bluuebunny/crossref_metadata_2025 huggingface dataset:
BirdVox: Machine listening for bird migration monitoring
Authors: Lostanlen Vincent , Cramer Aurora , Salamon Justin , Farnsworth Andrew , Van Doren Benjamin M. , Kelling Steve , Bello Juan Pablo
Published: June 2022

The steady decline of avian populations worldwide urgently calls for a cyber-physical system to monitor bird migration at the continental scale. Compared to other sources of information (radar and crowdsourced observations), bioacoustic sensor networks combine low latency with a high taxonomic specificity. However, the scarcity of flight calls in bioacoustic monitoring scenes (below 0.1% of total recording time) requires the automation of audio content analysis. In this article, we address the problem of scaling up the detection and classification of flight calls to a full-season dataset: 6672 hours across nine sensors, yielding around 480 million neural network predictions. Our proposed pipeline, BirdVox, combines multiple machine learning modules to produce per-species flight call counts. We evaluate BirdVox on an annotated subset of the full season (296 hours) and discuss the main sources of estimation error which are inherent to a real-world deployment: mechanical sensor failures, sensitivity to background noise, misdetection, and taxonomic confusion. After developing dedicated solutions to mitigate these sources of error, we demonstrate the usability of BirdVox by reporting a species-specific temporal estimate of flight call activity for the Swainson’s Thrush(Catharus ustulatus).
R/LinkedCharts: A novel approach for simple but powerful interactive data analysis
Authors: Ovchinnikova Svetlana , Anders Simon
Published: June 2022

In any research project involving data-rich assays, exploratory data analysis is a crucial step. Typically, this involves jumping back and forth between visualizations that provide overview of the whole data and others that dive into details. In data quality assessment, for example, it might be very helpful to have one chart showing a summary statistic for all samples, and clicking on one of the data points would display details on this sample in a second plot. Setting up such interactively linked charts is usually too cumbersome and time-consuming to use them inad hocanalysis. We present R/LinkedCharts, a framework that renders this task radically simple: Producing linked charts is as quickly done as is producing conventional static plots in R, requiring a data scientist to write only very few lines of simple R code to obtain complex and general visualization. We expect that the convenience of our new tool will enable data scientists and bioinformaticians to perform much deeper and more thorough EDA with much less effort. Furthermore, R/LinkedCharts apps, typically first written as quick-and-dirty hacks, can also later be polished to provide interactive data access in publication quality, thus contributing to open science.
Preparing glycomics data for robust statistical analysis with GlyCompareCT
Authors: Zhang Yujie , Krishnan Sridevi , Bao Bokan , Chiang Austin W.T. , Sorrentino James T. , Schinn Song-Min , Kellman Benjamin P. , Lewis Nathan E.
Published: June 2022

Glycomics data are rapidly increasing in scale and diversity. Complexities in glycan biosynthesis (hierarchy, competition, and compartmentalization) make preprocessing critical to address resulting sparsity (many similar glycosylation profiles may share few common glycans) and non-independence (substrate-competition in glycan biosynthesis results in non-independence incompatible with many statistical methods). Here, we present GlyCompareCT, a portable command-line tool, to address these challenges thereby facilitating downstream analyses. Given glycan abundances, GlyCompareCT conducts substructure decomposition to quantify hidden biosynthetic intermediate abundance and relationships between measured glycans. Thus, GlyComparCT mitigates sparsity and makes interdependence explicit thereby increasing statistical power. Ultimately, GlyComparCT is a user-friendly implementation of substructure analysis designed to increase accessibility, interoperability, and scope and consistency in glycomics analysis.

Availability and implementation

Source code, test data, and instructions for GlyCompareCT v1.1.0 are available at: https://github.com/LewisLabUCSD/GlyCompareCT

Supplementary information

https://github.com/LewisLabUCSD/GlyCompareCT/raw/main/Supplementary%20Material.pdf
Time-lapse Imaging of Microglial Activity and Astrocytic Calcium Signaling Reveals a Neuroprotective Effect of Cannabidiol in the Subacute Phase of Stroke
Authors: Meyer Erika , Rieder Phillip , Gobbo Davide , Cândido Gabriela , Scheller Anja , de Oliveira Rúbia Maria Weffort , Kirchhoff Frank
Published: June 2022

Pharmacological agents that limit secondary tissue loss and/or improve functional outcomes after stroke are still limited. Cannabidiol, the major non-psychoactive component of Cannabis sativa, has been proposed as a neuroprotective agent against experimental focal cerebral ischemia. The effects of cannabidiol have generally been related to the modulation of neuroinflammation, including the control of glial activation and the toxicity exerted by pro-inflammatory mediators. However, so far, most information concerning cannabidiol neuroprotective effects was obtained from histological and biochemical post-mortem assays. To test whether the effects of cannabidiol on glial cells could be also detected in vivo, we performed time-lapse imaging of microglial activity and astrocytic calcium signaling in the subacute phase of stroke using two-photon laser-scanning microscopy. First, C57BL/6N wild-type mice underwent either sham or transient middle cerebral artery occlusion surgery. The animals received intraperitoneal injection of vehicle or cannabidiol (10 mg/kg) 30 min, 24 h, and 48 h after surgery. One day later the neurological score test was performed. Brain tissue was processed to evaluate the neuronal loss and microglial activation. Transgenic mice with microglial expression of the enhanced green fluorescent protein and astrocyte-specific expression of the calcium sensor GCaMP3 were used to access in vivo microglial activity and astrocytic calcium signaling, respectively. The animals were submitted to the same experimental design described above and to imaging sessions before, 30 min, 24 h and, 48 h after surgery. Astrocytic calcium signaling was also assessed in acutely isolated slices 5 h after transient middle cerebral artery occlusion surgery in the presence of perfusion or cannabidiol solution. Cannabidiol prevented ischemia-induced neurological impairments as well as protected against neuronal loss in ischemic mice. Cannabidiol also reduced ischemia-induced microglial activation, as demonstrated in fixed tissue as well in in vivo conditions. No difference in the amplitude and duration of astrocytic calcium signals was detected before and after the middle cerebral artery occlusion in vivo. Similarly, no significant difference was found in the astrocytic calcium signals between contra and ipsilateral side of acutely isolated brain slices. The present results suggest that the neuroprotective effects of cannabidiol after stroke may occur in the subacute phase of ischemia and reinforce the strong anti-inflammatory property of this compound.
Within-species control reveals novel immune response genes in Caenorhabditis elegans
Authors: Ranawade Ayush , Hartman Emma , Levine Erel
Published: June 2022

The nematode Caenorhabditis elegans is a simple model host for studying the interaction between bacterial pathogens and the metazoan innate immune system. In the last two decades, much focus has been given to studying intestinal infection in the worm by the clinical strain Pseudomonas aeruginosa PA14. Powerful genetic and molecular tools in both species facilitate the identification and analysis of bacterial virulence factors as well as host defense factors.

However, findings from these studies are confounded by the use of the genetically, metabolically, and physiologically divergent E. coli OP50 as a food source and as the non-virulent control. Here we report the use of P. aeruginosa z11 strain as a preferable control for PA14 infection studies. We demonstrate that many aspects of worm behavior, health-span, longevity, food attraction, brood size, lifespan, and pathogen avoidance and survival are not affected in z11. We show that the use of z11 as a control for transcriptomics analysis can increase the discovery power. In particular, we identified three novel pathogenesis-related genes in C. elegans. Our findings accentuate the importance of choosing an appropriate control environment for host-pathogen studies.
Whole-genome sequencing identifies interferon induced protein IFI6 as a strong candidate gene for VNN resistance in European sea bass
Authors: Delpuech Emilie , Vandeputte Marc , Morvezen Romain , Bestin Anastasia , Besson Mathieu , Brunier Joseph , Bajek Aline , Imarazene Boudjema , François Yoannah , Bouchez Olivier , Cousin Xavier , Poncet Charles , Morin Thierry , Bruant Jean-Sébastien , Chatain Béatrice , Haffray Pierrick , Phocas Florence , Allal François
Published: June 2022

Background

Viral Nervous Necrosis (VNN) is major disease affecting of European sea bass. Understanding the biological mechanisms that underlie VNN resistance is thus important for the welfare of farmed fish and the sustainability of production systems. This study aimed at identifying key genomic regions and genes that determine VNN resistance in sea bass.

Results

We generated a dataset of around 900,000 single nucleotide polymorphisms (SNPs) identified from whole-genome sequencing (WGS) in the parental generation in two different commercial populations (pop A and pop B) comprising 2371 and 3428 European sea bass with phenotypic records for binary survival in a VNN challenge. In each commercial population, three cohorts were submitted to the redspotted grouper nervous necrosis virus (RGNNV) challenge by immersion and genotyped on a 57K SNP chip. After imputation of WGS SNPs from their parents, QTL mapping was performed using a Bayesian Sparse Linear Mixed Model (BSLMM). We found several QTL regions on different linkage groups (LG), most of which are specific to a single population, but a QTL region on LG12 was shared by both commercial populations. This QTL region is only 127 kB wide, and we identified IFI6, an interferon induced protein at only 1.9 kB of the most significant SNP. An unrelated validation population with 4 large families was used to validate the effect of the QTL, for which the survival of the susceptible genotype ranges from 39.8 to 45.4%, while that of the resistant genotype ranges from 63.8 to 70.8%.

Conclusions

We could precisely locate the genomic region implied in the main resistance QTL at less than 1.9 kb of the interferon alpha inducible protein 6 (IFI6), which has already been identified as a key player for other viral infections such as hepatitis B and C. This will lead to major improvements for sea bass breeding programs, allowing for greater genetic gain by using marker-assisted genomic selection to obtain more resistant fish. Further functional analyses are needed to evaluate the impact of the variant on the expression of this gene.
Incubation of palatable food craving is associated with brain-wide neuronal activation in mice
Authors: Madangopal Rajtarun , Szelenyi Eric R. , Nguyen Joseph , Brenner Megan B. , Drake Olivia R. , Pham Diana , Shekara Aniruddha , Jin Michelle , Choong Jia Jie , Heins Connor , Komer Lauren E. , Weber Sophia J. , Hope Bruce T. , Shaham Yavin , Golden Sam A.
Published: June 2022

Studies using rodent models have shown that relapse to drug or food seeking increases progressively during abstinence, a phenomenon termed ‘incubation of craving’. Mechanistic studies of incubation of craving have focused on specific neurobiological targets within pre- selected brain areas. Recent methodological advances in whole-brain immunohistochemistry, clearing, and imaging now enable unbiased brain-wide cellular resolution mapping of regions and circuits engaged during learned behaviors. However, these whole brain imaging approaches were developed for mouse brains while incubation of drug craving has primarily been studied in rats and incubation of food craving has not been demonstrated in mice. Here, we established a mouse model of incubation of palatable food craving and examined food reward seeking after 1, 15, and 60 abstinence days. We then used the neuronal activity marker Fos with intact brain mapping procedures to identify corresponding patterns of brain-wide activation. Relapse to food seeking was significantly higher after 60 abstinence days than after 1 or 15 days. Using unbiased ClearMap analysis, we identified increased activation of multiple brain regions, particularly corticostriatal structures, following 60, but not 15 abstinence days. We used orthogonal SMART2 analysis to confirm these findings within corticostriatal and thalamocortical subvolumes and applied expert-guided registration to investigate subdivision and layer-specific activation patterns. Overall, we (1) identified novel brain-wide activity patterns during incubation of food seeking using complementary analytical approaches, and (2) provide a single-cell resolution whole-brain atlas that can be used to identify functional networks and global architecture underlying incubation of food craving.

Significance Statement

Relapse to reward seeking progressively increases during abstinence, a phenomenon termed incubation of craving. Mechanistic studies of incubation can lead to novel relapse treatments. However, previous studies have primarily used rat models and targeted region-by-region analyses and a brain-wide functional atlas of incubation of reward seeking is lacking. We established a behavioral procedure for incubation of palatable food seeking in mice and applied whole-brain activity mapping with Fos as a neuronal activity marker to identify the functional connectome of this incubation. Like rats, mice showed incubation of food seeking during abstinence. Using two complementary activity mapping approaches, we identified a brain-wide pattern of increased neural activation that mirrored incubation of food seeking after 60, but not 15, days of abstinence.
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

The 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

Herpes 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

During 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

Amyotrophic 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

The 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

CRISPR/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

Skilled 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.

Highlights

Cortico-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

Mycobacterial 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.

Importance

Despite 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

Clostridioides 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.

Importance

Germination 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

Viruses, 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.

Importance

The 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

Listeria 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.

IMPORTANCE

Listeria 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

The 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.

IMPORTANCE

The 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.
The Protein Interaction Networks of Catalytically-Active and Catalytically-Inactive PqsE in Pseudomonas aeruginosa
Authors: Taylor Isabelle R. , Murray-Nerger Laura A. , Cristea Ileana M. , Bassler Bonnie L.
Published: June 2022

Pseudomonas aeruginosa is a human pathogen that relies on quorum sensing to establish infections. The PqsE quorum-sensing protein is required for P. aeruginosa virulence factor production and infection. PqsE has a reported enzymatic function in the biosynthesis of the quorum-sensing autoinducer called PQS. However, this activity is redundant because, in the absence of PqsE, this role is fulfilled by alternative thioesterases. Rather, PqsE drives P. aeruginosa pathogenic traits via a protein-protein interaction with the quorum-sensing receptor/transcription factor, RhlR, an interaction that enhances affinity of RhlR for target DNA sequences. PqsE catalytic activity is dispensable for interaction with RhlR. Thus, PqsE virulence function can be decoupled from its catalytic function. Here, we present an immunoprecipitation-mass spectrometry method employing eGFP-PqsE fusions to define the protein interactomes of wildtype PqsE and the catalytically inactive PqsE(D73A) variant in P. aeruginosa and their dependence on RhlR. Several proteins were identified to have specific interactions with wildtype PqsE, while not forming associations with PqsE(D73A). In the ΔrhlR strain, an increased number of specific PqsE interactors were identified, including the partner autoinducer synthase to RhlR, called RhlI. Collectively, these results suggest that specific protein-protein interactions depend on PqsE catalytic activity and that RhlR may prevent proteins from interacting with PqsE, possibly due to competition between RhlR and other proteins for PqsE binding. Our results provide a foundation for the identification of the in vivo PqsE catalytic function and, potentially, new proteins involved in P. aeruginosa quorum sensing.

IMPORTANCE

Pseudomonas aeruginosa causes hospital-borne infections in vulnerable patients, including in immunocompromised individuals, burn victims, and cancer patients undergoing chemotherapy. There are no effective treatments for P. aeruginosa infections, which are usually broadly resistant to antibiotics. Animal models show that to establish infection and to cause illness, P. aeruginosa relies on an interaction between two proteins: PqsE and RhlR. There could be additional protein-protein interactions involving PqsE, which, if defined, could be exploited for the design of new therapeutic strategies to combat P. aeruginosa. Here, we reveal previously unknown protein interactions in which PqsE participates that will be investigated for potential roles in pathogenesis.
Modeling the Orthosteric Binding Site of the G Protein-Coupled Odorant Receptor OR5K1
Authors: Nicoli Alessandro , Haag Franziska , Marcinek Patrick , He Ruiming , Kreißl Johanna , Stein Jörg , Marchetto Alessandro , Dunkel Andreas , Hofmann Thomas , Krautwurst Dietmar , Di Pizio Antonella
Published: June 2022

With approximately 400 encoding genes in humans, odorant receptors (ORs) are the largest subfamily of class A G protein-coupled receptors (GPCRs). Despite its high relevance and representation, the odorant-GPCRome is structurally poorly characterized: no experimental structures are available, and the low sequence identity of ORs to experimentally solved GPCRs is a significant challenge for their modeling. Moreover, the receptive range of most ORs is unknown. The odorant receptor OR5K1 was recently and comprehensively characterized in terms of cognate agonists. Here we report two additional agonists and functional data of the most potent compound on two mutants, L1043.32and L2556.51. Experimental data was used to guide the investigation of the binding modes of OR5K1 ligands into the orthosteric binding site using structural information from AI-driven modeling, as recently released in the AlphaFold Protein Structure Database, and from homology modeling. Induced-fit docking simulations were used to sample the binding site conformational space for ensemble docking. Mutagenesis data guided side chain residue sampling and model selection. We obtained models that could better rationalize the different activity of active (agonist) versus inactive molecules with respect to starting models, and also capture differences in activity related to minor structural differences. Therefore, we provide a model refinement protocol that can be applied to model the orthosteric binding site of ORs as well as that of GPCRs with low sequence identity to available templates.
Precise modelling and correction of a spectrum of β-thalassemic mutations in human erythroid cells by base editors
Authors: Prasad Kirti , Devaraju Nivedhitha , George Anila , Ravi Nithin Sam , Mahalingam Gokulnath , Rajendiran Vignesh , Panigrahi Lokesh , Venkatesan Vigneshwaran , Lakhotiya Kartik , Moorthy Yogapriya , Pai Aswin Anand , Nakamura Yukio , Kurita Ryo , Balasubramanian Poonkuzhali , Thangavel Saravanabhavan , Velayudhan Shaji R , Marepally Srujan , Srivastava Alok , Mohankumar Kumarasamypet M
Published: June 2022

β-thalassemia and HbE result from mutations in the β-globin locus that impedes the production of functional β-hemoglobin and represents one of the most common genetic disorders worldwide. Recent advances in genome engineering have opened up new therapeutic opportunities to directly correct these pathogenic mutations using base editors that install transition mutations (A>G and C>T) in the target region with minimal generation of indels. Herein, for the first time, we demonstrate the usage of base editor in the correction of point mutations spanning multiple regions of the HBB gene, including promoter, intron and exon. To this end, we have engineered human erythroid cells harbouring the diverse HBB mutations, thus eliminating the requirement of patient CD34+ HSPCs with desired mutations for the primary screening by base editors. We further performed precise creation and correction of individual HBB point mutations in human erythroid cells using base editors, which were effectively corrected in the HBB-engineered erythroid model. Intriguingly, most bystander effects produced by the base editor at the target site were reported to exhibit normal hemoglobin variants. Overall, our study provides the proof-of-concept for the precise, efficient and scarless creation and correction of various pathogenic mutations at the coding and non-coding regions of HBB gene in human erythroid cells using base editors and establishes a novel therapeutic platform for the treatment of β-thalassemia/HbE patients. This study can be further explored in correcting the other monogenic disorders caused due to single base substitutions.
Rapid Activation of Hematopoietic Stem Cells
Authors: Thapa Roshina , Elfassy Erez , Olender Leonid , Sharabi Omri , Gazit Roi
Published: June 2022

Adult Hematopoietic Stem Cells (HSCs) in the bone marrow (BM) are quiescent. Following perturbations, such as blood loss or infection, HSCs may undergo activation to accelerate the production of needed effector blood and immune cells. Surprisingly, little is known about the earliest stages of HSC activation. We utilize surface markers of HSC activation, CD69, and CD317, revealing response as early as 2 h after stimulation. The dynamic expression of HSC activation markers varies between viral-like (poly-Inosinic-poly-Cytidylic) or bacterial-like (Lipopolysaccharide) immune stimuli. We further quantify the dose response, demonstrating a low-threshold and similar sensitivity of HSCs and progenitors in the BM. Finally, we find a positive correlation between the expression of surface activation markers and early exit from quiescence into proliferation. Our data show that the premier response of adult stem cells to immune stimulation is rapid, sensitive, and directly leads to proliferation.
Targeting MALAT1 Augments Sensitivity to PARP Inhibition by Impairing Homologous Recombination in Prostate Cancer.
Authors: Yadav Anjali , Biswas Tanay , Praveen Ayush , Ganguly Promit , Verma Ayushi , Datta Dipak , Ateeq Bushra
Published: June 2022

Poly(ADP-ribose) polymerase inhibitors (PARPi) have emerged as the most promising targeted therapeutic intervention for the treatment of metastatic castrate-resistant prostate cancer (mCRPC). However, the clinical utility of PARPi has been limited to a subset of patients who harbor aberrations in the homologous recombination (HR) pathway. Here, we report that targeting MALAT1, an oncogenic lncRNA, known to be elevated in advanced-stage prostate cancer (PCa) demonstrates contextual synthetic lethality with PARPi. We show that MALAT1 silencing reprograms the HR transcriptome, contriving BRCAness-like phenotype, thus enhancing sensitivity towards PARPi. Moreover, transcriptome profiles of mCRPC patients exhibit convergence between expression of MALAT1, HR pathway, and neuroendocrine markers. Mechanistically, we show that targeting MALAT1 leads to a decrease in EZH2, a member of polycomb repressor complex-2 (PRC2), which in turn upregulates the expression of RE1 Silencing Transcription Factor (REST), a key repressor of neuroendocrine differentiation. Overall, we showed that MALAT1 plays a pivotal role in maintaining genomic integrity, thereby promoting disease progression. Conclusively, our findings suggest that inhibiting MALAT1 confers PARPi sensitization in patient's resistant to anti-androgens and conventional chemotherapeutics.