Biology subjects

Clark, J.

Publications and source records attributed to Clark, J..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Estimating the seroprevalence of tuberculosis (Mycobacterium bovis) infection in a wild deer population in southwest England

Bovine tuberculosis (bTB) is a major disease of cattle that is subject to an eradication strategy in England. To inform control policies and manage the epidemic, all potential sources of infection for cattle must be identified and understood. The causative agent of bTB, Mycobacterium bovis, has a wide host range including several deer species. While transmission between cattle and deer has been implicated in some localised endemic regions, the role of deer in the epidemiology of bTB in England is poorly understood. This paper presents the results of a serological survey to estimate the prevalence of M. bovis in a large wild deer population in the High bTB Risk Area of southwest England. Blood samples were collected post-mortem over a 12-month period from wild deer during annual deer management controls in the Exmoor area and tested for M. bovis serum antibodies. Overall, 432 samples were collected and 69 (16.0%) were seropositive. The true seroprevalence in our sample was estimated to be 29.2% (95% CrI 21.1-38.6%), using Bayesian Latent Class Analysis to account for imperfect diagnostic test accuracy. Prevalence did not appear to differ between sexes nor between species, although the sample was predominantly red deer. The lowest prevalence was observed in animals aged under 1-year. Whilst these results provide valuable insights into M. bovis seroprevalence in this wild deer population, they should be interpreted alongside other relevant information such as ecology, species-specific epidemiology and disease pathology, deer density and cattle management to inform potential transmission risks between cattle and wild deer.

zoology↗

bioRxiv2024

Modulation of blood-tumor barrier transcriptional programs improves intra-tumoral drug delivery and potentiates chemotherapy in GBM

Glioblastoma (GBM) is the most common malignant primary brain tumor. GBM has an extremely poor prognosis and new treatments are badly needed. Efficient drug delivery to GBM is a major obstacle as the blood-brain barrier (BBB) prevents passage of the majority of cancer drugs into the brain. It is also recognized that the blood-brain tumor barrier (BTB) in the growing tumor represents a challenge. The BTB is heterogeneous and poorly characterized, but similar to the BBB it can prevent therapeutics from reaching effective intra-tumoral doses, dramatically hindering their potential. Here, we identified a 12-gene signature associated with the BTB, with functions related to vasculature development, morphogenesis and cell migration. We identified CDH5 as a core molecule in this set and confirmed its over-expression in GBM vasculature using spatial transcriptomics of GBM patient specimens. We found that the indirubin-derivative, 6-bromoindirubin acetoxime (BIA), could downregulate CDH5 and other BTB signature genes, causing endothelial barrier disruption in endothelial monolayers and BBB 3D spheroids in vitro. Treatment of tumor-bearing mice with BIA enabled increased intra-tumoral accumulation of the BBB non-penetrant chemotherapeutic drug cisplatin and potentiated cisplatin-mediated DNA damage by targeting DNA repair pathways. Finally, using an injectable BIA nanoparticle formulation, PPRX-1701, we significantly improved the efficacy of cisplatin in patient-derived GBM xenografts and prolonged their survival. Overall, our work reveals potential targets at the BTB for improved chemotherapy delivery and the bifunctional properties of BIA as a BTB modulator and potentiator of chemotherapy, supporting its further development.

cancer biology↗

bioRxiv2024

Dissecting human monoclonal antibody responses from mRNA- and protein-based XBB.1.5 COVID-19 monovalent vaccines

The emergence of highly contagious and immune-evasive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has required reformulation of coronavirus disease 2019 (COVID-19) vaccines to target those new variants specifically. While previous infections and booster vaccinations can enhance variant neutralization, it is unclear whether the monovalent version, administered using either mRNA or protein-based vaccine platforms, can elicit de novo B-cell responses specific for Omicron XBB.1.5 variants. Here, we dissected the genetic antibody repertoire of 603 individual plasmablasts derived from five individuals who received a monovalent XBB.1.5 vaccination either with mRNA (Moderna or Pfizer/BioNtech) or adjuvanted protein (Novavax). From these sequences, we expressed 100 human monoclonal antibodies and determined binding, affinity and protective potential against several SARS-CoV-2 variants, including JN.1. We then select two vaccine-induced XBB.1.5 mAbs, M2 and M39. M2 mAb was a de novo, antibody, i.e., specific for XBB.1.5 but not ancestral SARS-CoV-2. M39 bound and neutralized both XBB.1.5 and JN.1 strains. Our high-resolution cryo-electron microscopy (EM) structures of M2 and M39 in complex with the XBB.1.5 spike glycoprotein defined the epitopes engaged and revealed the molecular determinants for the mAbs specificity. These data show, at the molecular level, that monovalent, variant-specific vaccines can elicit functional antibodies, and shed light on potential functional and genetic differences of mAbs induced by vaccinations with different vaccine platforms. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=43 SRC="FIGDIR/small/602781v1_ufig1.gif" ALT="Figure 1000"> View larger version (16K): org.highwire.dtl.DTLVardef@7c4708org.highwire.dtl.DTLVardef@11b66acorg.highwire.dtl.DTLVardef@1f1cec7org.highwire.dtl.DTLVardef@3e72fe_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2024

Perfusion-Independent Tissue Hypoxia in Cardiac Hypertrophy in Mice Measured by 64Cu-CTS PET Imaging

BackgroundHypoxia is central to many cardiac pathologies, but clinically its presence can only be inferred by indirect biomarkers including hypoperfusion and energetic compromise. Imaging hypoxia directly could offer new opportunities for the diagnosis and sub-stratification of cardiovascular diseases. ObjectivesTo determine whether [64Cu]CuCTS Positron Emission Tomography (PET) can identify hypoxia in a murine model of cardiac hypertrophy. MethodsMale C57BL/6 mice underwent abdominal aortic constriction (AAC) to induce cardiac hypertrophy, quantified by echocardiography over 4 weeks. Hypoxia and perfusion were quantified in vivo using [64Cu]CuCTS and [64Cu]CuGTSM PET, respectively, and radiotracer biodistribution was quantified post-mortem. Cardiac radiotracer retention was correlated with contractile function (measured by echocardiography), cardiac hypertrophy (measured by histology), HIF-1 stabilization and NMR-based metabolomics. The effect of anesthesia on [64Cu]CuCTS uptake was additionally investigated in a parallel cohort of mice injected with radiotracer while conscious. ResultsHearts showed increased LV wall thickness, reduced ejection fraction and fractional shortening following AAC. [64Cu]CuCTS retention was 317% higher in hypertrophic myocardium (p<0.001), despite there being no difference in perfusion measured by 64CuGTSM. Radiotracer retention correlated on an animal-by-animal basis with severity of hypertrophy, contractile dysfunction, HIF1 stabilization and metabolic signatures of hypoxia. [64Cu]CuCTS uptake in hypertrophic hearts was significantly higher when administered to conscious animals. Conclusions[64Cu]CuCTS PET can quantify cardiac hypoxia in hypertrophic myocardium, independent of perfusion, suggesting the hypoxia is caused by increased oxygen diffusion distances at the subcellular level. Alleviation of cardiac workload by anesthesia in preclinical models partially alleviates this effect.

bioengineering↗

bioRxiv2024

TRIP12 governs DNA Polymerase β involvement in DNA damage response and repair

The multitude of DNA lesion types, and the nuclear dynamic context in which they occur, present a challenge for genome integrity maintenance as this requires the engagement of different DNA repair pathways. Specific repair controllers that facilitate DNA repair pathway crosstalk between double strand break (DSB) repair and base excision repair (BER), and regulate BER protein trafficking at lesion sites, have yet to be identified. We find that DNA polymerase {beta} (Pol{beta}), crucial for BER, is ubiquitylated in a BER complex-dependent manner by TRIP12, an E3 ligase that partners with UBR5 and restrains DSB repair signaling. Here we find that, TRIP12, but not UBR5, controls cellular levels and chromatin loading of Pol{beta}. Required for Pol{beta} foci formation, TRIP12 regulates Pol{beta} involvement after DNA damage. Notably, excessive TRIP12-mediated shuttling of Pol{beta} affects DSB formation and radiation sensitivity, underscoring its precedence for BER. We conclude that the herein discovered trafficking function at the nexus of DNA repair signaling pathways, towards Pol{beta}-directed BER, optimizes DNA repair pathway choice at complex lesion sites. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=165 SRC="FIGDIR/small/588474v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@70c5bdorg.highwire.dtl.DTLVardef@1c60dfborg.highwire.dtl.DTLVardef@1c00ef6org.highwire.dtl.DTLVardef@1677f2_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2024

Protective effect and molecular mechanisms of human non-neutralizing cross-reactive spike antibodies elicited by SARS-CoV-2 mRNA vaccination

Neutralizing antibodies correlate with protection against SARS-CoV-2. Recent studies, however, show that binding antibody titers, in the absence of robust neutralizing activity, also correlate with protection from disease progression. Non-neutralizing antibodies cannot directly protect from infection but may recruit effector cells thus contribute to the clearance of infected cells. Also, they often bind conserved epitopes across multiple variants. We characterized 42 human mAbs from COVID-19 vaccinated individuals. Most of these antibodies exhibited no neutralizing activity in vitro but several non-neutralizing antibodies protected against lethal challenge with SARS-CoV-2 in different animal models. A subset of those mAbs showed a clear dependence on Fc-mediated effector functions. We determined the structures of three non-neutralizing antibodies with two targeting the RBD, and one that targeting the SD1 region. Our data confirms the real-world observation in humans that non-neutralizing antibodies to SARS-CoV-2 can be protective.

immunology↗

bioRxiv2023

Exploring the impact of microbial manipulation on the early development of kelp (Saccharina latissima) using an ecological core microbiome framework.

Kelp cultivation is a rapidly expanding economic sector, as kelp are valued for a wide range of commercial products and for restoration of kelp forest ecosystems. Microbes associated with kelp and other macroalgae play a critical role in processes such as nutrient exchange, chemical signaling, and defense against pathogens. Thus, manipulating the microbiome to enhance macroalgal growth and resilience is a promising, but largely untested, tool in sustainable kelp cultivation. The core microbiome hypothesis suggests that bacteria that are consistently found on a host (the core microbes) are likely to have a disproportionate impact on host biology, making them an attractive target for microbiome manipulation. Here, we surveyed wild Saccharina latissima and their surrounding environment to identify core bacterial taxa, compared to cultivated kelp, and experimentally tested the effect of cultured bacterial isolates on kelp development. We find that core bacteria are nearly absent in cultivated juvenile sporophytes in nurseries but eventually colonized after outplanting kelp to ocean farm sites. We find that bacterial inoculants can have both positive and negative effects on kelp development. In line with predictions from the core microbiome hypothesis, we find a positive correlation between the frequency of the bacterial genus in the wild and the bacterial effect on the number of sporophytes in kelp co-culture experiments. IMPORTANCEThe core microbiome hypothesis suggests that symbiotic microorganisms consistently associated with hosts have functional effects on host biology and health. However, there is a lack of evidence to either support or refute this idea. This study surveys the distribution of bacteria on wild and cultivated kelp to identify the core microbiome and tests the ability of bacterial isolates cultured from the surface of wild kelp to influence kelp growth and development in laboratory microbial manipulation experiments. The frequency of bacterial genera on wild kelp was positively correlated with influence on kelp development in laboratory experiments, providing support for the core microbiome hypothesis.

ecology↗

bioRxiv2023

SODA: Software to Support the Curation and Sharing of FAIR Autonomic Nervous System Data

Since 2014, the National Institutes of Health (NIH)s Stimulating Peripheral Activity to Relieve Conditions (SPARC) Program has been supporting research and development of therapeutic devices that modulate electrical activity in the autonomic nervous system (ANS) to improve organ function, also known as bioelectronic medicine. To optimize the reusability of data resulting from ANS-related research, the SPARC Program also supported the development of guidelines for curating and sharing data in line with the FAIR (Findable, Accessible, Interoperable, Reusable) Principles. These guidelines are exhaustive to maximize FAIRness of data but as a result, they are difficult and time-consuming for researchers to implement. To address these challenges, we developed SODA (Software to Organize Data Automatically), an open source and free cross-platform desktop software that guides researchers step-by-step in preparing and sharing their ANS-related data according to the SPARC guidelines. SODA combines intuitive user interfaces with automation to streamline the process and reduce researchers time, effort, and error in making their data FAIR. We provide in this paper an overview of SODA and results of testing its performance. We also provide an overview of the impact of SODA which is, to our knowledge, the first researcher-oriented tool for making data FAIR.

bioinformatics↗

bioRxiv2023

An evolutionary timescale for Bacteria calibrated using the Great Oxidation Event

Most of lifes diversity and history is microbial but it has left a meagre fossil record, greatly hindering understanding of evolution in deep time. However, the co-evolution of life and the Earth system has left signatures of bacterial metabolism in the geochemical record, most conspicuously the Great Oxidation Event (GOE) [~]2.33 billion years ago (Ga, (Poulton et al. 2021)), in which oxygenic photosynthesis and tectonism (Eguchi, Seales, and Dasgupta 2019) transformed Earths biosphere from dominantly anaerobic to aerobic. Here, we combine machine learning and phylogenetic reconciliation to infer ancestral transitions to aerobic lifestyles during bacterial evolution. Linking these transitions to the GOE provides new constraints to infer the timetree of Bacteria. We find that extant bacterial phyla are truly ancient, having radiated in the Archaean and the Proterozoic: the oldest include Bacillota (Firmicutes), which radiated 3.1-3.7 Ga, Cyanobacteria (3.3-3.5 Ga) and Patescibacteria (3-3.5 Ga). We show that most bacterial phyla were ancestrally anaerobic and that most transitions to an aerobic lifestyle post-dated the GOE. Our analyses trace oxygen production and consumption back to Cyanobacteria. From that starting point, horizontal transfer seeded aerobic lifestyles across bacterial diversity over hundreds of millions of years. Our analyses demonstrate that the diversification of aerobes proceeded in two waves corresponding to the GOE and to a second sustained rise in atmospheric O2 at the dawn of the Palezoic (Krause et al. 2022).

evolutionary biology↗

bioRxiv2023

Functional Genomics of Gastrointestinal Escherichia coli Isolated from Patients with Cancer and Diarrhea

We describe the epidemiology and clinical characteristics of 29 patients with cancer and diarrhea in whom Enteroaggregative Escherichia coli (EAEC) was initially identified by GI BioFire panel multiplex. E. coli strains were successfully isolated from fecal cultures in 14 of 29 patients. Six of the 14 strains were identified as EAEC and 8 belonged to other diverse E. coli groups of unknown pathogenesis. We investigated these strains by their adherence to human intestinal organoids, cytotoxic responses, antibiotic resistance profile, full sequencing of their genomes, and annotation of their functional virulome. Interestingly, we discovered novel and enhanced adherence and aggregative patterns for several diarrheagenic pathotypes that were not previously seen when co-cultured with immortalized cell lines. EAEC isolates displayed exceptional adherence and aggregation to human colonoids compared not only to diverse GI E. coli, but also compared to prototype strains of other diarrheagenic E. coli. Some of the diverse E. coli strains that could not be classified as a conventional pathotype also showed an enhanced aggregative and cytotoxic response. Notably, we found a high carriage rate of antibiotic resistance genes in both EAEC strains and diverse GI E. coli isolates and observed a positive correlation between adherence to colonoids and the number of metal acquisition genes carried in both EAEC and the diverse E. coli strains. This work indicates that E. coli from cancer patients constitute strains of remarkable pathotypic and genomic divergence, including strains of unknown disease etiology with unique virulomes. Future studies will allow for the opportunity to re-define E. coli pathotypes with greater diagnostic accuracy and into more clinically relevant groupings.

microbiology↗

bioRxiv2023

Decoding Duchenne muscular dystrophy transcriptome to single nuclei level reveals clinical-genetic correlations

The cellular and molecular consequences of lack of dystrophin in humans are only partially known, which is crucial for the development of new therapies aiming to slow or stop the progression Duchenne and Becker muscular dystrophies. We analyzed muscle biopsies of DMD patients and controls using single nuclei RNA sequencing (snRNAseq) and correlated the results with clinical data. DMD samples displayed an increase in regenerative fibers, satellite cells and fibro-adipogenic progenitor cells (FAPs) and a decrease in slow fibers and smooth muscle cells. Samples from patients with stable mild weakness were characterized by an increase in regenerative fibers, while those from patients with progressive weakness had fewer muscle fibers and increased FAPs. DMD muscle fibers displayed a strong regenerative signature, while DMD FAPs upregulated genes producing extracellular matrix and molecules involved in several signaling pathways. An analysis of intercellular communication profile identified FAPs as a key regulator of cell signaling in DMD samples. We show significant differences in the gene expression profiled of the different cell populations present in DMD muscle samples compared to controls.

bioinformatics↗

bioRxiv2022

A simplified, amplicon-based method for whole genome sequencing of human respiratory syncytial viruses

Human Respiratory Syncytial Virus (RSV) infections pose a significant risk to human health worldwide, especially for young children. Whole genome sequencing (WGS) provides a useful tool for global surveillance to better understand the evolution and epidemiology of RSV and provide essential information that may impact on antibody treatments, antiviral drug sensitivity and vaccine effectiveness. Here we report the development of a rapid and simplified amplicon-based one-step multiplex reverse-transcription polymerase chain reaction (mRT-PCR) for WGS of both human RSV-A and RSV-B viruses. The method requires only two reactions for each sample, which significantly reduces the cost and time compared to other commonly used RSV WGS methods. In silico analysis and laboratory testing revealed that the primers used in the new method covered most of the currently circulating RSV-A and RSV-B. Amplicons generated were suitable for both Illumina and Oxford Nanopore Technologies (ONT) NGS platforms. This new method was tested on 200 clinical samples collected in Australia in 2020 and 2021 with RSV Ct values between 10 and 32. A success rate of 88% with a full coverage for the genome of 99 RSV-A and 77 RSV-B was achieved. This assay is simple to set up, robust, easily scalable in sample preparation and relatively inexpensive, and as such, provides a valuable addition to existing NGS RSV WGS methods.

genomics↗

bioRxiv2022

Immunity to seasonal coronavirus spike proteins does not protect from SARS-CoV-2 challenge in a mouse model but has no detrimental effect on protection mediated by COVID-19 mRNA vaccination

Seasonal coronaviruses have been circulating widely in the human population for many years. With increasing age, humans are more likely to have been exposed to these viruses and to have developed immunity against them. It has been hypothesized that this immunity to seasonal coronaviruses may provide partial protection against infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and it has also been shown that coronavirus disease 2019 (COVID-19) vaccination induces a back-boosting effects against the spike proteins of seasonal betacoronaviruses. In this study, we tested if immunity to the seasonal coronavirus spikes from OC43, HKU1, 229E or NL63 would confer protection against SARS-CoV-2 challenge in a mouse model, and whether pre-existing immunity against these spikes would weaken the protection afforded by mRNA COVID-19 vaccination. We found that mice vaccinated with the seasonal coronavirus spike proteins had no increased protection as compared to the negative controls. While a negligible back-boosting effect against betacoronavirus spike proteins was observed after SARS-CoV-2 infection, there was no negative original antigenic sin-like effect on the immune response and protection induced by SARS-CoV-2 mRNA vaccination in animals with pre-existing immunity to seasonal coronavirus spike proteins. ImportanceThe impact that immunity against seasonal coronaviruses has on both susceptibility to SARS-CoV-2 infection as well as on COVID-19 vaccination is unclear. This study provides insights into both questions in a mouse model of SARS-CoV-2.

microbiology↗

bioRxiv2022

Store-Operated Ca2+ Channels Mediate Microdomain Ca2+ Signals and Amplify Gq-Coupled Ca2+ Elevations in Capillary Pericytes

Pericytes are multifunctional cells of the vasculature that are vital to brain homeostasis, yet many of their fundamental physiological properties remain unexplored. Ca2+ is a ubiquitous second messenger across cell-types, where it mediates diverse functions such as contractility and gene transcription. It is therefore important to understand the cellular mechanisms underlying Ca2+ signalling in capillary pericytes. Here, we performed pharmacological and ion substitution experiments to investigate the mechanisms underlying pericyte Ca2+ signaling in acute cortical brain slices of PDGFR{beta}-GCaMP6f mice. We report that in mid-capillary bed pericytes ([≥] 4th branch order), spontaneous microdomain Ca2+ signals are dependent on extracellular Ca2+, but largely independent of depolarization, L- and T-type voltage-gated calcium channels (VGCCs), and TRPC3/6 channels. In contrast, these microdomain signals were inhibited by multiple Orai channel blockers, including the specific antagonist GSK-7975A. Furthermore, capillary pericytes exhibited classical store operated calcium entry (SOCE) following store depletion that was sensitive to GSK-7975A and required for amplification of intracellular Ca2+ increases evoked by the vasoconstrictor endothelin-1. These results suggest that Orai SOCE mediates microdomain Ca2+ signals at rest and amplifies Gq-GPCR coupled Ca2+ elevations in capillary pericytes. Thus, SOCE is a major regulator of pericyte Ca2+ and a target for manipulating their function in health and disease.

neuroscience↗

bioRxiv2022

Real-time dynamic single-molecule protein sequencing on an integrated semiconductor device

Proteins are the main structural and functional components of cells, and their dynamic regulation and post-translational modifications (PTMs) underlie cellular phenotypes. Next-generation DNA sequencing technologies have revolutionized our understanding of heredity and gene regulation, but the complex and dynamic states of cells are not fully captured by the genome and transcriptome. Sensitive measurements of the proteome are needed to fully understand biological processes and changes to the proteome that occur in disease states. Studies of the proteome would benefit greatly from methods to directly sequence and digitally quantify proteins and detect PTMs with single-molecule sensitivity and precision. However current methods for studying the proteome lag behind DNA sequencing in throughput, sensitivity, and accessibility due to the complexity and dynamic range of the proteome, the chemical properties of proteins, and the inability to amplify proteins. Here, we demonstrate single-molecule protein sequencing on a compact benchtop instrument using a dynamic sequencing by stepwise degradation approach in which single surface-immobilized peptide molecules are probed in real-time by a mixture of dye-labeled N-terminal amino acid recognizers and simultaneously cleaved by aminopeptidases. By measuring fluorescence intensity, lifetime, and binding kinetics of recognizers on an integrated semiconductor chip we are able to annotate amino acids and identify the peptide sequence. We describe the expansion of the number of recognizable amino acids and demonstrate the kinetic principles that allow individual recognizers to identify multiple amino acids in a highly information-rich manner that is sensitive to adjacent residues. Furthermore, we demonstrate that our method is compatible with both synthetic and natural peptides, and capable of detecting single amino acid changes and PTMs. We anticipate that with further development our protein sequencing method will offer a sensitive, scalable, and accessible platform for studies of the proteome.

biophysics↗

bioRxiv2021

Comparative genomics of Acinetobacter baumannii and therapeutic bacteriophages from a patient undergoing phage therapy

In 2016, a 68-year-old patient with a disseminated multi-drug resistant Acinetobacter baumannii infection was treated using lytic bacteriophages in one of the first modern human clinical uses of phage therapy in the United States. Due to the emergency nature of the treatment there was little time to thoroughly characterize the phages used in this intervention or the pathogen itself. Here we report the genomes of the nine phages used for treatment and three strains of A. baumannii isolated prior to and during treatment. The eight phages used in the initial treatment were found to be a group of closely related T4-like myophages; the ninth phage, AbTP3{Phi}1, was found to be an unrelated Fri1-like podophage. Analysis of 19 A. baumannii isolates collected before and during phage treatment showed that resistance to the T4-like phages appeared as early as two days following the start of treatment. Three A. baumannii strains (TP1, TP2 and TP3) collected before and during treatment were sequenced to closure, and all contained a 3.9 Mb chromosome of sequence type 570 with a KL116 capsule locus and identical 8.7 kb plasmids. Phage-insensitive mutants of A. baumannii strain TP1 were generated in vitro and the majority of identified mutations were located in the bacterial capsule locus. The presence of the same mutation in both the in vitro mutants and in phage-insensitive isolates TP2 and TP3, which evolved in vivo during phage treatment, indicate that in vitro investigations can produce results that are relevant and predictive for the in vivo environment.

microbiology↗

bioRxiv2021

An allelic series of spontaneous mutations in Rorb cause a gait phenotype, retinal abnormalities, and transcriptomic changes relevant to human neurodevelopmental conditions

Rorb encodes the Retinoic Acid Receptor-related orphan receptor beta. Mutations in either of the two transcripts of Rorb cause defects in multiple systems, including abnormal photoreceptor abundance and morphology in the retina and a characteristic "high-stepper" or "duck-like" gait arising from dysfunction of interneurons in the spinal cord. Rorb is also important for cortical development and cell fate specification in mice. Rorb variants segregate with epilepsy and comorbidities such as intellectual disability in numerous clinical cases. Here we describe five mouse strains with spontaneous mutations in Rorb identified by their gait phenotype. These mutations affect different domains and isoforms of Rorb, which correspond to the spectrum of anatomical and physiological phenotypes exhibited by these mice. Gene set analysis in Rorb mutants implicates pathways associated with development and nervous system function, and differential gene expression analysis indicates changes in numerous genes related to epilepsy, bipolar disorder, and autism spectrum disorder (ASD). Many of these genes and their protein products are known to interact during synapse formation and neuronal activity. These findings further illuminate the role of Rorb in nervous system development, provide further evidence for an association between Rorb and several neurological conditions, and describe an allelic series of Rorb mutant mice that will be useful for dissecting thalamocortical afferent (TCA) development, neural cell fate determination, and as animal models exhibiting transcriptomic shifts in neurological conditions such as epilepsy, bipolar disorder, and ASD. {blacksquare}Five mutant mice with a characteristic high-stepper gait phenotype have mutations in Rorb {blacksquare}These allelic series mutations manifest in a spectrum of anatomical and physiological abnormalities {blacksquare}Gene expression data suggest involvement of pathways related to neurodevelopmental disorders

genetics↗

bioRxiv2021

Salt-inducible kinase (SIK) inhibition is protective in a mouse model of asthma

Interleukin-13 (IL-13) and other Th2 cytokines are important regulators of airway hyper-responsiveness, immune cell infiltration and inflammation in allergic asthma, and are produced when immune cells, such as type 2 innate lymphoid cells (ILC2s) and mast cells, are stimulated with IL-33. Here, we report that the IL-33-dependent secretion of IL-13 from ILC2s is prevented by inhibition of the salt-inducible kinases (SIKs), as we have shown previously in mast cells (Darling NJ et al., 2021, J. Biol. Chem. doi: 10.1016/j.jbc.2021.100428). We also report that a new SIK inhibitor with improved pharmacokinetic properties suppresses the recruitment of eosinophils to the lungs and serum immunoglobulin E (IgE) levels in the Alternaria alternata-induced model of allergic asthma. Our results suggest that drugs targeting SIK isoforms may have therapeutic potential for the treatment of asthma.

immunology↗