title: Aβ toxicity rescued by protein retention in the ER

abstract: Accumulation of A{beta} in the brain is one of the hallmarks of Alzheimers disease (AD). In the adult Drosophila brain, human A{beta} over-expression is toxic and leads to deterioration of climbing ability and shortened lifespan. However, it remains unknown if A{beta} is inherently toxic or if it triggers toxic downstream pathways that lead to neurodegeneration. Here, we describe a novel, and previously unidentified, protective role of intracellular laminin chain accumulation. Despite high A{beta} levels, over-expression of the extracellular matrix protein subunit Laminin B1 (LanB1) resulted in a robust rescue of toxicity, highlighting a potential protective mechanism of resistance to A{beta}. Over-expression of other Laminin subunits and a Collagen IV subunit also significantly rescued A{beta} toxicity, while combining LanB1 with these subunits led to an even larger rescue. Imaging revealed that LanB1 was retained in the ER but had no effect on the secretion of A{beta} into the extracellular milieu. LanB1 rescued toxicity independently of the IRE1/XBP1-mediated branch of the ER stress response. Interestingly, over-expression of ER-targeted GFP also rescued A{beta} toxicity, indicating a potentially broader benefit of ER protein retention. Finally, in proof-of-principle lentiviral transduction experiments using murine organotypic hippocampal slice cultures, over-expression of mouse Lamb1 resulted in ER-retention in transduced cells, highlighting a conserved mechanism. Typically, retention of proteins in the ER is detrimental to cellular health, but in the context of neuronal A{beta} toxicity it may prove to be beneficial and a new therapeutic avenue for AD.

category: neuroscience


title: Prefrontal cortex neural compensation during an operant devaluation task

abstract:
Deficits in goal-directed action are reported in multiple neuropsychiatric conditions, including schizophrenia. However, dysfunction is not always apparent in early stages of schizophrenia, possibly due to neural compensation. We designed a novel devaluation task in which goal-directed action could be guided by stimulus-outcome (S-O) [presumably orbitofrontal cortex (OFC)-mediated] or response-outcome (R-O) associations [presumably prelimbic cortex (PL)-mediated]. We previously found suggestive evidence that OFC and PL could compensate for each other in this task, and we more directly assessed this potential compensation here. In Experiment 1, rats received OFC, PL, combined OFC+PL, or sham lesions and then completed our devaluation task. The OFC+PL lesion group exhibited impaired devaluation. In Experiment 2, rats received cholera-toxin-b (CTb) into OFC and either neurotoxic or sham PL lesions. Rats were then sacrificed on the last training day to double-label for Arc and CTb. We found increased Arc+CTb in mediodorsal thalamus (MD) and increased Arc+ neurons in OFC when PL was lesioned, suggesting that PL lesions lead to a compensatory increased activation of the MD[->]OFC circuit. Our results suggest that our devaluation task can model neural compensation between OFC and PL and this compensation may be regulated by MD.

Significance StatementTo detect compensatory responses, behavioral models that use different strategies must be developed to determine if the strategies shift when a brain area or circuit is incapacitated. Neural compensation is commonly observed in human research but only a few models of neural compensation exist, and few identify compensation within the prefrontal cortex. This research is among the first to show neural compensation between prefrontal cortex regions and implicate a thalamocortical circuit in modulating this compensation. Not only will this model provide a way to behaviorally identify subtle neurological shifts, it can also elucidate basic neurological mechanisms that mediate how circuits interact with each other and how dysfunction in one circuit can affect connectivity in other brain areas.


title: Functional cortical localization of the tongue using corticokinematic coherence with a deep learning-assisted motion capture system

abstract: Measuring the corticokinematic coherence (CKC) between magnetoencephalographic and movement signals using an accelerometer can evaluate the functional localization of the primary sensorimotor cortex (SM1) of the upper limbs. However, it is difficult to determine the tongue CKC because an accelerometer yields excessive magnetic artifacts. We introduce and validate a novel approach for measuring the tongue CKC using a deep learning-assisted motion capture system with videography, and compare it with an accelerometer in a control task measuring finger movement. Twelve healthy volunteers performed rhythmical side-to-side tongue movements in the whole-head magnetoencephalographic system, which were simultaneously recorded using a video camera and examined offline using a deep learning-assisted motion capture system. In the control task, right finger CKC measurements were simultaneously evaluated via motion capture and an accelerometer. The right finger CKC with motion capture was significant at the movement frequency peaks or its harmonics over the contralateral hemisphere; the motion-captured CKC was 84.9% similar to that with the accelerometer. The tongue CKC was significant at the movement frequency peaks or its harmonics over both hemispheres, with no difference between the left and right hemispheres. The CKC sources of the tongue were considerably lateral and inferior to those of the finger. Thus, the CKC based on deep learning-assisted motion capture can evaluate the functional localization of the tongue SM1. In this approach, because no devices are placed on the tongue, magnetic noise, disturbances due to tongue movements, risk of aspiration of the device, and risk of infection to the experimenter are eliminated.


title: Geometric Deep Learning of the Human Connectome Project Multimodal Cortical Parcellation

abstract: Understanding the topographic heterogeneity of cortical organisation is an essential step towards precision modelling of neuropsychiatric disorders. While many cortical parcellation schemes have been proposed, few attempt to model inter-subject variability. For those that do, most have been proposed for high-resolution research quality data, without exploration of how well they generalise to clinical quality scans. In this paper, we benchmark and ensemble four different geometric deep learning models on the task of learning the Human Connectome Project (HCP) multimodal cortical parcellation. We employ Monte Carlo dropout to investigate model uncertainty with a view to propagate these labels to new datasets. Models achieved an overall Dice overlap ratio of >0.85 {+/-} 0.02. Regions with the highest mean and lowest variance included V1 and areas within the parietal lobe, and regions with the lowest mean and highest variance included areas within the medial frontal lobe, lateral occipital pole and insula. Qualitatively, our results suggest that more work is needed before geometric deep learning methods are capable of fully capturing atypical cortical topographies such as those seen in area 55b. However, information about topographic variability between participants was encoded in vertex-wise uncertainty maps, suggesting a potential avenue for projection of this multimodal parcellation to new datasets with limited functional MRI, such as the UK Biobank.


title: Global and network functional connectivity of nucleus basalis of Meynert is strengthened in blind individuals

abstract: Plasticity in the brain is differentially affected by age of blindness onset. One possible, but not yet identified mechanism is that the cholinergic signals originating from the nucleus basalis of Meynert may underlie differential extent of plasticity in early and late blind individuals. This prospect is based on the fact that the nucleus basalis of Meynert modulates cortical processes such as plasticity and sensory encoding and that the degree of cross-modal plasticity varies depending on the age of blindness onset. However, this question yet remains largely unclear. Here, we tested whether the early and late blind individuals develop dissociable plasticity in the nucleus basalis of Meynert using multi-parametric magnetic resonance imaging. We found the relatively preserved volumetric size and cerebrovascular reactivity, but significant disruption in the white matter integrity of the nucleus basalis of Meynert in both early and late blind individuals. Critically, despite its reduction in the white matter integrity, the nucleus basalis of Meynert of early blind individuals presented greater global and network functional connectivity including visual, language, and default-mode networks. Such changes in the functional connectivity were not observed in the late-blind individuals. Further, less duration of the visual experience was associated with greater global and network functional connectivity. These results indicate that the nucleus basalis of Meynert is differentially involved in the plasticity of early and late blind individuals - a similar amount of reduction in microstructural integrity in early and late blind individuals, but stronger and more widespread functional connectivity of the NBM in the early blind individuals. Our findings suggest that the nucleus basalis of Meynert may develop greater cholinergic influence on the cortex of early blind individuals. Such change may explain why early blind individuals present stronger and more widespread cross-modal plasticity during non-visual tasks compared to late blind individuals.


title: Dissociable plasticity of the nucleus basalis of Meynert in early and late blind individuals


title: Excess dietary sugar impairs colonic epithelial regeneration in response to damage.

abstract: The colonic epithelium requires continuous renewal by intestinal stem cells (ISCs) to restore the barrier after damage and proliferation of epithelial cells is modulated by dietary metabolites. We demonstrate that mice fed a high sugar diet failed to repair colonic barrier damage, resulting in increased intestinal pathology. Culturing ISCs in excess sugar limited murine and human colonoid development, indicating that dietary sugar can directly affect colonic epithelial proliferation. Similarly, in vivo lineage tracing experiments and transcriptomic analysis indicated that dietary sugar impeded the proliferative potential of ISCs. ISCs and their immediate daughter cells predominantly rely on mitochondrial respiration for energy; however, metabolic analysis of colonic crypts revealed that a high sugar diet primed the epithelium for glycolysis without a commensurate increase in aerobic respiration. Colonoids cultured in high-glucose conditions accumulated glycolytic metabolites but not TCA cycle intermediates, indicating that the two metabolic pathways may not be coupled in proliferating intestinal epithelium. Accordingly, biochemically inducing pyruvate flux through the TCA cycle by inhibiting pyruvate dehydrogenase kinase rescued sugar-impaired colonoid development. Our results indicate that excess dietary sugar can directly inhibit epithelial proliferation in response to damage and may inform diets that better support the treatment of acute intestinal injury.

category: immunology


title: Impact of Human Serum Proteins on Susceptibility of Acinetobacter baumannii to Cefiderocol: role of iron transport

abstract: Cefiderocol is a siderophore antibiotic that co-opts iron transporters to facilitate cell entry. We show that genes related to iron uptake systems and resistance to {beta}-lactams in Acinetobacter baumannii have altered expression levels in the presence of human serum, human serum albumin, or human pleural fluid. Cefiderocol MICs are also raised in the presence of the mentioned fluids. Clinical response in A. baumannii infections may be related to the interplay of these human factors.

category: microbiology


title: Deploying an in vitro gut model to assay the impact of a mannan-oligosaccharide prebiotic, Bio-Mos(R) on the Atlantic salmon (Salmo salar) gut microbiome.

abstract:
Mannose-oligosaccharide (MOS) pre-biotics are widely deployed in animal agriculture as immunomodulators as well as to enhance growth and gut health. Their mode of action is thought to be mediated through their impact on host microbial communities and the associated metabolism. Bio-Mos is a commercially available pre-biotic currently used in the agri-feed industry. To assess Bio-Mos for potential use as a prebiotic growth promotor in salmonid aquaculture, we have modified an established Atlantic salmon in vitro gut model, SalmoSim, to evaluate its impact on the host microbial communities. Inoculated from biological triplicates of adult farmed salmon pyloric caeca compartments, the microbial communities were stabilised in SalmoSim followed by a twenty-day exposure to the prebiotic and in turn followed by an eight day  wash out period. Dietary inclusion of MOS resulted in a significant increase in formate (p=0.001), propionate (p=0.037) and isovalerate (p=0.024) levels, correlated with increased abundances of several, principally, anaerobic microbial genera (Fusobacterium, Agarivorans, Pseudoalteromonas). DNA metabarcoding with the 16S rDNA marker confirmed a significant shift in microbial community composition in response to MOS supplementation with observed increase in lactic acid producing Carnobacterium. In conjunction with previous in vivo studies linking enhanced volatile fatty acid production alongside MOS supplementation to host growth and performance, our data suggests that Bio-Mos may be of value in salmonid production. Furthermore, our data highlights the potential role of in vitro gut models to augment in vivo trials of microbiome modulators.

ImportanceIn this paper we report the results of the impact of prebiotic (MOS supplementation) on microbial communities within recently developed Atlantic salmon gut microbiome in vitro simulator. Our data suggest that Bio-Mos may be of value in salmonid production as it enhances volatile fatty acid production in the Atlantic salmon gut and correlates with a significant shift in microbial community composition with observed increase in lactic acid producing Carnobacterium. In conjunction with previous in vivo studies linking enhanced volatile fatty acid production alongside MOS supplementation to host growth and performance, our data suggest that Bio-Mos may be of value in salmonid production. Furthermore, our data highlights the potential role of in vitro gut models to augment in vivo trials of microbiome modulators.

category: bioengineering


title: Optimized detection of somatic allelic imbalances specific for homologous recombination deficiency improves the prediction of clinical outcomes in ovarian cancer

abstract: Homologous recombination DNA-repair deficiency (HRD) is a common driver of genomic instability and confers a therapeutic vulnerability in human cancer. Using a systematic pan- cancer characterization, we revealed distinct patterns of somatic allelic imbalances (AIs) in high-grade serous ovarian cancer (HGSC). Using machine learning on a multi-omics dataset, we generated an optimized algorithm to detect HRD in HGSC (ovaHRDscar). ovaHRDscar improved the prediction of clinical outcomes in three independent validation cohorts. Characterization of 98 spatiotemporally distinct tumor samples indicated ovary/adnex as the preferred site to assess HRD. Cancer-specific optimization improved the prediction of clinical outcomes also in triple-negative breast cancer (tnbcHRDscar). In conclusion, our systematic approach based on multi-omics data improves the detection of HRD with the premise to improve patient selection for HR-targeted therapies.

category: cancer biology


title: Optimized detection of allelic imbalances specific for homologous recombination deficiency improves the prediction of clinical outcomes in cancer


title: MEKK3-TGFβ crosstalk regulates inward arterial remodeling

abstract:
Arterial remodeling is an important adaptive mechanism that maintains normal fluid shear stress in a variety of physiologic and pathologic conditions. Inward remodeling, a process that leads to reduction in arterial diameter, plays a critical role in progression of such common diseases as hypertension and atherosclerosis. Yet despite its pathogenic importance, molecular mechanisms controlling inward remodeling remain undefined. Mitogen-activated protein kinases (MAPKs) perform a number of functions ranging from control of proliferation to migration and cell fate transitions. While the MAPK ERK1/2 signaling pathway has been extensively examined in the endothelium, less is known about the role of the MEKK3/ERK5 pathway in vascular remodeling. To better define the role played by this signaling cascade, we studied the effect of endothelial-specific deletion of its key upstream MAP3K, MEKK3, in adult mice. The genes deletion resulted in a gradual inward remodeling of both pulmonary and systematic arteries leading to spontaneous hypertension in both vascular circuits and accelerated progression of atherosclerosis in hyperlipidemic mice. Molecular analysis revealed activation of TGF{beta} signaling both in vitro and in vivo. Endothelial-specific TGF{beta}R1 knockout prevented inward arterial remodeling in MEKK3 endothelial knockout mice. These data point to the unexpected participation of endothelial MEKK3 in regulation of TGF{beta}R1-Smad2/3 signaling and inward arterial remodeling in artery diseases.

SignificanceInward remodeling of arteries to reduce lumen diameter is a major factor in disease progression and morbidity in multiple vascular diseases, including hypertension and atherosclerosis. However, molecular mechanisms controlling inward arterial remodeling remain largely undefined. In this study, we identify endothelial MEKK3 as an unexpected regulator of inward remodeling via inhibition of TGF{beta}-Smad2/3 signaling. Genetic deletion of MEKK3 in adult endothelium results in induction of TGF{beta}-Smad2/3 signaling, endothelial-to-mesenchymal transition and inward remodeling in both pulmonary and arterial circuits. The latter process results in pulmonary and systemic hypertension and accelerates atherosclerosis. These results provide a new basis for understanding the inward artery remodeling that leads to reduced blood flow to affected tissues and exacerbates hypertension in vascular disease.

category: pathology


title: Improved radiation expression profiling by sequential application of sensitive and specific gene signatures

abstract:
PurposeCombinations of expressed genes can discriminate radiation-exposed from normal control blood samples by machine learning based signatures (with 8 to 20% misclassification rates). These signatures can quantify therapeutically-relevant as well as accidental radiation exposures. The prodromal symptoms of Acute Radiation Syndrome (ARS) overlap those present in Influenza and Dengue Fever infections. Surprisingly, these human radiation signatures misclassified gene expression profiles of virally infected samples as false positive exposures. The present study investigates these and other confounders, and then mitigates their impact on signature accuracy.

MethodsThis study investigated recall by previous and novel radiation signatures independently derived from multiple Gene Expression Omnibus datasets on common and rare non-malignant blood disorders and blood-borne infections (thromboembolism, S. aureus bacteremia, malaria, sickle cell disease, polycythemia vera, and aplastic anemia). Normalized expression levels of signature genes are used as input to machine learning-based classifiers to predict radiation exposure in other hematological conditions.

ResultsExcept for aplastic anemia, these blood-borne disorders modify the normal baseline expression values of genes present in radiation signatures, leading to false-positive misclassification of radiation exposures in 8 to 54% of individuals. Shared changes, predominantly in DNA damage response and apoptosis-related gene transcripts in radiation and confounding hematological conditions, compromise the utility of these signatures for radiation assessment. These confounding conditions (sickle cell disease, thromboembolism, S. aureus bacteremia, malaria) induce neutrophil extracellular traps, initiated by chromatin decondensation, DNA damage response and fragmentation followed by programmed cell death. Riboviral infections (for example, Influenza or Dengue fever) have been proposed to bind and deplete host RNA binding proteins, inducing R-loops in chromatin. R-loops that collide with incoming replication forks can result in incompletely repaired DNA damage, inducing apoptosis and releasing mature virus. To mitigate the effects of confounders, we evaluated predicted radiation-positive samples with novel gene expression signatures derived from radiation-responsive transcripts encoding secreted blood plasma proteins whose expression levels are unperturbed by these conditions.

ConclusionsThis approach identifies and eliminates misclassified samples with underlying hematological or infectious conditions, leaving only samples with true radiation exposures. Diagnostic accuracy is significantly improved by selecting genes that maximize both sensitivity and specificity in the appropriate tissue using combinations of the best signatures for each of these classes of signatures.

category: systems biology


title: Improved radiation expression profiling in blood by sequential application of sensitive and specific gene signatures


title: Systemic host inflammation induces stage-specific transcriptomic modification and slower maturation in malaria parasites

abstract: Maturation rate of malaria parasites within red blood cells (RBC) can be influenced by host nutrient status or circadian rhythm. Here, we observed in mice that systemic host inflammation, induced by lipopolysaccharide (LPS) conditioning or ongoing acute malaria infection, slowed the progression of a single cohort of parasites from one generation of RBC to the next. LPS-conditioning and acute infection both triggered substantial changes to the metabolomic composition of plasma in which parasites circulated. This altered plasma directly slowed parasite maturation in a manner that could not be rescued by supplementation, consistent with the presence of inhibitory factors. Single-cell transcriptomic assessment of mixed parasite populations, exposed to a short period of systemic host inflammation in vivo, revealed specific impairment in the transcriptional activity and translational capacity of trophozoites compared to rings or schizonts. Thus, we provide in vivo evidence of transcriptomic and phenotypic plasticity of asexual blood-stage Plasmodium parasites when exposed to systemic host inflammation.


title: A method for high throughput image based antifungal screening

abstract: Robust antifungal screening is technically challenging particularly for filamentous fungi. We present a method for undertaking antifungal screening assays that builds upon existing broth dilution protocols and incorporates time resolved image-based assessment of fungal growth. We show that the method performs with different fungi, particularly those for which spores can be used as inoculum, and with different compound classes, can accurately assess susceptibility or otherwise in only few hours, performs well even without replication and can even account for differences in inherent growth properties of strains.


title: A bacterial genome and culture collection of gut microbial in weanling piglet

abstract: The microbiota hosted in the pig gastrointestinal tract are important for productivity of livestock. However, the individual species and functional repertoires that make up the pig gut microbiome remain largely undefined. Here we comprehensively investigated the genomes and functions of the piglet gut microbiome using culture-based and metagenomics approaches. A collection included 266 cultured genomes and 482 metagenome-assembled genomes (MAGs) that were clustered to 428 species across 10 phyla was established. Among these clustered species, 333 genomes represent potential new species. Less matches between cultured genomes and MAGs revealed a substantial bias for the acquisition of reference genomes by the two strategies. Glycoside hydrolases was the dominant category of carbohydrate-active enzymes. 445 secondary metabolite biosynthetic genes were predicted from 292 genomes with bacteriocin being the most. Pan genome analysis of Limosilactobacillus reuteri uncover the biosynthesis of reuterin was strain-specific and the production was experimentally determined. These genomic resources will enable a comprehensive characterization of the microbiome composition and function of pig gut.


title: Characterising the Photoplethysmography Pulse Waveform for Use in Human Neuroscience: The Hybrid Excess and Decay (HED) Model

abstract: Photoplethysmography (PPG) offers a widely-used, convenient and non-invasive approach to monitoring basic indices of cardiovascular function such as heart rate and blood oxygenation. However, while the pulse waveform, generated by PPG comprises features that are shaped by physiological and psychological factors, it is frequently overlooked in analyses of such data. We suggest that studies could be enriched by exploiting the possibilities afforded by a systematic analysis of PPG waveforms. To do this we initially require a robust and automated means of characterising it, thereby allowing us to examine variations across individuals and between different physiological and psychological contexts. We present a psychophysiologically-relevant model, the Hybrid Excess and Decay (HED) Model, which characterises pulse wave morphology in terms of three underlying pressure waves and a decay function. We show that these parameters capture PPG data with a high degree of precision and, moreover, are sensitive to specific, physiologically-relevant changes within individuals. We present the theoretical and practical basis for the model and demonstrate its performance when applied to a pharmacological dataset of 105 participants receiving intravenous administrations of the sympathomimetic drug isoproterenol (Isoprenaline). We conclude by discussing the possible value in using the HED model to complement standard measures of PPG outputs.


title: Neutralising antibodies against the SARS-CoV-2 Delta variant induced by Alhydroxyquim-II-adjuvanted trimeric spike antigens

abstract: Global control of COVID-19 will require the deployment of vaccines capable of inducing long-term protective immunity against SARS-CoV-2 variants. In this report, we describe an adjuvanted subunit candidate vaccine that affords elevated, sustained and cross-variant SARS-CoV-2 neutralising antibodies (NAbs) in multiple animal models. Alhydroxiquim-II is a TLR7/8 small-molecule agonist chemisorbed on aluminium hydroxide. Vaccination with Alhydroxiquim-II combined with a stabilized, trimeric form of the SARS-CoV-2 spike protein (termed CoVac-II) resulted in high-titre NAbs in mice, with no decay in responses over an 8-month period. NAbs from sera of CoVac-II-immunized mice, horses and rabbits were broadly neutralising against SARS-CoV-2 variants. Boosting long-term CoVac-II-immunized mice with adjuvanted spike protein from the Beta variant markedly increased levels of NAb titres against multiple SARS-CoV-2 variants; notably high titres against the Delta variant were observed. These data strongly support the clinical assessment of Alhydroxiquim-II-adjuvanted spike proteins to protect against SARS-CoV-2 variants of concern.


title: Development of an in vitro model for animal species susceptibility to SARS-CoV-2 replication based on expression of ACE2 and TMPRSS2 in avian cells

abstract:
The SARS-CoV-2 (SC2) virus has caused a worldwide pandemic because of the viruss ability to transmit efficiently human-to-human. A key determinant of infection is the attachment of the viral spike protein to the host receptor angiotensin-converting enzyme 2 (ACE2). Because of the presumed zoonotic origin of SC2, there is no practical way to assess every species susceptibility to SC2 by direct challenge studies. In an effort to have a better predictive model of animal host susceptibility to SC2, we expressed the ACE2 and/or transmembrane serine protease 2 (TMPRSS2) genes from humans and other animal species in the avian fibroblast cell line, DF1, that is not permissive to infection. We demonstrated that expression of both human ACE2 and TMPRSS2 genes is necessary to support SC2 infection and replication in DF1 and a non-permissive sub-lineage of MDCK cells. Titers of SC2 in these cell lines were comparable to those observed in control Vero cells. To further test the model, we developed seven additional transgenic cell lines expressing the ACE2 and TMPRSS2 derived from Felis (cat), Equus (horse), Sus (pig), Capra (goat), Mesocricetus (Golden hamster), Myotis lucifugus (Little Brown bat) and Hipposideros armiger (Great Roundleaf bat) in DF1 cells. Results demonstrate permissive replication of SC2 in cat, Golden hamster, and goat species, but not pig or horse, which correlated with the results of reported challenge studies. The development of this cell culture model allows for more efficient testing of the potential susceptibility of many different animal species for SC2 and emerging variant viruses.

IMPORTANCESARS-CoV-2 (SC2) is believed to have originated in animal species and jumped into humans where it has produced the greatest viral pandemic of our time. Identification of animal species susceptible to SC2 infection would provide information on potential zoonotic reservoirs, and transmission potential at the human-animal interface. Our work provides a model system to test the ability of the virus to replicate in an otherwise non-permissive cell line by transgenic insertion of the ACE2 and TMPRSS2 genes from human and other animal species. The results from our in vitro model positively correlate with animal infection studies enhancing the predicative capability of the model. Importantly, we demonstrate that both proteins are required for successful virus replication. These findings establish a framework to test other animal species for susceptibility to infection that may be critical zoonotic reservoirs for transmission, as well as to test variant viruses that arise over time.


title: Algorithm for the Quantitation of Variants of Concern for Rationally Designed Vaccines Based on the Isolation of SARS-CoV-2 Hawaii Lineage B.1.243

abstract: SARS-CoV-2 worldwide emergence and evolution has resulted in variants containing mutations resulting in immune evasive epitopes that decrease vaccine efficacy. We acquired clinical samples, analyzed SARS-CoV-2 genomes, used the most worldwide emerged spike mutations from Variants of Concern/Interest, and developed an algorithm for monitoring the SARS-CoV-2 vaccine platform. The algorithm partitions logarithmic-transformed prevalence data monthly and Pearsons correlation determines exponential emergence. The SARS-CoV-2 genome evaluation indicated 49 mutations. Nine of the ten most worldwide prevalent (>70%) spike protein changes have r-values >0.9. The tenth, D614G, has a prevalence >99% and r-value of 0.67. The resulting algorithm is based on the patterns these ten substitutions elucidated. The strong positive correlation of the emerged spike protein changes and algorithmic predictive value can be harnessed in designing vaccines with relevant immunogenic epitopes. SARS-CoV-2 is predicted to remain endemic and continues to evolve, so must SARS-CoV-2 monitoring and next-generation vaccine design.

category: molecular biology


title: Effects of Hydroxychloroquine and Azithromycin on iPSC-derived Cardiomyocytes: Considerations for the Treatment of COVID-19 Patients

abstract: Despite known adverse effects of hydroxychloroquine (HCQ) and azithromycin (AZM) on cardiac function, HCQ and AZM have been used as combination therapy in the treatment of COVID-19 patients. Recent clinical data indicate higher complication rates with HCQ/AZM combination treatment in comparison to monotherapy. Here, we used human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to systematically investigate the effects of HCQ and AZM individually and in combination. The clinically observed QT prolongation caused by treatment with HCQ could be recapitulated in iPSC-CMs based on prolonged field potential duration (FPDc). Interestingly, HCQ-induced FPDc prolongation was strongly enhanced by combined treatment with AZM, although AZM alone slightly shortened FPDc in iPSC-CMs. Furthermore, combined treatment with AZM and HCQ leads to higher cardiotoxicity, more severe structural disarrangement, and more pronounced contractile and electrophysiological dysfunctions, compared to respective mono-treatments. First mechanistic insights underlying the synergistic effects of AZM and HCQ on iPSC-CM functionality are provided based on increased Cx43- and Nav1.5-protein levels. Taken together, our results highlight that combined treatment with HCQ and AZM strongly enhances the adverse effects on cardiomyocytes, providing mechanistic evidence for the high mortality in patients receiving HCQ/AZM combination treatment.

category: pharmacology and toxicology


title: Pandemic, epidemic, endemic: B cell repertoire analysis reveals unique anti-viral responses to SARS-CoV-2, Ebola and Respiratory Syncytial Virus

abstract: Immunoglobulin gene heterogeneity reflects the diversity and focus of the humoral immune response towards different infections, enabling inference of B cell development processes. Detailed compositional and lineage analysis of long read IGH repertoire sequencing, combining examples of pandemic, epidemic and endemic viral infections with control and vaccination samples, demonstrates general responses including increased use of IGHV4-39 in both EBOV and COVID-19 infection cohorts. We also show unique characteristics absent in RSV infection or yellow fever vaccine samples: EBOV survivors show unprecedented high levels of class switching events while COVID-19 repertoires from acute disease appear underdeveloped. Despite the high levels of clonal expansion in COVID-19 IgG1 repertoires there is a striking lack of evidence of germinal centre mutation and selection. Given the differences in COVID-19 morbidity and mortality with age, it is also pertinent that we find significant differences in repertoire characteristics between young and old patients. Our data supports the hypothesis that a primary viral challenge can result in a strong but immature humoral response where failures in selection of the repertoire risks off-target effects.


title: Identification of HCN1 as a 14-3-3 client

abstract: Hyperpolarization activated cyclic nucleotide-gated channel 1 (HCN1) is expressed throughout the nervous system and is critical for regulating neuronal excitability, with mutations being associated with multiple forms of epilepsy. Adaptive modulation of HCN1 has been observed as has pathogenic dysregulation. While the mechanisms underlying this modulation remain incompletely understood, regulation of HCN1 has been shown to include phosphorylation. A candidate phosphorylation-dependent regulator of HCN1 channels is 14-3-3. We used bioinformatics to identify three potential 14-3-3 binding sites in HCN1. Isothermal titration calorimetry demonstrated that recombinant 14-3-3 binds all three phospho-peptides with low micromolar affinity. We confirmed that 14-3-3 could pull down HCN1 from multiple tissue sources and used HEK293 cells to detail the interaction. Two binding sites in the intrinsically disordered C-terminus of HCN1 were necessary and sufficient for a phosphorylation-dependent interaction with 14-3-3. The same region of HCN1 containing the 14-3-3 binding sites is required for phosphorylation-independent protein degradation. We propose a model in which phosphorylation of S810 and S867 (human S789 and S846) recruits 14-3-3 to inhibit a yet unidentified factor signaling for protein degradation, thus increasing the half-life of HCN1.

category: biochemistry


title: Modeling Coronavirus Spike Protein Dynamics: Implications for Immunogenicity and Immune Escape

abstract:
The ongoing COVID-19 pandemic is a global public health emergency requiring urgent development of efficacious vaccines. While concentrated research efforts are underway to develop antibody-based vaccines that would neutralize SARS-CoV-2, and several first-generation vaccine candidates are currently in Phase III clinical trials or have received emergency use authorization, it is forecasted that COVID-19 will become an endemic disease requiring second-generation vaccines. The SARS-CoV-2 surface Spike (S) glycoprotein represents a prime target for vaccine development because antibodies that block viral attachment and entry, i.e. neutralizing antibodies, bind almost exclusively to the receptor binding domain (RBD). Here, we develop computational models for a large subset of S proteins associated with SARS-CoV-2, implemented through coarse-grained elastic network models and normal mode analysis. We then analyze local protein domain dynamics of the S protein systems and their thermal stability to characterize structural and dynamical variability among them. These results are compared against existing experimental data, and used to elucidate the impact and mechanisms of SARS-CoV-2 S protein mutations and their associated antibody binding behavior. We construct a SARS-CoV-2 antigenic map and offer predictions about the neutralization capabilities of antibody and S mutant combinations based on protein dynamic signatures. We then compare SARS-CoV-2 S protein dynamics to SARS-CoV and MERS-CoV S proteins to investigate differing antibody binding and cellular fusion mechanisms that may explain the high transmissibility of SARS-CoV-2. The outbreaks associated with SARS-CoV, MERS-CoV, and SARS-CoV-2 over the last two decades suggest that the threat presented by coronaviruses is ever-changing and long-term. Our results provide insights into the dynamics-driven mechanisms of immunogenicity associated with coronavirus S proteins, and present a new approach to characterize and screen potential mutant candidates for immunogen design, as well as to characterize emerging natural variants that may escape vaccine-induced antibody responses.

STATEMENT OF SIGNIFICANCEWe present novel dynamic mechanisms of coronavirus S proteins that encode antibody binding and cellular fusion properties. These mechanisms may offer an explanation for the widespread nature of SARS-CoV-2 and more limited spread of SARS-CoV and MERS-CoV. A comprehensive computational characterization of SARS-CoV-2 S protein structures and dynamics provides insights into structural and thermal stability associated with a variety of S protein mutants. These findings allow us to make recommendations about the future mutant design of SARS-CoV-2 S protein variants that are optimized to elicit neutralizing antibodies, resist structural rearrangements that aid cellular fusion, and are thermally stabilized. The integrated computational approach can be applied to optimize vaccine immunogen design and predict escape of vaccine-induced antibody responses by SARS-CoV-2 variants.

category: biophysics


title: Specific BRCA and immune configurations determine optimal response to platinum-based chemotherapy in triple negative breast and ovarian carcinomas

abstract: Loss of homologous recombination repair (HRR) via germline and somatic BRCA1 or BRCA2 gene mutations and via BRCA1 promoter methylation has been associated with better response to platinum agents and PARP inhibitors, in both triple negative breast cancer (TNBC) and ovarian carcinoma (OvCa). A major conundrum arising from recent clinical studies is why cancers with BRCA1 promoter methylation (BRCA1meth) respond more poorly as compared to those bearing mutations in BRCA1 and BRCA2 (BRCAmut), given the biologically equivalent HRR deficiency in both states. We dissected this problem through detailed genomic analyses of primary TNBC and OvCa cohorts, as well as experimentation with patient-derived xenograft (PDX) models and genetically engineered cell lines. Using the precise genomic scar of the tandem duplicator phenotype as a precise genomic indicator of BRCA1 deficiency, we found that, in all cohorts, BRCA1mut and BRCA1meth cancers share an equivalent degree of BRCA1-linked genomic rearrangements. Nonetheless, we consistently found that patients with BRCAmut cancers, but not those with BRCA1meth cancers, had significantly better response outcomes when compared to those with BRCA proficient cancers. When fully promoter methylated BRCA1 PDX TNBCs were exposed to a single short course of platinum chemotherapy an unmethylated BRCA1 promoter allele emerged in resultant tumors associated with an increase in BRCA1 expression. A separate analysis of PDXs derived from treatment naive TNBCs featured complete methylation of the BRCA1 promoter, whereas those derived from post-chemotherapy TNBCs invariably had only partial methylation. PDXs with partial methylation were significantly associated with lower response rates to in vivo platinum-based therapy compared to those with complete promoter methylation. Using single cell clonal expansions from a partially BRCA1meth PDX, we confirmed that the reduced level of methylation was due to the demethylation of one of the BRCA1 promoter alleles and not to the outgrowth of a non-methylated clone. Clinically, analysis of primary OvCas confirmed that high levels of BRCA1 methylation were significantly associated with reduced BRCA1 gene expression whereas cancers with lower levels of BRCA1 methylation had expression levels approaching those found in BRCA1 proficient cancers. These data suggest that unlike BRCAmut cancers, where HRR deficiency is achieved via mutations that are genetically  fixed, BRCA1meth cancers are highly adaptive to genotoxin exposure and more likely to recover BRCA1 expression, which may explain their poorer therapeutic response. We further found that an increased immune transcriptional signal, especially an elevated M1 macrophage signature, is associated with enhanced response to platinum-based chemotherapy only in patients with BRCA proficient cancers, in both TNBC and OvCa cohorts underscoring the importance of characterizing molecular heterogeneity to enhance predictive precision in assigning response probabilities in TNBC and OvCa.


title: Subjective social isolation is linked to subregional alterations in hippocampus-default network co-variation

abstract: Social interaction complexity makes humans unique. But in times of social deprivation this strength risks to expose important vulnerabilities. Human social neuroscience studies have placed a premium on the default network (DN). In contrast, hippocampus (HC) subfields have been intensely studied in rodents and monkeys. To bridge these two literatures, we here quantified how DN subregions systematically co-vary with specific HC subfields in the context of subjective social isolation (i.e., loneliness). By co-decomposition using structural brain scans of [~]40,000 UK Biobank participants, loneliness was specially linked to midline subregions in the uncovered DN patterns. These association cortex signatures coincided with concomitant HC patterns implicating especially CA1 and molecular layer. These patterns also showed a strong affiliation with the fornix white-matter tract and the nucleus accumbens. In addition, separable signatures of structural HC-DN co-variation had distinct associations with the genetic predisposition for loneliness at the population level.


title: White matter microstructure shows sex differences in late childhood: Evidence from 6,797 children

abstract: Sex differences in white matter microstructure have been robustly demonstrated in the adult brain using both conventional and advanced diffusion-weighted magnetic resonance imaging (dMRI) approaches. However, sex differences in white matter microstructure prior to adulthood remain poorly understood; previous developmental work focused on conventional microstructure metrics and yielded mixed results. Here we rigorously characterized sex differences in white matter microstructure among over 6,000 children from the Adolescent Brain Cognitive Development (ABCD) Study who were between 9 and 10 years old. Microstructure was quantified using both the conventional model - diffusion tensor imaging (DTI) - and an advanced model, restriction spectrum imaging (RSI). DTI metrics included fractional anisotropy (FA) and mean, axial, and radial diffusivity (MD, AD, RD). RSI metrics included normalized isotropic, directional, and total intracellular diffusion (N0, ND, NT). We found significant and replicable sex differences in DTI or RSI microstructure metrics in every white matter region examined across the brain. Sex differences in FA were regionally specific. Across white matter regions, boys exhibited greater MD, AD, and RD than girls, on average. Girls displayed increased N0, ND, and NT compared to boys, on average, suggesting greater cell and neurite density in girls. Together, these robust and replicable findings provide an important foundation for understanding sex differences in health and disease.


title: Audiovisual integration in the Mauthner cell enhances escape probability and reduces response latency

abstract: Fast and accurate threat detection is critically important for animal survival. Reducing perceptual ambiguity by integrating multiple sources of sensory information can enhance threat detection and reduce response latency. However, studies showing a direct link between behavioral correlates of multisensory integration and its underlying neural basis are rare. In fish, an explosive escape behavior known as C-start is driven by an identified neural circuit centered on the Mauthner cell. The Mauthner cell can trigger C-starts in response to visual and auditory stimuli allowing to investigate how multisensory integration in a single neuron affects behavioral outcome after threat detection. Here we demonstrate that in goldfish visual looms and brief auditory stimuli can be integrated to increase C-start probability and that this enhancement is inversely correlated to the saliency of the cues with weaker auditory cues producing a proportionally stronger multisensory effect. We also show that multisensory stimuli reduce response latency locked to the presentation of the auditory cue. Finally, we make a direct link between behavioral data and its underlying neural mechanism by reproducing empirical data with an integrate-and-fire computational model of the Mauthner cell.


title: Dynamic representation of space in the hippocampus: spatial novelty detection and consolidation in CA1 and CA2

abstract: Hippocampal place cells are the functional units of spatial navigation and are present in all subregions-CA1, CA2, CA3 and CA4. Recent studies on CA2 have indicated its role in social and contextual memory, but its contribution towards spatial novelty detection and consolidation remains largely unknown. The current study aims to uncover how CA1 and CA2 detect, process, assimilate and consolidate spatial novelty. Accordingly, a novel 3-day paradigm was designed where the animal was introduced to a completely new environment on the first day and to varying degrees of familiarity and novelty on subsequent days, as the track was extended in length and modified in shape, keeping other environmental constraints fixed. Detection of spatial novelty was found to be a dynamic and complex phenomenon, characterized by different responses from hippocampal place cells, depending on when novelty was introduced. Therefore, the study concludes that early novelty detection (the first time a novel space is introduced in a relatively familiar environment) and subsequent novelty detection are not processed in the same way. Additionally, while neuronal responses to spatial novelty detection (early and subsequent) were found to be the same in CA1 and CA2 ensembles, their responses differed in spatial consolidation mechanisms during subsequent sleep replays. For CA1, spatial coverage of prior behaviour was found to be closely reflected in subsequent sleep for that particular day, but CA2 showed no such  coherent response, highlighting mnemonic processing differences between CA2 and CA1 with respect to spatial novelty.


title: Neuroblast migration along cellular substrates in the developing porcine brain

abstract: In the past decade it has become evident that neuroblasts continue to supply the human cortex with interneurons via unique migratory streams shortly following birth. Due to the size of the human brain, these newborn neurons must migrate long distances through complex cellular landscapes to reach their final locations. This process is poorly understood, largely due to technical difficulties in acquiring and studying neurotypical postmortem human samples along with diverging developmental features of well-studied mouse models. We reasoned that migratory streams of neuroblasts utilize cellular substrates, such as blood vessels, to guide their trek from the subventricular zone to distant cortical targets. Here we evaluate the association between young interneuronal migratory streams and their preferred cellular substrates in gyrencephalic piglets during the developmental equivalent of human birth, infancy, and toddlerhood.


title: Methods for recording and imaging spreading depolarizations and seizure-like events in mouse hippocampal slices

abstract: Spreading depolarization (SD) is a sudden and synchronized depolarization of principal cells followed by depression of activity, which slowly propagates across brain regions like cortex or hippocampus. SD is considered to be mechanistically relevant to migraine, epilepsy, and traumatic brain injury. Interestingly, research into SD typically uses SD triggered immediately after a focal stimulus. Here we optimize an in vitro experimental model allowing us to record SD without focal stimulation. This method uses electrophysiological recordings and intrinsic optical imaging in slices. The method is also relatively easy and inexpensive. Acute hippocampal slices from mice or rats were prepared and used for extracellular and whole-cell recordings. Recordings were made in a submerged-style chamber with flow of artificial cerebrospinal fluid (aCSF) above and below the slices. Flow was fast (> 5ml/min), and temperature was 32{degrees}C. As soon as slices were placed in the chamber, aCSF containing 0 mM Mg2+ and 5 mM K+ (0 Mg2+/5 K+ aCSF) was used. Two major types of activity were observed: SD and seizure-like events (SLEs). Both occurred after many minutes of recording. Although both mouse and rat slices showed SLEs, only mouse slices developed SD and did so in the first hour of 0 Mg2+/5 K+ aCSF exposure. Intrinsic optical imaging showed that most SDs initiated in CA3 and could propagate into CA1 and dentate gyrus. In dentate gyrus, SD propagated in two separate waves: (1) into the hilus and (2) into granule cell and molecular layers simultaneously. This in vitro model can be used to better understand the mechanisms and relationship between SD and SLEs. It could also be useful in preclinical drug screening.


title: Connectomic features underlying diverse synaptic connection strengths and subcellular computation

abstract: Connectomes generated from electron microscopy images of neural tissue unveil the complex morphology of every neuron and the locations of every synapse interconnecting them. These wiring diagrams may also enable inference of synaptic and neuronal biophysics, such as the functional weights of synaptic connections, but this requires integration with physiological data to properly parameterize. Working with a stereotyped olfactory network in the Drosophila brain, we make direct comparisons of the anatomy and physiology of diverse neurons and synapses with subcellular and subthreshold resolution. We find that synapse density and location jointly predict the amplitude of the somatic postsynaptic potential evoked by a single presynaptic spike. Biophysical models fit to data predict that electrical compartmentalization allows axon and dendrite arbors to balance independent and interacting computations. These findings begin to fill the gap between connectivity maps and activity maps, which should enable new hypotheses about how network structure constrains network function.


title: Abnormal Neurite Density and Orientation Dispersion in Frontal Lobe Link to Elevated Hyperactive/Impulsive Behaviors in Young Adults with Traumatic Brain Injury

abstract: Traumatic brain injury is a major public health concern. A significant proportion of individuals experience post-traumatic brain injury behavioral impairments, especially in attention and inhibitory control domains. Traditional diffusion-weighted MRI techniques, such as diffusion tensor imaging, have provided tools to assess white matter structural disruptions reflecting the long-term brain tissue alterations associated with traumatic brain injury. The recently developed neurite orientation dispersion and density imaging is a more advanced diffusion-weighted MRI modality, which provides more refined characterization of brain tissue microstructures by assessing the neurite orientation dispersion and neurite density properties. In this study, we investigated the morphometrical and microstructural alterations at chronic brain injury stage and their relationships with the functional outcomes. Neurite orientation dispersion and density imaging data from 44 young adults with chronic traumatic brain injury (ranging from 18 27 years of age; 23 males/21 females) who had no prior-traumatic brain injury history of attention deficits and/or hyperactivity and 45 group-matched normal controls (23 males /22 females) were collected. Maps of fractional anisotropy, neurite orientation dispersion index, and neurite density index were calculated. Vertex-wise and voxel-wise analyses were conducted for gray matter and white matter, respectively. Post-hoc region of interest-based analyses were also performed. Compared to the controls, the group of traumatic brain injury showed significantly increased orientation dispersion index in various gray matter regions and significantly decreased orientation dispersion index in several white matter regions. Brain-behavioral association analyses indicated that the reduced neurite density index of left precentral gyrus and the reduced orientation dispersion index of left superior longitudinal fasciculus were significantly associated with elevated hyperactive/impulsive symptoms in the patients with traumatic brain injury. These findings suggest that traumatic brain injury-induced chronic neurite orientation dispersion alterations of left superior longitudinal fasciculus and left precentral may significantly contribute to post-traumatic brain injury hyperactive/impulsive behaviors in young adults with traumatic brain injury.


title: Stabilising microbial communities by looped mass transfer

abstract: Creating structurally and functionally stable microbiomes would be greatly beneficial to biotechnology and human health but so far has proven challenging. Here, we propose a looped mass transfer design that keeps microbiomes constant over long periods of time. The effluent of five parallel reactors that began with the same inoculum, was mixed in a reactor that represented a regional pool. Part of this pool was transferred back to the five reactors. Community dynamics were monitored and visualized by quantitative microbial flow cytometry and selected taxonomic sequencing of whole communities and sorted subcommunities. The rescue effect, known from metacommunity theory, was the main stabilizing mechanism that led to the survival of subcommunities with zero netgrowth, especially at high mass transfer rates. The looped mass transfer approach promises to overcome notorious stochastic structural fluctuations in bioreactors and has the potential to design and stabilize communities that can perform desired functions.


title: A Lentiviral Envelope Signal Sequence is a Tetherin Antagonizing Protein

abstract: Signal sequences are N-terminal peptides, generally less than 30 amino acids in length, that direct translocation of proteins into the endoplasmic reticulum and secretory pathway. The envelope glycoprotein (Env) of the nonprimate lentivirus Feline immunodeficiency virus (FIV) contains the longest signal sequence of all eukaryotic, prokaryotic and viral proteins (175 amino acids). The reason is unknown. Tetherin is a dual membrane-anchored host protein that inhibits the release of enveloped viruses from cells. Primate lentiviruses have evolved three antagonists: the small accessory proteins Vpu and Nef, and in the case of HIV-2, Env. Here we identify the FIV Env signal sequence (Fess) as the FIV tetherin antagonist. A short deletion in the central portion of Fess had no effect on viral replication in the absence of tetherin but severely impaired virion budding in its presence. Fess is necessary and sufficient, acting as an autonomous accessory protein with the rest of Env dispensable. In contrast to primate lentivirus tetherin antagonists, it functions by stringently blocking the incorporation of this restriction factor into viral particles rather than by degrading it or downregulating it from the plasma membrane.


title: Critical Role for Isoprenoids in Apicoplast Biogenesis by Malaria Parasites

abstract: Isopentenyl pyrophosphate (IPP) is an essential metabolic output of the apicoplast organelle in Plasmodium falciparum malaria parasites and is required for prenylation-dependent vesicular trafficking and other cellular processes. We have elucidated a critical and previously uncharacterized role for IPP in apicoplast biogenesis. Inhibiting IPP synthesis blocks apicoplast elongation and inheritance by daughter merozoites, and apicoplast biogenesis is rescued by exogenous IPP and polyprenols. Knockout of the only known isoprenoid-dependent apicoplast pathway, tRNA prenylation by MiaA, has no effect on blood-stage parasites and thus cannot explain apicoplast reliance on IPP. However, we have localized an annotated polyprenyl synthase (PPS) to the apicoplast lumen. PPS knockdown is lethal to parasites, rescued by IPP, and blocks apicoplast biogenesis, thus explaining apicoplast dependence on isoprenoid synthesis. We hypothesize that PPS synthesizes long-chain polyprenols critical for apicoplast membrane fluidity and biogenesis. This work critically expands the paradigm for isoprenoid utilization in malaria parasites and identifies a novel essential branch of apicoplast metabolism suitable for therapeutic targeting.


title: GFAP splice variants fine-tune glioma cell invasion and tumour dynamics by modulating migration persistence.

abstract: Glioma is the most common form of malignant primary brain tumours in adults. Their highly invasive nature makes the disease incurable to date, emphasizing the importance of better understanding the mechanisms driving glioma invasion. Glial fibrillary acidic protein (GFAP) is an intermediate filament protein that is characteristic for astrocyte- and neural stem cell-derived gliomas. Glioma malignancy is associated with changes in GFAP alternative splicing, as the canonical isoform GFAP is downregulated in higher-grade tumours, leading to increased dominance of the GFAP{delta} isoform in the network. In this study, we used intravital imaging and an ex vivo brain slice invasion model. We show that the GFAP{delta} and GFAP isoforms differentially regulate the tumour dynamics of glioma cells. Depletion of either isoform increases the migratory capacity of glioma cells. Remarkably, GFAP{delta}-depleted cells migrate randomly through the brain tissue, whereas GFAP-depleted cells show a directionally persistent invasion into the brain parenchyma. This study shows that distinct compositions of the GFAP-network lead to specific migratory dynamics and behaviours of gliomas.

category: cell biology


title: GFAP splice variants fine-tune glioma cell invasion and tumour dynamics by modulating migration persistence


title: Lifelong single-cell profiling of cranial neural crest diversification

abstract:
The cranial neural crest generates a huge diversity of derivatives, including the bulk of connective and skeletal tissues of the vertebrate head. How neural crest cells acquire such extraordinary lineage potential remains unresolved. By integrating single-cell transcriptome and chromatin accessibility profiles of cranial neural crest-derived cells across the zebrafish lifetime, we observe region-specific establishment of enhancer accessibility for distinct fates. Neural crest-derived cells rapidly diversify into specialized progenitors, including multipotent skeletal progenitors, stromal cells with a regenerative signature, fibroblasts with a unique metabolic signature linked to skeletal integrity, and gill-specific progenitors generating cell types for respiration. By retrogradely mapping the emergence of lineage-specific chromatin accessibility, we identify a wealth of candidate lineage-priming factors, including a Gata3 regulatory circuit for respiratory cell fates. Rather than multilineage potential being an intrinsic property of cranial neural crest, our findings support progressive and region-specific chromatin remodeling underlying acquisition of diverse neural crest lineage potential.

HighlightsO_LISingle-cell transcriptome and chromatin atlas of cranial neural crest
C_LIO_LIProgressive emergence of region-specific cell fate competency
C_LIO_LIChromatin accessibility mapping identifies candidate lineage regulators
C_LIO_LIGata3 function linked to gill-specific respiratory program
C_LI

Graphical Abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=173 SRC="FIGDIR/small/456710v1_ufig1.gif" ALT="Figure 1">
View larger version (69K):
org.highwire.dtl.DTLVardef@1c4ca91org.highwire.dtl.DTLVardef@cf0d10org.highwire.dtl.DTLVardef@162bfe9org.highwire.dtl.DTLVardef@2565fd_HPS_FORMAT_FIGEXP  M_FIG C_FIG

category: developmental biology


title: Fatty acid-binding proteins and fatty acid synthase influence glial reactivity and promote the formation of Müller glia-derived progenitor cells in the avian retina

abstract: The capacity for retinal regeneration varies greatly across vertebrates species. A recent comparative epigenetic and transcriptomic investigation of Muller glial (MG) in the retinas of fish, birds and mice revealed that Fatty Acid Binding Proteins (FABPs) are among the most highly up-regulated genes in activated chick MG (Hoang et al., 2020). Herein we provide an in-depth follow-up investigation to describe patterns of expression and how FABPs and fatty acid synthase (FASN) influence glial cells in the chick retina. During development, FABP7 is highly expressed by embryonic retinal progenitor cells (eRPCs) and maturing MG, whereas FABP5 is gradually up-regulated in maturing MG and remains elevated in mature glial cells. PMP2 (FABP8) is expressed by oligodendrocytes and FABP5 is expressed by non-astrocytic inner retinal glial cells, and both of these FABPs are significantly up-regulated in activated MG in damaged or growth factor-treated retinas. In addition to suppressing the formation of MGPCs, we find that FABP-inhibition suppressed the accumulation of proliferating microglia, although the microglia appeared highly reactive. scRNA-seq analyses of cells treated with FABP-inhibitor revealed distinct changes in patterns of expression suggesting that FABPs are involved in the transitions of MG from a resting state to a reactive state and conversion from reactive MG to MGPCs. Inhibition of FABPs in undamaged retinas had a significant impact upon the transcriptomic profiles of MG, with up-regulation of genes associated with gliogenesis, decreases in genes associated with neurogenesis, and suppression of the ability of MG to become MGPCs. scRNA-seq analyses of microglia indicated that FABP inhibition enhances gene modules related to reactivity, proliferation and cytokine signaling. We find that the proliferation of retinal progenitors in the circumferential marginal zone (CMZ) is unaffected by FABP-inhibitor. Upstream of FABP activity, we inhibited FASN in damaged retinas, which reduced numbers of dying cells, increased the proliferation of microglia, and potently suppressed the formation MGPCs in damaged retinas. We conclude that the activity of FASN and FABPs are required early during the formation of proliferating MGPCs. Fatty acid metabolism and cell signaling involving fatty acids are important in regulating glial homeostasis in the retina, and the dedifferentiation and proliferation of microglia and MGPCs.


title: Heterozygous loss of ZBTB38 leads to early embryonic lethality in mice via suppressing Nanog and Sox2

abstract: Mammalian DNA methylation is an epigenetic modification which is involved in various biological processes, including gene expression regulation. In mice, methyltransferases are responsible for DNA methylation, which are critical for early embryogenesis. However, the significance of methyl-CpG binding proteins (MBPs) that bind methylated CpG remains largely unknown. We previously demonstrated that ZBTB38/CIBZ-a zinc finger type of MBP-is required for ES cell proliferation by positively regulating Nanog expression. However, the physiological function of ZBTB38 remains unclear. In this study, we generated conditional ZBTB38 knockout mice using Cre-loxP technology. Unexpectedly, our results showed that germline loss of the ZBTB38 single allele resulted in decreased epiblast cell proliferation and increased apoptosis shortly after implantation, leading to early embryonic lethality. We found that heterozygous loss of ZBTB38 reduced the expression of Nanog, Sox2, and the genes responsible for epiblast proliferation, differentiation, and cell viability. Despite this lethal phenotype, ZBTB38 is dispensable for ES cell establishment and identity. Together, these findings indicate that ZBTB38 is essential for early embryonic development, providing new insights into the roles of MBP in implantation.


title: CNest: A Novel Copy Number Association Discovery Method Uncovers 862 New Associations from 200,629 Whole Exome Sequence Datasets in the UK Biobank

abstract: Copy number variation (CNV) has long been known to influence human traits having a rich history of research into common and rare genetic disease and although CNV is accepted as an important class of genomic variation, progress on copy number (CN) phenotype associations from Next Generation Sequencing data (NGS) has been limited, in part, due to the relative difficulty in CNV detection and an enrichment for large numbers of false positives. To date most successful CN genome wide association studies (CN-GWAS) have focused on using predictive measures of dosage intolerance or gene burden tests to gain sufficient power for detecting CN effects. Here we present a novel method for large scale CN analysis from NGS data generating robust CN estimates and allowing CN-GWAS to be performed genome wide in discovery mode. We provide a detailed analysis in the large scale UK BioBank resource and a specifically designed software package for deriving CN estimates from NGS data that are robust enough to be used for CN-GWAS. We use these methods to perform genome wide CN-GWAS analysis across 78 human traits discovering 862 genetic associations that are likely to contribute strongly to trait distributions based solely on their CN or by acting in concert with other genetic variation. Finally, we undertake an analysis comparing CNV and SNP association signals across the same traits and samples, defining specific CNV association classes based on whether they could be detected using standard SNP-GWAS in the UK Biobank.

category: genomics


title: ReporterSeq reveals genome-wide determinants of proteasome expression

abstract: The ubiquitin-proteasome system (UPS) is critical for cellular and organismal health. To uncover mechanisms regulating the UPS in normal and stress conditions, we systematically probed the genome of the eukaryotic model system Saccharomyces cerevisiae for modulators of the UPS master regulator Rpn4 under basal and stress conditions using the reverse genetic method ReporterSeq. The top UPS regulators were the thioredoxin reductase Trr1 and proteins of the large ribosomal subunit, both of which had no previously known role in UPS regulation. Unlike all known mechanisms for Rpn4 regulation which regulate Rpn4 levels, we found that Trr1 modulates the molecular activity of Rpn4 and does so in response to oxidative stress. Our work illuminates the genetic landscape through which cells regulate the UPS, and provides insight into how cells combat proteotoxicity.


title: Variability of social interactions in new versus established relationships in female degus

abstract: When an organism explores a new environment or stimulus it varies its behavior to ensure proper sampling. As contingencies are learned, behavioral variance can give-way to routines and stereotypies. This phenomenon is common across animal species and non-biological learning systems but has not been well studied in the social domain, in which the stimulus an animal investigates, another individual, may react negatively to unexpected behaviors. Here we develop a set of generalizable measures of interaction variability to test the hypothesis that social relationship formation is associated with initially high variability, followed by stabilization of dyadic interaction patterns. We focus these tests on female degus (Octodon degus), rodents predisposed to form cooperative peer relationships. Degus were presented with a series of 20 minute, dyadic "reunion" sessions across days, interleaving exposures to familiar and unfamiliar same-sex conspecifics. We found that degu pairs could be distinguished from one-another by their session-to-session interactive behaviors, suggesting dyad-specific social relationships. Consistent with predictions, stranger interactions changed from the first to second exposure, after which they became less variable across days. Unexpectedly, however, long-term cagemates showed overall more interaction variability across sessions than strangers. Taking into account all interaction variability measures, differences between strangers and cagemates were only observed when considering many (> 10) interactions, or longer (>5 min, or session-to-session) time periods. Shorter scales, including interaction transitions, showed meaningful patterns but no reliable group differences. The results suggest that while variability of social behavior over minutes or across sessions may change as relationships develop, interactions between unfamiliar adult female degus are not systematically more variable than those of cagemates. Given known ecological pressures on female degus to form large, stable social networks, these patterns are consistent with the notion that interaction variability may be maladaptive for social coordination.

category: animal behavior and cognition


title: Variability of social interactions in new and established relationships in female degus


title: Super-Resolution Electrochemical Impedance Imaging with a 100 x 100 CMOS Sensor Array

abstract: This paper presents a 100 x 100 super-resolution integrated sensor array for microscale electrochemical impedance spectroscopy (EIS) imaging. The system is implemented in 180 nm CMOS with 10 m x 10 m pixels. Rather than treating each electrode independently, the sensor is designed to measure the mutual capacitance between programmable sets of pixels. Multiple spatially-resolved measurements can then be computationally combined to produce super-resolution impedance images. Experimental measurements of sub-cellular permittivity distributions within single algae cells demonstrate the potential of this new approach.


title: epiRomics: a multi-omics R package to identify and visualize enhancers

abstract:
SummaryepiRomics is an R package designed to integrate multi-omics data in order to identify and visualize enhancer regions alongside gene expression and other epigenomic modifications. Regulatory network analysis can be done using combinatory approaches to infer regions of significance such as enhancers, when combining ChIP and histone data. Downstream analysis can identify co-occurrence of these regions of interest with other user-supplied data, such as chromatin availability or gene expression. Finally, this package allows for results to be visualized at high resolution in a stand-alone browser.

Availability and ImplementationepiRomics is released under Artistic-2.0 License. The source code and documents are freely available through Github (https://github.com/Huising-Lab/epiRomics).

Contactammawla@ucdavis.edu or mhuising@ucdavis.edu

Supplementary informationSupplementary data, and methods are available online on biorxiv or Github.

category: bioinformatics


title: Pancreatic ductal adenocarcinoma cells employ integrin α6β4 to form hemidesmosomes and regulate cell proliferation

abstract:
Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis due to its aggressive progression, late detection and lack of druggable driver mutations, which often combine to result in unsuitability for surgical intervention. Together with activating mutations of the small GTPase KRas, which are found in over 90% of PDAC tumours, a contributory factor for PDAC tumour progression is formation of a rigid extracellular matrix (ECM) and associated desmoplasia. This response leads to aberrant integrin signalling, and accelerated proliferation and invasion. To identify the integrin adhesion systems that operate in PDAC, we analysed a range of pancreatic ductal epithelial cell models using 2D, 3D and organoid culture systems. Proteomic analysis of isolated integrin receptor complexes from human pancreatic ductal epithelial (HPDE) cells predominantly identified integrin 6{beta}4 and hemidesmosome components, rather than classical focal adhesion components. Electron microscopy, together with immunofluorescence, confirmed the formation of hemidesmosomes by HPDE cells, both in 2D and 3D culture systems. Similar results were obtained for the human PDAC cell line, SUIT-2. Analysis of HPDE cell secreted proteins and cell-derived matrices (CDM) demonstrated that HPDE cells secrete a range of laminin subunits and form a hemidesmosome-specific, laminin 332-enriched ECM. Expression of mutant KRas (G12V) did not affect hemidesmosome composition or formation by HPDE cells. Cell-ECM contacts formed by mouse and human PDAC organoids were also assessed by electron microscopy. Organoids generated from both the PDAC KPC mouse model and human patient-derived PDAC tissue displayed features of acinar-ductal cell polarity, and hemidesmosomes were visible proximal to prominent basement membranes. Furthermore, electron microscopy identified hemidesmosomes in normal human pancreas. Depletion of integrin {beta}4 using siRNA reduced cell proliferation in both SUIT-2 and HPDE cells, reduced the number of SUIT-2 cells in S-phase, and induced G1 cell cycle arrest, indicating a requirement for 6{beta}4-mediated adhesion for cell cycle progression and growth. Taken together, these data suggest that laminin-binding adhesion mechanisms in general, and hemidesmosome-mediated adhesion in particular, may be under-appreciated in the context of PDAC.

Proteomic data are available via ProteomeXchange with the identifiers PXD027803, PXD027823 and PXD027827.


title: Oculomotor freezing tracks perception and is immune to decision bias

abstract:
The appearance of a salient stimulus rapidly inhibits saccadic eye movements. Curiously, this "oculomotor freezing" reflex is triggered only by stimuli that the observer reports seeing. It remains unknown, however, if oculomotor freezing is linked to the observers sensory experience, or their decision that a stimulus was present. To dissociate between these possibilities, we manipulated decision criterion via monetary payoffs and stimulus probability in a detection task. These manipulations greatly shifted observers decision criteria but did not affect the degree to which microsaccades were inhibited by stimulus presence. Moreover, the link between oculomotor freezing and explicit reports of stimulus presence was stronger when the criterion was conservative rather than liberal. We conclude that the sensory threshold for oculomotor freezing is independent of decision bias. Provided that conscious experience is also unaffected by such bias, oculomotor freezing is an implicit indicator of sensory awareness.

New & NoteworthySometimes a visual stimulus reaches awareness, and sometimes it does not. To understand why, we need objective, bias-free measures of awareness. We discovered that a reflexive freezing of small eye movements indicates when an observer detects a stimulus. Furthermore, when we biased observers decisions to report seeing the stimulus, the oculomotor reflex was unaltered. This suggests that the threshold for conscious perception is independent of the decision criterion and is revealed by oculomotor freezing.


title: Oculomotor freezing indicates conscious detection free of decision bias


title: Accurate predictions of individual differences in task-evoked brain activity from resting-state fMRI using a sparse ensemble learner

abstract: Modelling and predicting individual differences in task-evoked FMRI activity can have a wide range of applications from basic to clinical neuroscience. It has been shown that models based on resting-state activity can have high predictive accuracy. Here we propose several improvements to such models. Using a sparse ensemble leaner, we show that (i) features extracted using Stochastic Probabilistic Functional Modes (sPROFUMO) outperform the previously proposed dual-regression approach, (ii) that the shape and overall intensity of individualised task activations can be modelled separately and explicitly, (iii) training the model on predicting residual differences in brain activity further boosts individualised predictions. These results hold for both surface-based analyses of the Human Connectome Project data as well as volumetric analyses of UK-biobank data. Overall, our model achieves state of the art prediction accuracy on par with the test-retest reliability of tfMRI scans, suggesting that it has potential to supplement traditional task localisers.


title: Iron oxidation is regulated by the two-component system, RegSR, and plays a role in photolithoheterotrophic growth in Rhodopseudomonas palustris

abstract:
Purple nonsulfur bacteria (PNSB) are metabolically versatile organisms generate energy through both aerobic and anaerobic respiration as well as anoxygenic photosynthesis. In many PNSB, the redox-sensing, two-component system RegBA is a global regulator of energy generating and consuming pathways, such as photosynthesis, carbon fixation, and nitrogen fixation, when cells are shifted from an aerobic to an anaerobic environment. However, in the PNSB Rhodopseudomonas palustris, the role of the RegBA homolog, RegSR, was unclear since global regulation of these same pathways involves the oxygen-sensing signal transduction system, FixJL-K, in R. palustris. Using RNA-seq analysis, we found that RegSR plays a role in regulating the operon pioABC, which encodes genes required for Fe(II) oxidation. We found that transcript levels of pioABC under photoheterotrophic conditions was dependent on the oxidation state of the carbon substrate and whether the cells were fixing nitrogen. We also found that R. palustris can carry out photolithoheterotrophic growth using Fe(II) oxidation when grown with the oxidized carbon substrate, malate, requiring regSR and pioABC. We present a model in which RegSR regulates pioABC in response to a cellular redox signal, allowing R. palustris to use Fe(II) oxidation to access more electrons when there is an increased cellular demand for reducing equivalents.

SignificanceMixotrophy is thought to be widespread in aquatic environments, yet little is understood about how mixotrophy affects biogeochemical cycles. Fe(II)-oxidizing anoxygenic phototrophs likely play an important role in iron cycling since they are thought to have thrived in the anoxic, iron-rich oceans of early Earth and can be found in both freshwater and marine environments. Although Fe(II) oxidation by anoxygenic phototrophs is largely studied in the context of photoautotrophic growth, these organisms can also grow photoheterotrophically. We present the first evidence linking photolithoheterotrophic growth using Fe(II) to the pathway required for photoautotrophic Fe(II) oxidation in an anoxygenic phototroph. Understanding this metabolism will be important for understanding how mixotrophic metabolism contributes to iron cycling in anoxic environments.


title: Stromal-cell deletion of STAT3 protects mice from kidney fibrosis by inhibiting pericytes trans-differentiation and migration

abstract:
Signal transducer and activator of transcription 3 (STAT3) is a key transcription factor implicated in the pathogenesis of kidney fibrosis. Although tubular Stat3 deletion in tubular epithelial cells is known to protect mice from kidney fibrosis, the exact function of STAT3 in stromal cells remains unknown. We utilized stromal-cell Stat3 knock-out (KO) mice, CRISPR and pharmacologic activators and inhibitors of STAT3 to investigate its function in pericyte-like cells. STAT3 is phosphorylated in tubular epithelial cells in acute kidney injury whereas its activation expanded to interstitial cells in chronic kidney disease in mice and humans. Stromal cell-specific deletion of Stat3 protects mice from folic acid- and aristolochic acid-induced kidney fibrosis. Mechanistically, STAT3 directly regulates the inflammatory pathway, differentiation of pericytes into myofibroblasts. Specifically, STAT3 activation leads to an increase in migration and profibrotic signaling in genome-edited pericyte-like cells, 10T1/2. Conversely, Stat3 KO or blocking STAT3 function inhibits detachment, spreading, migration, and profibrotic signaling. Furthermore, STAT3 binds to Collagen1a1 promoter of fibrotic mouse kidneys and in pericyte-like cells. Together, our study identifies a previously unknown function of STAT3 in stromal cells that promotes kidney fibrosis and may have therapeutic value in fibrotic kidney disease.

Graphical Abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=73 SRC="FIGDIR/small/456872v1_ufig1.gif" ALT="Figure 1">
View larger version (27K):
org.highwire.dtl.DTLVardef@35dcbforg.highwire.dtl.DTLVardef@2fc055org.highwire.dtl.DTLVardef@fc0d92org.highwire.dtl.DTLVardef@1def2e6_HPS_FORMAT_FIGEXP  M_FIG C_FIG


title: Ethology of morphogenesis reveals the design principles of cnidarian size and shape development

abstract: During development, organisms interact with their natural habitats while undergoing morphological changes, yet it remains unclear whether the interplay between developing systems and their environments impacts animal morphogenesis. Here, we use the cnidarian Nematostella vectensis as a developmental model to uncover a mechanistic link between organism size, shape and behavior. Using quantitative live imaging, including extensive behavioral profiling, combined with molecular and biophysical experiments, we demonstrate that the muscular hydraulic machinery that controls body movement directly drives larva-polyp morphogenesis. Unexpectedly, size and shape development are differentially controlled by antagonistic muscles. A simple theoretical model shows how a combination of slow-priming and fast-pumping pressures generated by muscular hydraulics acts as a global mechanical regulator that coordinates tissue remodeling. Altogether, our findings illuminate how dynamic behavioral modes in the environment can be harnessed to drive morphogenetic trajectories, establishing ethology as a critical component of organismal morphogenesis - termed ethology of morphogenesis.


title: Ecological conditions experienced by bat reservoir hosts predict the intensity of Hendra virus excretion over space and time

abstract: The ecological conditions experienced by wildlife reservoir hosts affect the amount of pathogen they excrete into the environment. This then shapes pathogen pressure, the amount of pathogen available to recipient hosts over space and time, which affects spillover risk. Few systems have data on both long-term ecological conditions and pathogen pressure, yet such data are critical for advancing our mechanistic understanding of ecological drivers of spillover risk. To identify these ecological drivers, we here reanalyze shedding data from a spatially replicated, multi-year study of Hendra virus excretion from Australian flying foxes in light of 25 years of long-term data on changing ecology of the bat reservoir hosts. Using generalized additive mixed models, we show that winter virus shedding pulses, previously considered idiosyncratic, are most pronounced after recent food shortages and in bat populations that have been displaced to novel habitats. We next derive the area under each annual shedding curve (representing cumulative virus excretion) and show that pathogen pressure is also affected by the ecological conditions experienced by bat populations. Finally, we illustrate that pathogen pressure positively predicts observed spillover frequency. Our study suggests that recent ecological conditions of flying fox hosts are shifting the timing, magnitude, and cumulative intensity of Hendra virus shedding in ways that shape the landscape of spillover risk. This work provides a mechanistic approach to understanding and estimating risk of spillover from reservoir hosts in complex ecological systems and emphasizes the importance of host ecological context in identifying the determinants of pathogen shedding.

category: ecology


title: A flexible point and variance estimator to assess bird/bat fatality from carcass searches

abstract: Estimation of bird and bat fatalities due to collision with anthropogenic structures (such as power lines or wind turbines) is an important ecological issue. However, searching for collision victims usually only detects a proportion of the true number of collided individuals. Various mortality estimators have previously been proposed to correct for this incomplete detection, based on regular carcass searches and additional field experiments. However, each estimator implies specific assumptions/restrictions, which may easily be violated in practice. In this study, we extended previous approaches and developed a versatile algorithm to compute point and variance estimates for true carcass numbers. The presented method allows for maximal flexibility in the data structure. Using simulated data, we showed that our point and variance estimators ensured unbiased estimates under various challenging data conditions. The presented method may improve the estimation of true collision numbers, as an important pre-condition for calculating collision rates and evaluating measures to reduce collision risks, and may thus provide a basis for management decisions and/or compensation actions with regard to planned or existing wind turbines and power lines.


title: Mapping of genotype-by-environment interactions in phenology identifies two cues for flowering in switchgrass (Panicum virgatum)

abstract:
Plant phenological timings are major fitness components affected by multiple environmental cues; thus, phenological traits can have important genotype-by-environment interactions (GxE). We use a flexible, data-driven method to characterize GxE in the timing of vegetative growth ( green-up) and flowering across eight environments and in two highly divergent switchgrass (Panicum virgatum) populations. We classified polygenic GxE patterns as suggestive of modulation of genetic effects via weather-based cues--or other, unknown modulators. >26% of Gulf population SNPs affecting flowering had effects that covaried with photoperiodicity and >34% of Midwest upland population SNPs affecting flowering had effects that covaried with cumulative growing degree days. 76% of SNP effects on green-up showed antagonistic pleiotropy, a change in effect sign, between environments native to Gulf plants (Texas) and environments native to Midwest plants (North). In contrast, <2% of flowering effects showed antagonistic pleiotropy; the majority (>64%) showed no GxE. Top GxE-associated SNPs were highly enriched in the top associations from an independent pseudo-F2 cross of individuals from the same two populations. Breeding for particular alleles at GxE-associated loci could change flowering responsiveness to photoperiod cues in switchgrass. More broadly, this approach could be used to flexibly characterize patterns of GxE across species, populations and environments.

Significance StatementThe timing of plant seasonal development (phenology) has major impacts on fitness because of the negative consequences of plant-environment mismatches. Here we map the genetic basis of two phenological events, the start of above-ground growth and flowering, in two genetically and phenologically distinct populations of switchgrass. We do this at eight field locations spanning the latitudinal range of both populations. Our approach allows us to identify regions of the genome with effects that covary with weather-related environmental features at every location. For flowering, these features differed by population: the Midwest population had genetic effects that primarily covaried with cumulative growing degree days, a temperature-related measure, while the Gulf population had genetic effects that primarily covaried with photoperiod, a day-length-related measure.

category: genetics


title: Structures of the Human SPAK and OSR1 Conserved C-Terminal (CCT) Domains

abstract: STE20/SPS1-related proline/alanine-rich kinase (SPAK) and Oxidative Stress Responsive 1 (OSR1) kinase are two serine/threonine protein kinase that regulate the function of ion co-transporters through phosphorylation. The highly conserved C-terminal (CCT) domains of SPAK and OSR1 bind to RFx[V/I] peptide sequences from their upstream With No Lysine Kinases (WNKs), facilitating their activation via phosphorylation. Thus, the inhibition of SPAK and OSR1 binding, via their CCT domains, to WNK kinases is a plausible strategy for inhibiting SPAK and OSR1 kinases. To facilitate structure-guided drug design of such inhibitors, we expressed and purified human SPAK and OSR1 CCT domains and solved their crystal structures. We also employed a biophysical strategy and determined the affinity of SPAK and OSR1 CCT domains to an 18-mer peptide derived from WNK4. Together, the crystal structures and affinity data reported herein provide a robust platform to facilitate the design of CCT domain specific small molecule inhibitors of SPAK-activation by WNK kinases, potentially leading to new improved treatments for hypertension and ischemic stroke.


title: Somatic mutation rates scale with lifespan across mammals

abstract: The rates and patterns of somatic mutation in normal tissues are largely unknown outside of humans. Comparative analyses can shed light on the diversity of mutagenesis across species and on long-standing hypotheses regarding the evolution of somatic mutation rates and their role in cancer and ageing. Here, we used whole-genome sequencing of 208 intestinal crypts from 56 individuals to study the landscape of somatic mutation across 16 mammalian species. We found somatic mutagenesis to be dominated by seemingly endogenous mutational processes in all species, including 5-methylcytosine deamination and oxidative damage. With some differences, mutational signatures in other species resembled those described in humans, although the relative contribution of each signature varied across species. Remarkably, the somatic mutation rate per year varied greatly across species and exhibited a strong inverse relationship with species lifespan, with no other life-history trait studied displaying a comparable association. Despite widely different life histories among the species surveyed, including [~]30-fold variation in lifespan and [~]40,000-fold variation in body mass, the somatic mutation burden at the end of lifespan varied only by a factor of [~]3. These data unveil common mutational processes across mammals and suggest that somatic mutation rates are evolutionarily constrained and may be a contributing factor in ageing.


title: Microbial communities in retail draft beers and the biofilms they produce

abstract:
In the beer brewing industry, microbial spoilage presents a consistent threat that must be monitored and controlled to ensure the palatability of a finished product. Many of the predominant beer spoilage microbes have been identified and characterized, but the mechanisms of contamination and persistence remain an open area of study. Post-production, many beers are distributed as kegs that are attached to draft delivery systems in retail settings where ample opportunities for microbial spoilage are present. As such, restaurants and bars can experience substantial costs and downtime for cleaning when beer draft lines become heavily contaminated. Spoilage monitoring on the retail side of the beer industry is often overlooked, yet this arena may represent one of the largest threats to the profitability of a beer if its flavor profile becomes substantially distorted. In this study, we sampled and cultured microbial communities found in beers dispensed from a retail draft system to identify the contaminating bacteria and yeasts. We also evaluated their capability to establish new biofilms in a controlled setting. Among four tested beer types, we identified over a hundred different contaminant bacteria and nearly twenty wild yeasts. The culturing experiments demonstrated that most of these microbes were viable and capable of joining new biofilm communities. From these data, we provide an important starting point for the efficient monitoring of beer spoilage in draft systems and provide suggestions for cleaning protocol improvements that can benefit the retail community.

ImportanceBeer production, packaging, and service are each vulnerable to contamination by microbes that metabolize beer chemicals and impart undesirable flavors, which can result in the disposal of entire batches. Therefore, great effort is taken by brewmasters to reduce and monitor contamination during production and packaging. A commonly overlooked quality control stage of a beer supply chain is at the retail service end, where beer kegs supply draft lines in bars and restaurants under non-sterile conditions. We found that retail draft line contamination is rampant and that routine line cleaning methods are insufficient to efficiently suppress beer spoilage. Thus, many customers unknowingly experience spoiled versions of the beers they consume. This study identified the bacteria and yeast that were resident in draft beer samples and also assessed their abilities to colonize tubing material as members of stable biofilm communities.


title: IRE1/XBP1 signaling promotes skeletal muscle regeneration through a cell non-autonomous mechanism

abstract: Skeletal muscle regeneration is regulated by coordinated activation of multiple signaling pathways activated in both injured myofibers and satellite cells. The unfolded protein response (UPR) is a major mechanism that detects and alleviates protein-folding stresses in ER. However, the role of individual arms of the UPR in skeletal muscle regeneration remains less understood. In the present study, we demonstrate that IRE1 (also known as ERN1) and its downstream target, XBP1, are activated in skeletal muscle of mice upon injury. Myofiber-specific ablation of IRE1 or XBP1 in mice diminishes skeletal muscle regeneration that is accompanied with reduced number of satellite cells and their fusion to injured myofibers. Ex vivo cultures of myofiber explants demonstrate that ablation of IRE1 reduces the proliferative capacity of myofiber- associated satellite cells. Myofiber-specific deletion of IRE1 dampens Notch signaling and canonical NF-{kappa}B pathway in skeletal muscle of mice. Our results also demonstrate that targeted ablation of IRE1 reduces skeletal muscle regeneration in the mdx mice, a model of Duchenne muscular dystrophy. Collectively, our results reveal that the IRE1-mediated signaling promotes muscle regeneration through augmenting the proliferation of satellite cells in a cell non- autonomous manner.


title: The IRE1/XBP1 signaling axis promotes skeletal muscle regeneration through a cell non-autonomous mechanism


title: DPPA2 and DPPA4 are dispensable for mouse zygotic genome activation and preimplantation development

abstract: How maternal factors in oocytes initiate zygotic genome activation (ZGA) remains elusive. Recent studies indicate that DPPA2 and DPPA4 are required for establishing a 2-cell embryo-like (2C-like) state in mouse embryonic stem cells (ESCs) in a DUX-dependent manner. These results suggest that DPPA2 and DPPA4 are essential maternal factors that regulate Dux and ZGA in embryos. By analyzing maternal knockout and maternal-zygotic knockout embryos, we unexpectedly found that Dux activation, ZGA, and preimplantation development are normal in embryos without DPPA2 or DPPA4. Thus, unlike in ESCs/2C-like cells, DPPA2 and DPPA4 are dispensable for ZGA and preimplantation development.


title: Identification of Potential inhibitors for Hematopoietic Prostaglandin D2 Synthase: Computational Modeling and Molecular Dynamics Simulations

abstract: To design a new therapeutic agent for Hematopoietic Prostaglandin D2 synthase (hPGDS), a set of 60 molecules with different molecular scaffolds were (range of pIC50 values are from 8.301 to 3.932) considered to create a pharmacophore model. Further, identification of potential hPGDS inhibitors were carried out by using virtual screening with different databases (from 15,74,182 molecules). The Molecular screening was performed using different sequential methods right from Pharmacophore based virtual screening, molecular docking, MM-GBSAstudies, ADME property analysis and molecular dynamics simulations using Maestro11.9 software. Based on the best pharmacophore model (ADRR_1), the resultant set of 18,492 molecules were screened. The preliminarily screened molecules were subjected to molecular docking (PDB_ID: 2CVD) methods. A set of 27 molecules was screened from the resultant molecular docking outcomes (360 molecules) based on binding free energy ({Delta}Gbind) and Lipinskis rule of five. Out of 27 molecules, 4 were selected visual data analysis and further subjected to molecular dynamics (MD) simulation study. Outcomes of the present study conclude with three new proposed molecules (SP1, SP2 and SP10) which show a good range of interaction with human hPGDS enzyme in comparison to the marketed compounds i.e., HQL-79, TFC-007, HPGDS inhibitor I and TAS-204.