bluuebunny/crossref_metadata_2025 huggingface dataset:
Single-cell and nucleus RNA-seq in a mouse model of AD reveal activation of distinct glial subpopulations in the presence of plaques and tangles
Authors: Balderrama-Gutierrez Gabriela , Liang Heidi , Rezaie Narges , Carvalho Klebea , Forner Stefania , Matheos Dina , Rebboah Elisabeth , Green Kim N. , Tenner Andrea J. , LaFerla Frank , Mortazavi Ali
Published: October 2021

Multiple mouse models have been generated that strive to recapitulate human Alzheimer’s disease (AD) pathological features to investigate disease mechanisms and potential treatments. The 3xTg-AD mouse presents the two major hallmarks of AD, which are plaques and tangles that increase during aging. While behavioral changes and the accumulation of plaques and tangles have been well described in the 3xTg-AD mice, the subpopulations of neurons and glial cells present throughout disease progression have not been characterized. Here, we used single-cell RNA-seq to investigate changes in subpopulations of microglia, and single-nucleus RNA-seq to explore subpopulations of neurons, astrocytes, and oligodendrocytes in the hippocampus and cortex of aging 3xTg-AD as well as 5xFAD mice for comparison. We recovered a common path of age-associated astrocyte activation between the 3xTg-AD and the 5xFAD models and found that 3xTg-AD-derived astrocytes seem to be less activated. We identified multiple subtypes of microglia, including a subpopulation with a distinct transcription factor expression profile that showed an early increase inCsf1expression before the switch to disease associated microglia (DAM). We used bulk RNA-seq in the hippocampus of 3xTg-AD mice across their lifespan to identify distinct modules of genes whose expression increases with aging and worsening pathology. Finally, scATAC-seq revealed multiple subpopulations of cells with accessible chromatin in regions around genes associated with glial activation. Overall, differences between the main glial groups point to a slower activation process in the 3xTg-AD model when compared to the 5xFAD. Our study contributes to the identification of progressive transcriptional changes of glial cells in a mouse model that has plaques and tangles, thus providing information to aid in targeted AD therapeutics that could translate into positive clinical outcomes.
Loss of the cleaved-protamine 2 domain leads to incomplete histone-to-protamine exchange and infertility in mice
Authors: Arévalo Lena , Merges Gina Esther , Schneider Simon , Oben Franka Enow , Neumann Isabelle , Schorle Hubert
Published: October 2021

Protamines are unique sperm-specific proteins that package and protect paternal chromatin until fertilization. A subset of mammalian species expresses two protamines (PRM1 and PRM2), while in others PRM1 is sufficient for sperm chromatin packaging. Alterations of the species-specific ratio between PRM1 and PRM2 are associated with infertility. Unlike PRM1, PRM2 is generated as a precursor protein consisting of a highly conserved N-terminal domain, termed cleaved PRM2 (cP2), which is consecutively trimmed off during chromatin condensation. The carboxyterminal part, called mature PRM2 (mP2), interacts with DNA and together with PRM1, mediates chromatin-hypercondensation. The removal of the cP2 domain is believed to be imperative for proper chromatin condensation, yet, the role of cP2 is not yet understood. We generated mice lacking the cP2 domain while the mP2 is still expressed. We show that the cP2 domain is indispensable for complete sperm chromatin protamination and male mouse fertility. cP2 deficient sperm show incomplete PRM2 incorporation, resulting in a severely altered protamine ratio, retention of transition proteins and aberrant retention of the testis specific histone variant H2A.L.2. During epididymal transit, cP2 deficient sperm seem to undergo ROS mediated degradation leading to complete DNA fragmentation. The cP2 domain therefore seems to be a key aspect in the complex crosstalk between histones, transition proteins and protamines during sperm chromatin condensation. Overall, we present the first step towards understanding the role of the cP2 domain in paternal chromatin packaging and open up avenues for further research.
The organization of individually mapped structural and functional semantic networks in aging adults
Authors: Ketchabaw W. Tyler , DeMarco Andrew T. , Paul Sachi , Dvorak Elizabeth , van der Stelt Candace , Turkeltaub Peter E.
Published: October 2021

Language function in the brain, once thought to be highly localized, is now appreciated as relying on a connected but distributed network. The semantic system is of particular interest in the language domain because of its hypothesized integration of information across multiple cortical regions. Previous work in healthy individuals has focused on group-level functional connectivity (FC) analyses of the semantic system, which may obscure interindividual differences driving variance in performance. These studies also overlook the contributions of white matter networks to semantic function. Here, we identified semantic network nodes with a semantic decision fMRI task in 53 typically-aging adults, characterized network organization using structural connectivity (SC), and quantified the segregation and integration of the network using FC. Hub regions were identified in left inferior frontal gyrus. The individualized semantic network was composed of three interacting modules: 1) default-mode module characterized by bilateral medial prefrontal and posterior cingulate regions and also including right-hemisphere homotopes of language regions; 2) left frontal module extending dorsally from inferior frontal gyrus to pre-motor area; and 3) left temporoparietal module extending from temporal pole to inferior parietal lobule. FC within Module3 and integration of the entire network related to a semantic verbal fluency task, but not a matched phonological task. These results support and extend the tri-network semantic model (Xu et al., 2017) and the controlled semantic cognition model (Chiou et al., 2018) of semantic function.
Gaussian Network Model Revisited: Effects of Mutation and Ligand Binding on Protein Behavior
Authors: Erman Burak
Published: October 2021

The coarse-grained Gaussian Network model, GNM, considers only the alpha carbons of the folded protein. Therefore it is not directly applicable to the study of mutation or ligand binding problems where atomic detail is required. This shortcoming is improved by including the local effect of heavy atoms in the neighborhood of each alpha carbon into the Kirchoff Adjacency Matrix. The presence of other atoms in the coordination shell of each alpha carbon diminishes the magnitude of fluctuations of that alpha carbon. But more importantly, it changes the graph-like connectivity structure, i.e., the Kirchoff Adjacency Matrix of the whole system which introduces amino acid specific and position specific information into the classical coarse-grained GNM which was originally modelled in analogy with phantom network theory of rubber elasticity. With this modification, it is now possible to make predictions on the effects of mutation and ligand binding on residue fluctuations and their pair-correlations. We refer to the new model as ‘all-atom GNM’. Using examples from published data we show that the all-atom GNM applied to in silico mutated proteins and to their laboratory mutated structures gives similar results. Thus, loss and gain of correlations, which may be related to loss and gain of function, may be studied by using simple in silico mutations only.
PEAK2VEC ENABLES INFERRENCE OF TRANSCRIPTIONAL REGULATION FROM ATAC-SEQ
Authors: Liang Lifan , Lu Xinghua , Lu Songjian
Published: October 2021

Transcription factor (TF) binding sites in ATAC-seq are typically determined by footprint analysis. However, the performance of footprint analysis remains unsatisfying and most TFs do not exhibit footprint patterns. In this study, we modified the convolutional neural network to project sequences into an embedding space. Sequences with similar nucleotide patterns will stay close together in the embedding. The dimensionality of this embedding space represents binding specificities of various TFs. In the simulation experiment, peak2vec accurately distinguished the three TFs in the embedding space while conventional deep learning cannot. When applied to the ATAC-seq profiles of hepatitis carcinoma, peak2vec recovered multiple motifs curated in database, while significant portion of sequences corresponding to the TF are located at the promoter region of its regulated genes.
A model screening pipeline for bile acid converting anti-Clostridioides difficile bacteria reveals unique biotherapeutic potential of Peptacetobacter hiranonis
Authors: Vinithakumari Akhil A. , Hernandez Belen G. , Ghimire Sudeep , Adams Seidu , Stokes Caroline , Jepsen Isaac , Brezina Caleb , Sahin Orhan , Li Ganwu , Tangudu Chandra , Andreasen Claire , Philips Gregory J. , Wannemuehler Michael , Jergens Albert E. , Scaria Joy , Sponseller Brett , Mooyottu Shankumar
Published: October 2021

Clostridioides difficile is an antibiotic-resistant bacterium that causes serious, toxin-mediated enteric disease in humans and animals. Gut dysbiosis and resultant alterations in the intestinal bile acid profile play an important role in the pathogenesis of C. difficile infection (CDI). Restoration of the gut microbiota and re-establishment of bacterial bile acid metabolism using fecal microbiota transplantation (FMT) has been established as a promising strategy against this disease, although this method has several limitations. Thus, a more defined and precise microbiota-based approach using bacteria that biotransform primary bile acids into secondary bile acids could effectively overcome these limitations and control CDI. Therefore, a screening pipeline was developed to isolate bile acid converting bacteria from fecal samples. Dogs were selected as a model CDI-resistant microbiota donor for this pipeline, which yielded a novel Peptacetobacter hiranonis strain that possesses unique anti-C. difficile properties, and both bile acid deconjugation and 7-α dehydroxylating activities to perform bile acid conversion. The screening pipeline included a set of in vitro tests along with a precision in vivo gut colonization and bile acid conversion test using altered Schadler flora (ASF) colonized mice. In addition, this pipeline also provided essential information on the growth requirements for screening and cultivating the candidate bacterium, its survival in a CDI predisposing environment, and potential pathogenicity. The model pipeline documented here yielded multiple bile acid converting bacteria, including a P. hiranonis isolate with unique anti-C. difficile biotherapeutic potential, which can be further tested in subsequent preclinical and human clinical trials.
ReadZS detects cell type-specific and developmentally regulated RNA processing programs in single-cell RNA-seq
Authors: Meyer Elisabeth , Chaung Kaitlin , Dehghannasiri Roozbeh , Salzman Julia
Published: October 2021

RNA processing (RNAP), including splicing and alternative polyadenylation, is crucial to gene function and regulation, but methods to detect RNAP from single-cell RNA sequencing data are limited by reliance on pre-existing annotations, peak-calling heuristics, and collapsing measurements by cell type. We introduce ReadZS, the first annotation-free statistical approach to identify regulated RNAP in single cells. ReadZS discovers cell type-specific RNAP in the human lung and conserved, developmentally regulated RNAP in mammalian spermatogenesis - including global 3’ UTR shortening in human spermatogenesis. ReadZS also discovers global 3’ UTR lengthening in Arabidopsis root development, highlighting the usefulness of this method in under-annotated transcriptomes.
Fibroblast-derived HGF integrates muscle and nerve development during morphogenesis of the mammalian diaphragm
Authors: Sefton Elizabeth M. , Gallardo Mirialys , Tobin Claire E. , Colasanto Mary P. , Kardon Gabrielle
Published: October 2021

The diaphragm is a domed muscle between the thorax and abdomen essential for breathing in mammals. Diaphragm development requires the coordinated development of muscle, connective tissue, and nerve, which are derived from different embryonic sources. Defects in diaphragm development cause the common and often lethal birth defect, Congenital Diaphragmatic Hernias (CDH). HGF/MET signaling is required for diaphragm muscularization, but the source of HGF and the specific functions of this pathway in muscle progenitors or potentially the phrenic nerve have not been explicitly tested. Using conditional mutagenesis and pharmacological inhibition of MET, we demonstrate that the pleuroperitoneal folds (PPFs), transient embryonic structures that give rise to the connective tissue, are the source of HGF critical for diaphragm muscularization and phrenic nerve primary branching. HGF not only is required for recruitment of muscle progenitors to the diaphragm, but is continuously required for maintenance and motility of the pool of progenitors to enable full muscularization. Thus, the connective tissue fibroblasts and HGF coordinately regulate diaphragm muscularization and innervation. Defects in PPF-derived HGF result in muscleless regions that are susceptible to CDH.

Summary Statement

Fibroblast-derived HGF signals to Met+ muscle progenitors and nerve to control the expansion of diaphragm muscle and primary branching of phrenic nerve axons - structures critical for breathing in mammals.
Intravital imaging of cerebral microinfarct reveals an astrocyte reaction led to glial scar
Authors: Lee Jingu , Kim Joon-Goon , Hong Sujung , Kim Young Seo , Ahn Soyeon , Kim Ryul , Chun Heejung , Park Ki Duk , Jeong Yong , Kim Dong-Eog , Lee C. Justin , Ku Taeyun , Kim Pilhan
Published: October 2021

Cerebral microinfarct increases the risk of dementia. But how microscopic cerebrovascular disruption affects the brain tissue in cellular-level are mostly unknown. Herein, with a longitudinal intravital imaging, we serially visualized in vivo dynamic cellular-level changes in astrocyte, pericyte and neuron as well as microvascular integrity after the induction of cerebral microinfarction for 1 month in mice. At day 2-3, it revealed a localized edema with acute astrocyte loss, neuronal death, impaired pericyte-vessel coverage and extravascular leakage indicating blood-brain barrier (BBB) dysfunction. At day 5, edema disappeared with recovery of pericyte-vessel coverage and BBB integrity. But brain tissue continued to shrink with persisted loss of astrocyte and neuron in microinfarct until 30 days, resulting in a collagen-rich fibrous scar surrounding the microinfarct. Notably, reactive astrocytes appeared at the peri-infarct area early at day 2 and thereafter accumulated in the peri-infarct. Oral administration of a reversible monoamine oxidase B inhibitor significantly decreased the astrocyte reactivity and fibrous scar formation. Our result suggests that astrocyte reactivity may be a key target to alleviate the impact of microinfarction.
DOMINANT AWN INHIBITORencodes the ALOG protein originating from gene duplication and inhibits awn elongation by suppressing cell proliferation and elongation in sorghum
Authors: Takanashi Hideki , Kajiya-Kanegae Hiromi , Nishimura Asuka , Yamada Junko , Ishimori Motoyuki , Kobayashi Masaaki , Yano Kentaro , Iwata Hiroyoshi , Tsutsumi Nobuhiro , Sakamoto Wataru
Published: October 2021

The awn, a needle-like structure extending from the tip of the lemma in grass species, plays a role in environmental adaptation and fitness. In some crops, awns appear to have been eliminated during domestication. Although numerous genes involved in awn development have been identified, several dominant genes that eliminate awns are also known to exist. For example, in sorghum (Sorghum bicolor), the dominant awn inhibiting gene has been known since 1921; however, its molecular features remain uncharacterized. In this study, we conducted quantitative trait locus analysis and a genome-wide association study of awn-related traits in sorghum and identifiedDOMINANT AWN INHIBITOR(DAI), which encodes the ALOG family protein on chromosome 3.DAIappeared to be present in most awnless sorghum cultivars, likely because of its effectiveness. Detailed analysis of the ALOG protein family in cereals revealed thatDAIoriginated from duplication of its twin paralog (DAIori) on chromosome 10. Observations of immature awns in near-isogenic lines revealed that DAI inhibits awn elongation by suppressing both cell proliferation and elongation. We also found that onlyDAIgained a novel function to inhibit awn elongation through an awn-specific expression pattern distinct from that ofDAIori. Interestingly, heterologous expression ofDAIwith its own promoter in rice inhibited awn elongation in the awned cultivar Kasalath. We found thatDAIoriginated from gene duplication, providing an interesting example of gain-of-function that occurs only in sorghum but shares its functionality with rice and sorghum.
SARS-CoV-2 Beta variant infection elicits potent lineage-specific and cross-reactive antibodies
Authors: Reincke S Momsen , Yuan Meng , Kornau Hans-Christian , Corman Victor M , van Hoof Scott , Sánchez-Sendin Elisa , Ramberger Melanie , Yu Wenli , Hua Yuanzi , Tien Henry , Schmidt Marie Luisa , Schwarz Tatjana , Jeworowski Lara Maria , Brandl Sarah E , Rasmussen Helle Foverskov , Homeyer Marie A , Stöffler Laura , Barner Martin , Kunkel Désirée , Huo Shufan , Horler Johannes , von Wardenburg Niels , Kroidl Inge , Eser Tabea M , Wieser Andreas , Geldmacher Christof , Hoelscher Michael , Gänzer Hannes , Weiss Günter , Schmitz Dietmar , Drosten Christian , Prüss Harald , Wilson Ian A. , Kreye Jakob
Published: October 2021

SARS-CoV-2 Beta variant of concern (VOC) resists neutralization by major classes of antibodies from non-VOC COVID-19 patients and vaccinated individuals. Here, serum of Beta variant infected patients revealed reduced cross-neutralization of non-VOC virus. From these patients, we isolated Beta-specific and cross-reactive receptor-binding domain (RBD) antibodies. The Beta-specificity results from recruitment of novel VOC-specific clonotypes and accommodation of VOC-defining amino acids into a major non-VOC antibody class that is normally sensitive to these mutations. The Beta-elicited cross-reactive antibodies share genetic and structural features with non-VOC-elicited antibodies, including a public VH1-58 clonotype targeting the RBD ridge independent of VOC mutations. These findings advance our understanding of the antibody response to SARS-CoV-2 shaped by antigenic drift with implications for design of next-generation vaccines and therapeutics.

One sentence summary

SARS-CoV-2 Beta variant elicits lineage-specific antibodies and antibodies with neutralizing breadth against wild-type virus and VOCs.
Juvenile hormones direct primordial germ cell migration to the embryonic gonad
Authors: Lacy J Barton , Justina Sanny , Emily P Dawson , Marcela Nouzova , Fernando Gabriel Noriega , Matthias Stadtfeld , Ruth Lehmann
Published: October 2021

Germ cells are essential to sexual reproduction. Across the animal kingdom, extracellular signaling isoprenoids, such as retinoic acids (RAs) in vertebrates and juvenile hormones (JHs) in invertebrates, facilitate multiple processes in the germline lifecycle. Here we investigated the role of these potent signaling molecules in embryonic germ cell migration, using JHs inDrosophila melanogasteras a model system. In contrast to their established endocrine roles during larval and adult germline development, we found that JH signaling acts locally during embryonic development. Using anin vivobiosensor, we found JH signaling is first active near primordial germ cells (PGCs) as they migrate to the developing somatic gonad. Throughin vivoandin vitroassays, we found that JHs are both necessary and sufficient for PGC migration. Analysis into the mechanisms of this newly uncovered paracrine JH function revealed that PGC migration was compromised when JHs were reduced or increased, suggesting that specific titers or spatiotemporal JH dynamics are required for robust PGC colonization to the gonad. Compromised PGC migration can impair fertility and cause germ cell tumors in many species, including humans. In mammals, retinoids, a JH-related family of signaling isoprenoids, has many roles in development and reproduction. We found that retinoic acid, like JH, was sufficient to impact PGC migrationin vitro. Together, our study reveals a previously unanticipated role of isoprenoids as local effectors of pre-gonadal PGC development and suggests a broadly shared mechanism in PGC migration.

Highlights

Juvenile hormones (JH) are necessary & sufficient for Primordial Germ Cell (PGC) migration.

JH signaling acts directly in and around migrating PGCs prior to its endocrine function.

Compensatory feedback sensitive to JH receptor function maintains JH homeostasis.

JH-like retinoic acids may have similar roles during mammalian germ cell migration.
Hypoxia-directed tumor targeting of CRISPR/Cas9 and HSV-TK suicide gene therapy using lipid nanoparticles
Authors: Davis Alicia , Morris Kevin V. , Shevchenko Galina
Published: October 2021

Hypoxia is a characteristic feature of solid tumors that contributes to tumor aggressiveness and is associated with resistance to cancer therapy. The hypoxia inducible factor-1 (HIF-1) transcription factor complex mediates hypoxia-specific gene expression by binding to hypoxia responsive element (HRE) sequences within the promoter of target genes. HRE driven expression of therapeutic cargo has been widely explored as a strategy to achieve cancer-specific gene expression. By utilizing this system, we achieve hypoxia-specific expression of two therapeutically relevant cargo elements: the Herpes Simplex Virus thymidine kinase (HSV-tk) suicide gene and the CRISPR/Cas9 nuclease. Using an expression vector containing five copies of the HRE derived from the vascular endothelial growth factor gene, we are able to show high transgene expression in cells in a hypoxic environment, similar to levels achieved using the CMV and CBh promoters. Furthermore, we are able to deliver our therapeutic cargo to tumor cells with high efficiency using plasmid packaged lipid nanoparticles (LNPs) to achieve specific killing of tumor cells in hypoxic conditions, while maintaining tight regulation with no significant changes to cell viability in normoxia.
Comprehensive simulation and interpretation of single nucleotide substitutions in GJB2 reveals the genetic and phenotypic landscape of GJB2-related hearing loss
Authors: Xiang Jiale , Sun Xiangzhong , Song Nana , Chen Lisha , Ramaswamy Sathishkumar , Tayoun Ahmad Abou , Peng Zhiyu
Published: October 2021

Genetic variants in the GJB2 gene are the most frequent causes of congenital and childhood hearing loss worldwide. In addition to nonsyndromic hearing loss, GJB2 pathogenic variants are also correlated with syndromic phenotypes, showing high genetic and phenotypic heterogeneity. To comprehensively delineate the genetic and phenotypic landscape of GJB2 variants, we interpreted and manually curated all the 2043 possible single-nucleotide substitution (SNS) coding variants in this gene following the hearing loss-specific ACMG/AMP guidelines. As a result, 61 (3.0%), 188 (9.2%), 1487 (72.8%), 301 (14.7%) and 6 (0.3%) variants were classified as pathogenic, likely pathogenic, variant of uncertain significance, likely benign and benign, respectively. Interestingly, 54% (84/156) of pathogenic/likely pathogenic missense variants were not recorded in ClinVar. Further analysis showed that the second transmembrane domain (TM2) and the 310 helix are highly enriched for pathogenic missense variants. The N-terminal tail and the extracellular loop (E1) showed a high density of variants that are associated with syndromic or dominant nonsyndromic hearing loss. On the other hand, the intracellular loops (CL and CT) were extremely tolerant to variation. Based on this new information, we propose refinements of the guidelines for variant interpretation in GJB2. In summary, our study interpreted all possible SNS variants in the coding region of the GJB2 gene, characterized novel clinically significant (N = 249) and benign or likely benign (N = 307) in this gene, and revealed significant genotype-phenotype correlations at this common hearing loss locus. The interpretation of GJB2 SNS variants in the coding region provides a prototype for genes with similarly high genetic and phenotypic heterogeneity.
Astrocytic extracellular vesicles modulate neuronal calcium homeostasis via transglutaminase-2
Authors: Tonoli Elisa , Verduci Ivan , Prada Ilaria , Gabrielli Martina , Forcaia Greta , Coveney Clare , Savoca Maria Pia , Boocock David J. , Sancini Giulio , Mazzanti Michele , Verderio Claudia , Verderio Elisabetta A.M.
Published: October 2021

We have uncovered a novel role for astrocytes-derived extracellular vesicles (EVs) in controlling intraneuronal Ca2+concentration ([Ca2+]i) and identified transglutaminase-2 (TG2) as a surface-cargo of astrocytes-derived EVs. Incubation of hippocampal neurons with primed astrocyte-derived EVs have led to an increase in [Ca2+]i, unlike EVs from TG2-knockout astrocytes. Exposure of neurons or brain slices to extracellular TG2 promoted a [Ca2+]irise, which was reversible upon TG2 removal and was dependent on Ca2+influx through the plasma membrane. Patch-clamp and calcium imaging recordings revealed TG2-dependent neuronal membrane depolarisation and activation of inward currents, due to the opening of L-type-VOCCs and to Na+/Ca2+-exchanger (NCX) operation in the reverse mode, as indicated by VOCCs/NCX pharmacological inhibitors. A subunit of Na+/K+-ATPase was selected by comparative proteomics and identified as being functionally inhibited by extracellular TG2, implicating Na+/K+-ATPase inhibition in NCX reverse mode-switching leading to Ca2+influx and higher basal [Ca2+]i. These data suggest that reactive astrocytes control intraneuronal [Ca2+]ithrough release of EVs with TG2 as responsible cargo, which could have a significant impact on synaptic activity in brain inflammation.
Plant-insect chemical communication in ecological communities: an information theory perspective
Authors: Zu Pengjuan , García-García Reinaldo , Schuman Meredith , Saavedra Serguei , Melián Carlos J.
Published: October 2021

Cross-species communication, where signals are sent by one species and perceived by others, is one of the most intriguing types of communication that functionally links different species to form complex ecological networks. Yet, global changes and human activities can affect communication by increasing the fluctuations of species composition and phenology, altering signal profiles and intensity, and introducing noises. So far, most studies on cross-species communication have focused on a few specific species isolated from ecological communities. Scaling up investigations of cross-species communication to the community level is currently hampered by a lack of conceptual and practical methodologies. Here, we propose an interdisciplinary framework based on information theory to investigate mechanisms shaping cross-species communication at the community level. We use plants and insects, the cornerstones of most ecosystems, as a showcase; and focus on chemical communication as the key communication channel. We first introduce some basic concepts of information theory, then we illustrate information patterns in plant-insect chemical communication, followed by a further exploration of how to integrate information theory into ecological and evolutionary processes to form testable mechanistic hypotheses. We conclude by highlighting the importance of community-level information as a vehicle to better understand the maintenance of ecological systems, especially when facing rapid global changes.
Enteric nervous system modulation of luminal pH modifies the microbial environment to promote intestinal health
Authors: Hamilton M. Kristina , Wall Elena S. , Guillemin Karen , Eisen Judith S.
Published: October 2021

The enteric nervous system (ENS) controls many aspects of intestinal homeostasis, including parameters that shape the habitat of microbial residents. Previously we showed that zebrafish lacking an ENS, due to deficiency of the sox10 gene, develop intestinal inflammation and bacterial dysbiosis, with an expansion of proinflammatory Vibrio strains. To understand the primary defects resulting in dysbiosis in sox10 mutants, we investigated how the ENS shapes the intestinal environment in the absence of microbiota and associated inflammatory responses. We found that intestinal transit, intestinal permeability, and luminal pH regulation are all aberrant in sox10 mutants, independent of microbially induced inflammation. Treatment with the proton pump inhibitor, omeprazole, corrected the more acidic luminal pH of sox10 mutants to wild type levels. Omeprazole treatment also prevented overabundance of Vibrio and ameliorated inflammation in sox10 mutant intestines. Treatment with the carbonic anhydrase inhibitor, acetazolamide, caused wild type luminal pH to become more acidic, and increased both Vibrio abundance and intestinal inflammation. We conclude that a primary function of the ENS is to regulate luminal pH, which plays a critical role in shaping the resident microbial community and regulating intestinal inflammation.

Author Summary

The intestinal microbiota is an important determinant of health and disease and is shaped by the environment of the gut lumen. The nervous system of the intestine, the enteric nervous system (ENS), helps maintain many aspects of intestinal health including a healthy microbiota. We used zebrafish with a genetic mutation that impedes ENS formation to investigate how the ENS prevents pathogenic shifts in the microbiota. We found that mutants lacking an ENS have a lower luminal pH, higher load of pathogenic bacteria, and intestinal inflammation. We showed that correcting the low pH, using the commonly prescribed pharmacological agent omeprazole, restored the microbiota and prevented intestinal inflammation. Conversely, we found that lowering the luminal pH of wild type animals, using the drug acetazolamide, caused expansion of pathogenic bacteria and increased intestinal inflammation. From these experiments, we conclude that a primary function of the ENS is to maintain normal luminal pH, thereby constraining intestinal microbiota community composition and promoting intestinal health.
Transition of Vibrio cholerae through a natural host induces resistance to environmental changes
Authors: Depelteau Jamie S. , Limpens Ronald , Nag Dhrubajyoti , Koch Bjørn E. V. , Withey Jeffrey H. , Meijer Annemarie H. , Briegel Ariane
Published: October 2021

The pandemic-related strains of Vibrio cholerae are known to cause diarrheal disease in animal hosts. These bacteria must overcome rapid changes in their environment, such as the transition from fresh water to the gastrointestinal system of their host. To study the morphological adjustments during environmental transitions, we used zebrafish as a natural host. Using a combination of fluorescent light microscopy, cryogenic electron tomography and serial block face scanning electron microscopy, we studied the structural changes that occur during the infection cycle. We show that the transition from an artificial nutrient-rich environment to a nutrient-poor environment has a dramatic impact on the cell shape, most notably membrane dehiscence. In contrast, excreted bacteria from the host retain a uniform distance between the membranes as well as their vibrioid shape. Inside the intestine, V. cholerae cells predominantly colonized the anterior to mid-gut, forming micro-colonies associated with the microvilli as well as within the lumen. The cells retained their vibrioid shape but changed their cell-length depending on their localization. Our results demonstrate dynamic changes in morphological characteristics of V. cholerae during the transition between the different environments, and we propose that these structural changes are critical for the pathogen’s ability to colonize host tissues.
Ad26.COV2.S Prevents SARS-CoV-2 Induced Pathways of Inflammation and Thrombosis in Hamsters
Authors: Aid Malika , Vidal Samuel J. , Piedra-Mora Cesar , Ducat Sarah , Chan Chi N. , Bondoc Stephen , Colarusso Alessandro , Starke Carly E. , Nekorchuk Michael , Busman-Sahay Kathleen , Estes Jacob D. , Martinot Amanda J. , Barouch Dan H.
Published: October 2021

Syrian golden hamsters exhibit features of severe disease after SARS-CoV-2 challenge and are therefore useful models of COVID-19 pathogenesis and prevention with vaccines. Recent studies have shown that SARS-CoV-2 infection stimulates type I interferon, myeloid, and inflammatory signatures similar to human disease, and that weight loss can be prevented with vaccines. However, the impact of vaccination on transcriptional programs associated with COVID-19 pathogenesis and protective adaptive immune responses is unknown. Here we show that SARS-CoV-2 challenge in hamsters stimulates antiviral, myeloid, and inflammatory programs as well as signatures of complement and thrombosis associated with human COVID-19. Notably, single dose immunization with Ad26.COV2.S, an adenovirus serotype 26 vector (Ad26)-based vaccine expressing a stabilized SARS-CoV-2 spike protein, prevents the upregulation of these pathways such that the gene expression profiles of vaccinated hamsters are comparable to uninfected animals. Finally, we show that Ad26.COV2.S vaccination induces T and B cell signatures that correlate with binding and neutralizing antibody responses. These data provide further insights into the mechanisms of Ad26.COV2.S based protection against severe COVID-19 in hamsters.

Author Summary

In this study, we show that vaccination with Ad26.COV2.S protected SARS-CoV-2 challenged hamsters from developing severe COVID-19 disease by attenuating excessive proinflammatory responses, such as IL-6 and IL-1, macrophages and neutrophils signaling. Ad26 vaccination also prevented the upregulation of pathways associated with thrombosis such coagulation and clotting cascades associated with infection, and the transcriptomic profiles of vaccinated animals were largely comparable to control uninfected hamsters. In contrast, SARS-CoV-2 challenged unvaccinated hamsters showed significant increase of these proinflammatory and prothrombotic pathways and significant weight loss compared to vaccinated hamsters.
Female fruit flies cannot protect stored sperm from high temperature damage
Authors: Walsh Benjamin S. , Parratt Steven R. , Snook Rhonda R. , Bretman Amanda , Atkinson David , Price Tom A. R.
Published: October 2021

Recently, it has been demonstrated that heat-induced male sterility is likely to shape population persistence as climate change progresses. However, an under-explored possibility is that females may be able to successfully store and preserve sperm at temperatures that sterilise males, which could ameliorate the impact of male infertility on populations. Here, we test whether females from two fruit fly species can protect stored sperm from a high temperature stress. We find that sperm carried by female Drosophila virilis are almost completely sterilised by high temperatures, whereas sperm carried by female Zaprionus indianus show only slightly reduced fertility. Heat-shocked D. virilis females can recover fertility when allowed to remate, suggesting that the delivered heat-shock is destroying stored sperm and not directly damaging females in this species. The temperatures required to reduce fertility of mated females are substantially lower than the temperatures required to destroy mature sperm in males, suggesting that females are worse than males at protecting mature sperm. This suggests that female sperm storage is unlikely to ameliorate the impacts of high temperature fertility losses in males, and instead exacerbates fertility costs of high temperatures, representing an important determinant of population persistence during climate change.
Efficient single copy integration via homology-directed repair (scHDR) by 5’modification of large DNA donor fragments in mice
Authors: Medert Rebekka , Thumberger Thomas , Tavhelidse Tinatini , Hub Tobias , Kellner Tanja , Oguchi Yoko , Dlugosz Sascha , Zimmermann Frank , Wittbrodt Joachim , Freichel Marc
Published: October 2021

CRISPR/Cas approaches have largely replaced conventional gene targeting strategies. However, homology-directed repair (HDR) in the mouse genome is not very efficient, and precisely inserting longer sequences using HDR remains challenging, given that donor constructs preferentially integrate as concatemers. Here, we show that injecting 5’biotinylated donor DNA in mouse embryos at the two-cell stage leads to efficient single-copy HDR (scHDR) alleles. Our dedicated genotyping strategy showed that these alleles occurred with a frequency of 19%, 20%, and 26%, respectively, in three independent gene loci, indicating that scHDR is dramatically boosted by 5’biotinylation. Thus, we suggest that a combination of a 5’biotinylated donor and diligent analysis of concatemer integration are prerequisites for efficiently and reliably generating conditional alleles or other large fragment knock-ins into the mouse genome.
Standardised method for cardiomyocyte isolation and purification from individual murine neonatal, infant, and adult hearts
Authors: Nicks Amy M , Holman Sara R , Chan Andrea Y , Tsang Michael , Young Paul E , Humphreys David T , Naqvi Nawazish , Husain Ahsan , Li Ming , Smith Nicola J , Iismaa Siiri E , Graham Robert M
Published: October 2021

Primary cardiomyocytes are invaluable for understanding postnatal heart development. However, a universal method to obtain freshly purified cardiomyocytes without using different agedependent isolation procedures and cell culture, is lacking. Here, we report the development of a standardised method that allows rapid isolation and purification of high-quality cardiomyocytes from individual neonatal through to adult C57BL/6J murine hearts. Langendorff retrograde perfusion, which is currently limited to adult hearts, was adapted for use in neonatal and infant hearts by developing an easier in situ aortic cannulation technique. Tissue digestion conditions were optimised to achieve efficient digestion of hearts of all ages in a comparable timeframe (<14 min). This resulted in a high yield (1.56-2.2×106 cells/heart) and viability (~70-100%) of cardiomyocytes post-isolation. An immunomagnetic cell separation step was then applied to yield highly purified cardiomyocytes (~95%) as confirmed by immunocytochemistry, flow cytometry, and qRT-PCR. For cell-type specific studies, cardiomyocyte DNA, RNA, and protein could be extracted in sufficient yields to conduct molecular experiments. We generated transcriptomic datasets for neonatal cardiomyocytes from individual hearts, for the first time, which revealed nine sex-specific genes (FDR<0.05) encoded on the sex chromosomes. Finally, we also developed an in situ fixation protocol that preserved the native cytoarchitecture of cardiomyocytes (~94% rodshaped post-isolation), and used it to evaluate cell morphology during cardiomyocyte maturation, as well as capture spindle-shaped neonatal cells undergoing cytokinesis. Together, these procedures allow molecular and morphological profiling of high-quality cardiomyocytes from individual hearts of any postnatal age.
Structural insights into the BRAF monomer-to-dimer transition mediated by RAS binding
Authors: Martinez Fiesco Juliana A. , Durrant David E. , Morrison Deborah K. , Zhang Ping
Published: October 2021

RAF kinases are essential effectors of RAS, but how RAS binding initiates the conformational changes needed for autoinhibited RAF monomers to form active dimers has remained unclear. Here, we present cryo-electron microscopy structures of full-length BRAF complexes derived from mammalian cells: autoinhibited, monomeric BRAF:14-3-32:MEK and BRAF:14-3-32 complexes, and an inhibitor-bound, dimeric BRAF2:14-3-32 complex, at 3.7, 4.1, and 3.9 Å resolution, respectively. In both autoinhibited, monomeric structures, the RAS binding domain (RBD) of BRAF is resolved, revealing that the RBD forms an extensive contact interface with the 14-3-3 protomer bound to the BRAF C-terminalsite and that key basic residues required for RBD-RAS binding are exposed. Moreover, through structure-guided mutational studies, our findings indicate that RAS-RAF binding is a dynamic process and that RBD residues at the center of the RBD:14-3-3 interface have a dual function, first contributing to RAF autoinhibition and then to the full spectrum of RAS-RBD interactions.
The parallel-stranded d(CGA) duplex is a highly predictable structural motif with two conformationally distinct strands
Authors: Luteran Emily M. , Paukstelis Paul J.
Published: October 2021

DNA can adopt non-canonical structures that have important biological functions while also providing structural diversity for nanotechnology applications. Here, we describe the crystal structures of two oligonucleotides composed of d(CGA) triplet repeats in the parallel-stranded duplex form. The structure determination of four unique d(CGA)-based parallel-stranded duplexes across two crystal structures has allowed us to characterize and establish structural parameters of d(CGA) triplets in the parallel-stranded duplex form. Our results show that d(CGA) units are highly uniform, but that each strand in the duplex is structurally unique and has a distinct role in accommodating structural asymmetries induced by the C-CH+ base pair.
In situ Nuclear Matrix preparation in Drosophila melanogaster and its use in studying the components of nuclear architecture
Authors: Pathak Rashmi U , Sureka Rahul , Bihani Ashish , Varma Parul , Mishra Rakesh K
Published: October 2021

The study of Nuclear Matrix (NuMat) over the last 40 years has been limited to either isolated nuclei from tissues or cells grown in culture. Here, we provide a protocol for NuMat preparation in intact Drosophila melanogaster embryos and its use in dissecting the components of nuclear architecture. The protocol does not require isolation of nuclei and therefore maintains the three-dimensional milieu of an intact embryo, which is biologically more relevant compared to cells in culture. One of the advantages of this protocol is that only a small number of embryos are required. The protocol can be extended to larval tissues like salivary glands and imaginal discs with little modification. Taken together, it becomes possible to carry out such studies in parallel to genetic experiments using mutant and transgenic flies. This protocol, therefore, opens the powerful field of fly genetics to cell biology in the study of nuclear architecture.

Nuclear Matrix is a biochemically defined entity and a basic component of the nuclear architecture. Here we present a protocol to isolate and visualize Nuclear Matrix in situ in the intact embryos and tissues of Drosophila melanogaster and its potential applications.
Simulations of flow over a bio-inspired undulated cylinder with dynamically morphing topography
Authors: Yuasa Mikihisa , Lyons Kathleen , Franck Jennifer A.
Published: October 2021

Inspired by the geometric properties of seal whiskers, the addition of spanwise undulations on a cylinder have been shown to lower the mean and oscillatory forces and modify the frequency of flow-induced vibration when compared to smooth cylinders. Previously, computational fluid dynamics (CFD) has been used to characterize the hydrodynamic response with respect to specific geometric features. However simulations are time intensive due to complex three-dimensional meshing and computation time, limiting the number of geometric perturbations explored. A method is proposed in which the specific geometric features of this complex topography can be modified during a simulation thereby decreasing the time per geometric modification and removing the need for manual meshing of the complex structure. The surface of the seal whisker inspired geometry is parameterized into seven defining parameters, each of which is directly controlled in order to morph the surface into any realization within the defined parameter space. Once validated, the algorithm is used to explore the undulation amplitudes in the chord and thickness directions, by varying each independently from 0 to 0.3 thicknesses in increments of 0.05 at a Reynolds number of 500. The force and frequency response are examined for this matrix of geometric parameters, yielding detailed force trends not previously investigated. The impact of the bio-inspired morphing algorithm will allow for further optimization and development of force-mitigating underwater devices and other engineering applications in need of vibration suppression, frequency tuning, or force reduction.
Evaluating live microbiota biobanking using an ex vivo microbiome assay and metaproteomics
Authors: Zhang Xu , Walker Krystal , Mayne Janice , Li Leyuan , Ning Zhibin , Stintzi Alain , Figeys Daniel
Published: October 2021

Biobanking of live microbiota is becoming indispensable for mechanistic and clinical investigations of drug-microbiome interactions and fecal microbiota transplantation. However, there is a lack of methods to rapidly and systematically evaluate whether the biobanked microbiota maintains their cultivability and functional activity. In this study, we use a rapid ex vivo microbiome assay and metaproteomics to evaluate the cultivability and the functional responses of biobanked microbiota to treatment with a prebiotic (fructo-oligosaccharide, FOS). Our results indicate that the microbiota cultivability and their functional responses to FOS treatment were well maintained by freezing in a deoxygenated glycerol buffer at -80°C for 12 months. We also demonstrate that the fecal microbiota is functionally stable for 48 hours on ice in a deoxygenated glycerol buffer, allowing off-site fecal sample collection and shipping to laboratory for live microbiota biobanking. This study provides a method for rapid evaluation of the cultivability of biobanked live microbiota. Our results show minimal detrimental influences of long-term freezing in deoxygenated glycerol buffer on the cultivability of fecal microbiota.
Fast enzymatic HCO3- dehydration supports photosynthetic water oxidation in Photosystem II from pea
Authors: Shitov Alexandr V. , Terentyev Vasily V. , Govindjee Govindjee
Published: October 2021

Carbonic anhydrase (CA) activity, associated with Photosystem II (PSII) from Pisum sativum, has been shown to enhance water oxidation. But, the nature of the CA activity, its origin and role in photochemistry has been under debate, since the rates of CA reactions, measured earlier, were less than the rates of photochemical reactions. Here, we demonstrate high CA activity in PSII from Pisum sativum, measured by HCO3- dehydration at pH 6.5 (i.e. under optimal condition for PSII photochemistry), with kinetic parameters Km of 2.7 mM; Vmax of 2.74·10-2 mM·sec-1; kcat of 1.16·103 sec-1 and kcat/Km of 4.1·105 M-1 sec-1, showing the enzymatic nature of this activity, which kcat exceeds by ∼13 times the rate of PSII, as measured by O2 evolution. The similar dependence of HCO3- dehydration, of the maximal quantum yield of photochemical reactions and of O2 evolution on the ratio of chlorophyll/photochemical reaction center II demonstrate the interconnection of these processes on the electron donor side of PSII. Since the removal of protons is critical for fast water oxidation, and since HCO3- dehydration consumes a proton, we suggest that CA activity, catalyzing very fast removal of protons, supports efficient water oxidation in PSII and, thus, photosynthesis in general.