent system to investigate the evolutionary effects of interactions between the microbiome and the environment. To address this question, we created field mesocosms ofD. melanogasterundergoing seasonal adaptation with and without the vertically transmitted bacteria,Wolbachia pipientis.Sampling temporal patterns in the microbiome revealed thatWolbachiaconstrained microbial diversity. Furthermore, interactions betweenWolbachiaand the microbiome contributed to fitness-associated traits.Wolbachiaoften exerted negative fitness effects on hosts, and the microbiome modulated these effects. Our work supports recent theoretical advances suggesting that hosts in temporally fluctuating environments benefit from flexible microbial associations with low transmission fidelity—specifically when changes in the microbiome can better enable host phenotypes to match environment change. We conclude by exploring the consequences of complex interactions betweenWolbachiaand the microbiome for our understanding of eco-evolutionary processes and the utility ofWolbachiain combating vector-borne disease.
Prime editing in chicken fibroblasts and primordial germ cells
Authors: Atsuta Yuji , Suzuki Katsuya , Yaguchi Haruna , Saito Daisuke 
Published: June 2022

ABSTRACTCRISPR/Cas9-based genome editing technologies are revolutionizing developmental biology. One of the advanced CRISPR-based techniques is prime editing (PE), which enables precise gene modification in multiple model organisms. In the current study, we describe a method to apply PE to the genome of chicken fibroblasts and primordial germ cells (PGCs). By combining PE with a transposon-mediated genomic integration, drug selection, and the single-cell culture method, we successfully generated prime-edited chick PGCs. The chicken PGC is widely used as an experimental model to study germ cell formation and as a vector for gene transfer to produce transgenic chickens. Such experimental models are useful in the developmental biology field and as potential bioreactors to produce pharmaceutical and nutritious proteins. Thus, the method presented here will provide not only a powerful tool to investigate gene function in germ cell development but also a basis for generating prime-edited transgenic birds.
Molecular specification of cortico-brainstem versus corticospinal projection neurons in development
Authors: Kaiser Julia , Patel Payal , Dündar Friederike , Perez-Tetuan Jimena , Angira Nirupama , Sieger Eytan , Sahni Vibhu 
Published: June 2022

AbstractSkilled motor control requires precise connections between subcerebral projection neurons (SCPN) in the cerebral cortex and their appropriate subcerebral targets in the brainstem or spinal cord. The brainstem is an important motor control center and cortical projections to the brainstem serve distinct motor control functions than corticospinal projections. However, mechanisms controlling cortico-brainstem versus corticospinal projections during development remain unknown. Here, we show that the transition between the brainstem and cervical cord distinguishes cortico-brainstem from corticospinal neurons from the earliest stages of SCPN axon extension in white matter. We used high throughput single-cell RNA sequencing of FACS-purified SCPN, retrogradely labeled from either the cerebral peduncle (labeling both cortico-brainstem and corticospinal neurons) or the cervical cord (labeling corticospinal neurons only) at critical times of axon extension. We identify that cortico-brainstem and corticospinal neurons are molecularly distinct: We establish Neuropeptide Y (Npy) as specifically enriched in cortico-brainstem neurons in lateral cortex, while CART prepropeptide (Cartpt) delineates cervical-projecting corticospinal neurons. Our results highlight molecular specification of cortico-brainstem vs. corticospinal projections well before these axons reach their appropriate segmental target and suggest a broad molecular program over SCPN axon targeting to distinct subcerebral targets early in development. These findings are likely to inform future investigations of motor circuit development, as well as approaches aimed at enhancing motor recovery after central nervous system damage.HighlightsCortico-brainstem neurons (CBN) limit their axon extension to supraspinal levels from the earliest time points of white matter axon extension in development.CBN can be molecularly delineated from corticospinal neurons (CSN) even at these initial time points.Molecular diversification of developing subcerebral projection neurons occurs across at least two axes: cortical location (medial vs. lateral) and projection targeting specificity (brainstem vs. spinal)Within lateral cortex, Neuropeptide Y (Npy) is expressed by CBN, while CART prepropeptide (Cartpt) expression delineates cervical-projecting CSN.Abstract Figure
Polar protein Wag31 both activates and inhibits cell wall metabolism at the poles and septum
Authors: Habibi Arejan Neda , Patel Parthvi Bharatkumar , Quintanilla Samantha Y. , Emami Saleh Arash , Boutte Cara C. 
Published: June 2022

AbstractMycobacterial cell elongation occurs at the cell poles; however, it is not clear how cell wall insertion is restricted to the pole and organized. Wag31 is a pole-localized cytoplasmic protein that is essential for polar growth, but its molecular function has not been described. In this study we used alanine scanning mutagenesis to identify Wag31 residues involved in cell morphogenesis. Our data show that Wag31 has separate functions in not only new and old pole elongation, but also inhibition of both septation and new pole elongation. Our examination of phospho-ablative and phospho-mimetic mutants of Wag31 suggests that phosphorylation of Wag31 promotes old pole elongation, while the unphosphorylated form of Wag31 may promote resolution of the septum. This work establishes new regulatory functions of Wag31 in the mycobacterial cell cycle and clarifies the role of phosphorylation on Wag1.ImportanceDespite many previous studies, the molecular mechanisms of polar growth in mycobacteria is unclear. Wag31 is required for this polar elongation. In this work, we dissect Wag1 function by phenotyping wag31 point mutants. We find that Wag31 promotes elongation at both poles in different ways, and it can also inhibit cell wall metabolism at both the new pole and the septum. This work is important because it clarifies that Wag31 is doing several different things in the cell and gives us genetic tools to disentangle its functions.
Imaging Clostridioides difficile spore germination and germination proteins
Authors: Baloh Marko , Nerber Hailee N. , Sorg Joseph A. 
Published: June 2022

AbstractClostridioides difficile spores are the infective form for this endospore-forming organism. The vegetative cells are intolerant to oxygen and poor competitors with a healthy gut microbiota. Therefore, in order for C. difficile to establish infection, the spores have to germinate in an environment that supports vegetative growth. To initiate germination, C. difficile uses Csp-type germinant receptors that consist of the CspC and CspA pseudoproteases as the bile acid and co-germinant receptors, respectively. CspB is a subtilisin-like protease that cleaves the inhibitory pro-peptide from the pro-SleC cortex lytic enzyme thereby activating it and initiating cortex degradation. Though several locations have been proposed for where these proteins reside within the spore (i.e., spore coat, outer spore membrane, cortex, inner spore membrane), these have been based, mostly, on hypotheses or prior data in C. perfringens. In this study, we visualize the germination process using TEM and SEM, and, using immunogold labeling of the spore proteins, find that these proteins are localized to the spore cortex, consistent with the observed, rapid, changes to the spore structure during germination.ImportanceGermination by C. difficile spores is the first step in the establishment of potentially life-threatening CDI. A deeper understanding of the mechanism by which spores germinate may provide insight for how to either prevent spore germination into a disease-causing vegetative form, or trigger germination prematurely when the spore is either in the outside environment or in a host environment that is non-conducive to the establishment of colonization / disease.
SUMOylation of SARS-CoV-2 Nucleocapsid protein enhances its interaction affinity and plays a critical role for its nuclear translocation
Authors: Madahar Vipul , Rodgers Victor G. J. , Liao Jiayu 
Published: June 2022

AbstractViruses, such as SARS-CoV-2, infect hosts and take advantages of host cellular machinery for their genome replication and new virion production. Identification and elucidation of host pathways for viral infection are critical for understanding the viral life cycle and novel therapeutics development. SARS-CoV-2 N protein is critical for viral RNA(vRNA) genome packaging in new virion formation, Here, we report that identification of SUMOylation sites of SARS-CoV-2 N protein and role of SUMO modification in N protein interaction affinity with itself using our qFRET/MS coupled method. We found, for the first time, that the SUMO modification of N protein can significantly increase its interaction affinity with itself and may support its oligomer formation. One of the identified Lys residues, K65 was critical for N protein translocation to nucleus, where the vRNA replication and packaging take place. The in vitro assessment of the affinity of N protein to N protein with SUMO mutants provides insight of the oligomerized N protein formation after SUMO modification. These results suggest that the host human SUMOylation pathway may be very critical for N protein functions in viral replication. The host SUMOylation pathway may be a critical host factor for the SARS-CoV-2 virus life cycle. Identification and inhibition of critical host SUMOyaltion could provide a novel strategy for future anti-viral therapeutics development, such as SARS-CoV-2 and other viruses.ImportanceThe SARS-CoV-2 virus N protein plays a critical role critical for viral RNA(vRNA) genome packaging in host cell nucleus for new virion formation. Therefore, deciphering the molecular mechanisms modulating N activity could be a strategy to identify potential targets amenable to therapeutics. Here, we identify a comprehensive SUMOylation sites of N proteins using an in vitro reconstitute SUMOyaltion assay containing SUMO E1 activating enzyme, E2 conjugating enzyme, and E3 ligase. We find that SUMOylation modification of N protein can significantly enhance it interaction affinity with itself, indicating an increased oligomerization capability, which is critical for N protein activity for vRNA genome packaging. In addition, we find one of SUMOylation sites of N protein is critical for its nucleus translocation, which is a critical for viral genome packaging. The SUMOylation modification may represent novel potential approach to design new antivirals with the ability to modulate SARS-CoV-2 virus replication.
The transcriptional regulator SpxA1 influences the morphology and virulence of Listeria monocytogenes
Authors: Cesinger Monica R. , Daramola Oluwasegun I. , Kwiatkowski Lucy M. , Reniere Michelle L. 
Published: June 2022

ABSTRACTListeria monocytogenes is a gram-positive facultative anaerobe and an excellent model pathogen for investigating regulatory changes that occur during infection of a mammalian host. SpxA1 is a widely conserved transcriptional regulator that induces expression of peroxide-detoxifying genes in L. monocytogenes and is thus required for aerobic growth. SpxA1 is also required for L. monocytogenes virulence, although the SpxA1-dependent genes important in this context remain to be identified. In this work, we sought to investigate the role of SpxA1 in a tissue culture model of infection and made the surprising discovery that ΔspxA1 cells are dramatically elongated during growth in the host cytosol. Quantitative microscopy revealed ΔspxA1 also forms elongated filaments extracellularly during early exponential phase in rich medium. Scanning and Transmission Electron Microscopy (STEM) analysis found the likely cause of this morphological phenotype is aberrantly placed division septa localized outside of cell midpoints. Quantitative mass spectrometry of whole cell lysates identified SpxA1-dependent changes in protein abundance, including a significant number of motility and flagellar proteins that were depleted in the ΔspxA1 mutant. Accordingly, we found that both the filamentation and the lack of motility contributed to decreased phagocytosis of ΔspxA1 by macrophages. Overall, we identify a novel role for SpxA1 in regulating cell elongation and motility, both of which impact L. monocytogenes virulence.IMPORTANCEListeria monocytogenes is an environmental saprophyte and the causative agent of the serious foodborne disease listeriosis. L. monocytogenes uses a highly complex transcriptional network to rapidly respond to the transition from living in the environment to infecting a host. One of the regulators important for L. monocytogenes pathogenesis is SpxA1, although the SpxA1-regulated genes necessary for virulence remain undefined. This study set out to examine the role of SpxA1 during infection and discovered that SpxA1 regulates cell shape and motility during both extracellular and intracellular growth. Together, our analyses find that SpxA1 has a multifaceted role in virulence, as SpxA1 regulation of both cell morphology and motility are important for infection.
A nuclear export signal in KHNYN required for its antiviral activity evolved as ZAP emerged in tetrapods
Authors: Lista Maria Jose , Ficarelli Mattia , Wilson Harry , Kmiec Dorota , Youle Rebecca L , Wanford Joseph , Winstone Helena , Odendall Charlotte , Taylor Ian A , Neil Stuart J D , Swanson Chad M 
Published: June 2022

ABSTRACTThe zinc finger antiviral protein (ZAP) inhibits viral replication by directly binding CpG dinucleotides in cytoplasmic viral RNA to inhibit protein synthesis and target the RNA for degradation. ZAP evolved in tetrapods and there are clear orthologs in reptiles, birds and mammals. When ZAP emerged, other proteins may have evolved to become cofactors for its antiviral activity. KHNYN is a putative endoribonuclease that is required for ZAP to restrict retroviruses. To determine its evolutionary path after ZAP emerged, we compared KHNYN orthologs in mammals and reptiles to those in fish, which do not encode ZAP. This identified residues in KHNYN that are highly conserved in species that encode ZAP, including several in the CUBAN domain. The CUBAN domain interacts with NEDD8 and Cullin-RING E3 ubiquitin ligases. Deletion of the CUBAN domain decreased KHNYN antiviral activity, increased protein expression and increased nuclear localization. However, mutation of residues required for the CUBAN domain-NEDD8 interaction increased KHNYN abundance but did not affect its antiviral activity or cytoplasmic localization, indicating that Cullin- mediated degradation may control its homeostasis and regulation of protein turnover is separatable from its antiviral activity. By contrast, the C-terminal residues in the CUBAN domain form a CRM1-dependent nuclear export signal (NES) that is required for its antiviral activity. Deletion or mutation of the NES increased KHNYN nuclear localization and decreased its interaction with ZAP. The final two positions of this NES are not present in fish KHNYN orthologs and we hypothesize their evolution allowed KHNYN to act as a ZAP cofactor.IMPORTANCEThe interferon system is part of the innate immune response that inhibits viruses and other pathogens. This system emerged approximately 500 million years ago in early vertebrates. Since then, some genes have evolved to become antiviral interferon- stimulated genes (ISGs) while others evolved so their encoded protein could interact with proteins encoded by ISGs and contribute to their activity. However, this remains poorly characterized. ZAP is an ISG that arose during tetrapod evolution and inhibits viral replication. Because KHNYN interacts with ZAP and is required for its antiviral activity against retroviruses, we conducted an evolutionary analysis to determine how specific amino acids in KHNYN evolved after ZAP emerged. This identified a nuclear export signal that evolved in tetrapods and is required for KHNYN to traffic in the cell to interact with ZAP. Overall, specific residues in KHNYN evolved to allow it to act as a cofactor for ZAP antiviral activity.