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Background: Smith-Lemli-Opitz syndrome (SLOS) is an inherited disorder of cholesterol biosynthesis associated with congenital malformations, growth delay, intellectual disability and behavior problems. SLOS is caused by bi-allelic mutations in DHCR7, which lead to reduced activity of 7-dehydrocholesterol reductase that catalyzes the last step in cholesterol biosynthesis. Symptoms of SLOS are thought to be due to cholesterol deficiency and accumulation of its precursor 7-dehydrocholesterol (7-DHC) and 8-dehydrocholesterol (8-DHC), and toxic oxysterols. Therapy for SLOS often includes dietary cholesterol supplementation, but lipids are poorly absorbed from the diet, possibly due to impaired bile acid synthesis. We hypothesized that bile acid supplementation with cholic acid would improve dietary cholesterol absorption and raise plasma cholesterol levels. Methods: Twelve SLOS subjects (10 M, 2F, ages 2–27 years) who had plasma cholesterol ≤125 mg/dL were treated with cholic acid (10 mg/kg/day) divided twice daily for 2 months. Plasma cholesterol, 7-DHC and 8-DHC were measured by GC–MS. Oxysterols were measured by ultra-high-performance LC-MS/MS. Data were analyzed using paired t-tests. Results: At baseline, plasma cholesterol was 75 ± 24 mg/dL (mean ± SD; range 43–125, n = 12). After 2 months on cholic acid, mean plasma cholesterol increased to 97 ± 29 mg/dL (p = 0.011). Eleven of 12 subjects showed an increase in plasma cholesterol that varied from 3.8% to 85.7% (mean 38.7 ± 23.3%). 7-Hydroxycholesterol decreased by 20.6% on average (p = 0.013) but no significant changes were seen in 7-DHC or 8-DHC. Mean body weight tended to increase (3.6% p = 0.069). Subjects tolerated cholic acid well and experienced no drug-related adverse events. Conclusions: In this pilot study, cholic acid supplementation was well tolerated and safe and resulted in an increase in plasma cholesterol in most SLOS subjects. Further controlled longitudinal studies are needed to look for the sustainability of the biochemical effect and possible clinical benefits.

College promise programs, or place-based scholarships, are aimed at helping students attend and afford higher education. The American Dream Scholarship (ADS), offered by Miami Dade College (MDC), is a promise program that covers tuition and fees for the first 60 credits of an associate degree for students residing in and graduating from high school in Miami-Dade County. In this study, we relied on Bourdieu’s (1986) sociological framework to conceptualize the impact of the ADS on MDC’s student body. We posed the research question of whether the scholarship had any effect on total first-time, full-time college enrollment at MDC. Utilizing data primarily from the Integrated Postsecondary Education Data System, we used two comparison groups of colleges that were untreated by any county-level promise program: 26 colleges in the Florida College System and 37 public institutions nationally with the same Carnegie Classification as MDC. We applied difference-in-differences analyses and event studies to explore our research question. Results suggest that compared to untreated institutions in the Florida College System and institutions with the same Carnegie Classification, MDC enrolled 18.5% and 32% more first-time, full-time degree-seeking undergraduates, respectively, after ADS’s inception. This enrollment increase demonstrates that ADS positively contributed to MDC’s programmatic goals to increase student enrollments.

Here, by performing an integrative multi-omics analysis coupled to clinical features of COVID-19 patients prospectively followed for up to 6 month, the authors identify specific gut microbiome patterns associated with disease severity and development of post-acute COVID-19 syndrome.

BackgroundVirtual care was rapidly integrated into pediatric health services during the COVID-19 pandemic. While virtual care offers many benefits, it is necessary to better understand the experiences of those who receive, deliver, and coordinate virtual care in order to support sustainable, high-quality, and patient-centered health care. To date, methods implemented to evaluate users’ experiences of virtual care have been highly variable, making comparison and data synthesis difficult. ObjectiveThis study aims to describe evaluation strategies currently used to understand personal experiences with pediatric virtual care in Canada. MethodsIn this mixed methods environmental scan, we first distributed a web-based questionnaire to clinical, research, and operational leaders delivering and evaluating pediatric virtual care in Canada. The questionnaire collected information about how experiences with virtual care have been or are currently being evaluated and whether these evaluations included the perspectives of children or youth, families, providers, or support staff. Second, respondents were asked to share the questions they used in their evaluations, and a content analysis was performed to identify common question categories. Third, we conducted semistructured interviews to further explore our respondents’ evaluation experiences across 4 domains—evaluation approaches, distribution methods, response rates, and lessons learned—and interest in a core set of questions for future evaluations. ResultsThere were 72 respondents to the web-based questionnaire; among those who had conducted an evaluation, we identified 15 unique evaluations, and 14 of those provided a copy of the tools used to evaluate virtual care. These evaluations measured the virtual care experiences of parents or caregivers (n=15, 100%), children or youth (n=11, 73%), health care providers (n=11, 73%), and support staff (n=4, 27%). The most common data collection method used was electronic questionnaires distributed by email. Two respondents used validated tools; the remainder modified existing tools or developed new tools. Content analysis of the 14 submitted questionnaires revealed that the most common questions were about overall participant satisfaction, the comparison of virtual care to in-person care, and whether participants would choose virtual care options in the future. Interview findings indicate respondents frequently relied on methods used by peers and that a standardized, core set of questions to evaluate experiences with virtual care would be helpful to improve evaluation practices and support pediatric health care delivery. ConclusionsAt our institution and elsewhere in Canada, experiences with pediatric virtual care have been evaluated using a variety of methods. A more consistent evaluation approach using standardized tools may enable more regular comparisons of experiences with virtual care and the synthesis of findings across health care settings. In turn, this may better inform our approach to virtual care, improve its integration into health systems, and facilitate sustainable, high-quality, patient-centered care.

Abstract Background Glomerular endotheliosis is the pathognomonic glomerular lesion in pre-eclampsia that has also been described in those taking tyrosine kinase inhibitors for cancer treatment. Ibrutinib is a Bruton’s tyrosine kinase inhibitor used to treat chronic lymphocytic leukemia (CLL). We report the first known case of glomerular endotheliosis on kidney biopsy in a patient on ibrutinib monotherapy. Case presentation The patient presented with acute on chronic kidney disease, proteinuria, low C3 and C4 and a high rheumatoid factor titer. A kidney biopsy was performed to confirm a preliminary diagnosis of membranoproliferative glomerulonephritis (MPGN), the most common glomerular disease in patients with CLL. Unexpectedly, the kidney biopsy showed pre-eclampsia-like lesions on light and electron microscopy: occlusion of glomerular peripheral capillary lumens by swollen reactive endothelial cells. Findings of glomerulonephritis were not seen, and there were no specific glomerular immune deposits by immunofluorescence or electron microscopy. Conclusions CLL is known to cause glomerular lesions, mainly MPGN. There is increasing evidence that ibrutinib, a major treatment for CLL, can cause kidney disease, but the precise pathology is not characterized. We present a patient with CLL on ibrutinib with signs of glomerular endotheliosis. Based on the absence of CLL-induced kidney pathologies typically seen on the kidney biopsy and the non-selectivity of ibrutinib, we attributed the glomerular endotheliosis to ibrutinib. In pre-eclampsia, increased soluble fms-like tyrosine kinase 1 (sFlt1) levels induce endothelial dysfunction by decreasing vascular endothelial growth factor (VEGF). Ibrutinib has been demonstrated to have non-selective tyrosine kinase inhibition, including inhibition of VEGF receptor (VEGFR) and epidermal growth factor receptor (EGFR). VEGFR and EGFR inhibitors have recently been described in the literature to cause hypertension, proteinuria, and glomerular endotheliosis. Kidney biopsy should be performed in CLL patients on ibrutinib that present with acute kidney injury (AKI) or proteinuria to determine whether the clinical picture is attributable to the disease itself or a complication of the therapy.

Although lipids are known to affect Hedgehog (Hh) signalling, the underlying mechanisms remain unclear. Here, the authors show that Canopy4 regulates membrane sterol lipid levels, with knockout mouse embryos exhibiting digit number changes and other Hh signalling-related developmental defects.

Abstract Biobanking in health care has evolved over the last few decades from simple biological sample repositories to complex and dynamic units with multi‐organizational infrastructure networks and has become an essential tool for modern medical research. Cardiovascular tissue biobanking provides a unique opportunity to utilize cardiac and vascular samples for translational research into heart failure and other related pathologies. Current techniques for diagnosis, classification, and treatment monitoring of cardiac disease relies primarily on interpretation of clinical signs, imaging, and blood biomarkers. Further research at the disease source (i.e. myocardium and blood vessels) has been limited by a relative lack of access to quality human cardiac tissue and the inherent shortcomings of most animal models of heart disease. In this review, we describe a model for cardiovascular tissue biobanking and databasing, and its potential to facilitate basic and translational research. We share techniques to procure endocardial samples from patients with hypertrophic cardiomyopathy, heart failure with reduced ejection fraction, and heart failure with preserved ejection fraction, in addition to aortic disease samples. We discuss some of the issues with respect to data collection, privacy, biobank consent, and the governance of tissue biobanking. The development of tissue biobanks as described here has significant scope to improve and facilitate translational research in multi‐omic fields such as genomics, transcriptomics, proteomics, and metabolomics. This research heralds an era of precision medicine, in which patients with cardiovascular pathology can be provided with optimized and personalized medical care for the treatment of their individual phenotype.

Dilated Cardiomyopathy is a common form of heart failure. Determining how this disease affects the structure and organization of cardiomyocytes in the human heart is important in understanding how the heart becomes less effective at contraction. Here we isolated and characterised Affimers (small non-antibody binding proteins) to Z-disc proteins ACTN2 (α-actinin-2), ZASP (also known as LIM domain binding protein 3 or LDB3) and the N-terminal region of the giant protein titin (TTN Z1-Z2). These proteins are known to localise in both the sarcomere Z-discs and the transitional junctions, found close to the intercalated discs that connect adjacent cardiomyocytes. We use cryosections of left ventricles from two patients diagnosed with end-stage Dilated Cardiomyopathy who underwent Orthotopic Heart Transplantation and were whole genome sequenced. We describe how Affimers substantially improve the resolution achieved by confocal and STED microscopy compared to conventional antibodies. We quantified the expression of ACTN2, ZASP and TTN proteins in two patients with dilated cardiomyopathy and compared them with a sex- and age-matched healthy donor. The small size of the Affimer reagents, combined with a small linkage error (the distance from the epitope to the dye label covalently bound to the Affimer) revealed new structural details in Z-discs and intercalated discs in the failing samples. Affimers are thus useful for analysis of changes to cardiomyocyte structure and organisation in diseased hearts.

Dysbiosis of gut microbiota is well-described in patients with coronavirus 2019 (COVID-19), but the dynamics of antimicrobial resistance genes (ARGs) reservoir, known as resistome, is less known. Here, we performed longitudinal fecal metagenomic profiling of 142 patients with COVID-19, characterized the dynamics of resistome from diagnosis to 6 months after viral clearance, and reported the impact of antibiotics or probiotics on the ARGs reservoir. Antibiotic-naive patients with COVID-19 showed increased abundance and types, and higher prevalence of ARGs compared with non-COVID-19 controls at baseline. Expansion in resistome was mainly driven by tetracycline, vancomycin, and multidrug-resistant genes and persisted for at least 6 months after clearance of SARS-CoV-2. Patients with expanded resistome exhibited increased prevalence of Klebsiella sp. and post-acute COVID-19 syndrome. Antibiotic treatment resulted in further increased abundance of ARGs whilst oral probiotics (synbiotic formula, SIM01) significantly reduced the ARGs reservoir in the gut microbiota of COVID-19 patients during the acute infection and recovery phase. Collectively, these findings shed new insights on the dynamic of ARGs reservoir in COVID-19 patients and the potential role of microbiota-directed therapies in reducing the burden of accumulated ARGs.

Abstract Background A shortage of primary care physicians has been reported in many countries. Primary care systems are diverse and the challenges leading to a decline in workforce are at times context-specific and require tailored solutions. Inviting frontline clinicians to share their insights can help identify optimal strategies for a particular setting. To determine priorities for family physicians’ and general practitioners’ recruitment and retention in Singapore, we invited primary care physicians to rank pertinent strategies using PRIORITIZE, a transparent, systematic priority-setting approach. Methods The study advisory board, consisting of representatives of Singapore’s key primary care stakeholders, determined the criteria for prioritising of general practitioners (GPs) and family physicians (FPs) recruitment and retention strategies in Singapore. A comprehensive list of GPs and FPs recruitment and retention strategies was extracted from a recent systematic review of the relevant literature. A questionnaire listing the strategies and the scoring criteria was administered online to doctors practicing in public and private sector in Singapore. Respondents’ scores were combined to create a ranked list of locally most relevant strategies for improving GPs and FPs recruitment and retention. Results We recruited a diverse sample of 50 GPs and FPs practicing in a variety of primary care settings, many with a range of additional professional responsibilities. Around 60 and 66% of respondents thought that there was a problem with recruitment and retention of GPs and FPs in Singapore, respectively. Strategies focusing on promoting primary care by emphasizing the advantages and enhancing the status of the profession as well as training-related strategies, such as sub-specialisation and high-quality rotations were considered priorities for improving recruitment. For retention of GPs and FPs, improving working conditions by increasing GPs’ and FPs’ salary and recognition, as well as varying or reducing time commitment, were seen as the most important strategies. The ranking between physicians working in public and private sector was mostly similar, with nine out of the top ten recruitment and retention strategies being the same. Conclusion Primary care physicians’ ranking of recruitment and retention strategies for GPs and FPs in Singapore provide important insight into the challenges and the solutions as seen by the members of the profession themselves. This information can guide future policy and decision making in this area.

Defective 3β-hydroxysterol-Δ7 -reductase (DHCR7) in the developmental disorder, Smith-Lemli-Opitz syndrome (SLOS), results in a deficiency in cholesterol and accumulation of its precursor, 7-dehydrocholesterol (7-DHC). Here, we show that loss of DHCR7 causes accumulation of 7-DHC-derived oxysterol metabolites, premature neurogenesis from murine or human cortical neural precursors, and depletion of the cortical precursor pool, both in vitro and in vivo. We found that a major oxysterol, 3β,5α-dihydroxycholest-7-en-6-one (DHCEO), mediates these effects by initiating crosstalk between glucocorticoid receptor (GR) and neurotrophin receptor kinase TrkB. Either loss of DHCR7 or direct exposure to DHCEO causes hyperactivation of GR and TrkB and their downstream MEK-ERK-C/EBP signaling pathway in cortical neural precursors. Moreover, direct inhibition of GR activation with an antagonist or inhibition of DHCEO accumulation with antioxidants rescues the premature neurogenesis phenotype caused by the loss of DHCR7. These results suggest that GR could be a new therapeutic target against the neurological defects observed in SLOS.

Peripartum cardiomyopathy (PPCM) is a rare form of acute onset heart failure that presents in otherwise healthy pregnant women around the time of delivery. While most of these women respond to early intervention, about 20% progress to end-stage heart failure that symptomatically resembles dilated cardiomyopathy (DCM). In this study, we examined two independent RNAseq datasets from the left ventricle of end-stage PPCM patients and compared gene expression profiles to female DCM and non-failing donors. Differential gene expression, enrichment analysis and cellular deconvolution were performed to identify key processes in disease pathology. PPCM and DCM display similar enrichment in metabolic pathways and extracellular matrix remodeling suggesting these are similar processes across end-stage systolic heart failure. Genes involved in golgi vesicles biogenesis and budding were enriched in PPCM left ventricles compared to healthy donors but were not found in DCM. Furthermore, changes in immune cell populations are evident in PPCM but to a lesser extent compared to DCM, where the latter is associated with pronounced pro-inflammatory and cytotoxic T cell activity. This study reveals several pathways that are common to end-stage heart failure but also identifies potential targets of disease that may be unique to PPCM and DCM.

The disrupted organisation of the ryanodine receptors (RyR) and junctophilin (JPH) is thought to underpin the transverse tubule (t-tubule) remodelling in a failing heart. Here, we assessed the nanoscale organisation of these two key proteins in the failing human heart. Recently, an advanced feature of the t-tubule remodelling identified large flattened t-tubules called t-sheets, that were several microns wide. Previously, we reported that in the failing heart, the dilated t-tubules up to ~1 μm wide had increased collagen, and we hypothesised that the t-sheets would also be associated with collagen deposits. Direct stochastic optical reconstruction microscopy (dSTORM), confocal microscopy, and western blotting were used to evaluate the cellular distribution of excitation-contraction structures in the cardiac myocytes from patients with idiopathic dilated cardiomyopathy (IDCM) compared to myocytes from the non-failing (NF) human heart. The dSTORM imaging of RyR and JPH found no difference in the colocalisation between IDCM and NF myocytes, but there was a higher colocalisation at the t-tubule and sarcolemma compared to the corbular regions. Western blots revealed no change in the JPH expression but did identify a ~50% downregulation of RyR (p = 0.02). The dSTORM imaging revealed a trend for the smaller t-tubular RyR clusters (~24%) and reduced the t-tubular RyR cluster density (~35%) that resulted in a 50% reduction of t-tubular RyR tetramers in the IDCM myocytes (p < 0.01). Confocal microscopy identified the t-sheets in all the IDCM hearts examined and found that they are associated with the reticular collagen fibres within the lumen. However, the size and density of the RyR clusters were similar in the myocyte regions associated with t-sheets and t-tubules. T-tubule remodelling is associated with a reduced RyR expression that may contribute to the reduced excitation-contraction coupling in the failing human heart.

Off-target effects in CRISPR screens for essential regulatory elements have not been systematically evaluated. Here the authors find Cas9 nuclease, CRISPRi/a each have distinct off-target effects, and that these can be accurately identified and removed using the GuideScan sgRNA specificity score.

Resveratrol is a polyphenol that may improve weight loss outcomes in obese individuals. However, assessing the effectiveness of resveratrol supplementations as an appropriate intervention for weight loss in obesity across randomized control trials (RCTs) has been complicated by variability in their design. This study aims to evaluate design elements across RCTs of resveratrol interventions in obesity with weight loss as an end-point outcome, as recorded in ClinicalTrials.gov. We found discrepancies in participant inclusion criteria (sample size, age ranges, sex, BMI, medical conditions), interventional design (delivery modalities, dosages, duration) and primary outcomes measured (anthropomorphic, blood biomarkers). We identified a near three-fold variation in study sample size, two-fold variation in minimum inclusion age, five modalities of therapeutic resveratrol delivery with interventional durations ranging from two weeks to six months. Weight loss was only identified as a primary outcome in three of the seven studies evaluated. In conclusion, heterogeneity in trial design using resveratrol suggests that weight-loss-related outcomes are difficult to interpret and cross-validate. Indeed, conclusions drawn from human studies have been inconsistent, which may be attributed to study design heterogeneity including major differences in sample population, age, sex, BMI, underlying health conditions and end-point measures.

Background: The relationship between HIV (human immunodeficiency virus) and COVID-19 clinical outcome is uncertain, with conflicting data and hypotheses. We aimed to assess the prevalence of people living with HIV (PLWH) among COVID-19 cases and whether HIV infection affects the risk of severe COVID-19 or related death at the global and continental level. Methods: Electronic databases were systematically searched in July 2021. In total, 966 studies were screened following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Narratives were synthesised and data pooled for the global and continental prevalence of HIV–SARS-CoV-2 coinfection. The relative risks of severity and mortality in HIV-infected COVID-19 patients were computed using a random-effect model. Risk of bias was assessed using the Newcastle–Ottawa score and Egger’s test, and presented as funnel plots. Results: In total, 43 studies were included involving 692,032 COVID-19 cases, of whom 9097 (1.3%) were PLWH. The global prevalence of PLWH among COVID-19 cases was 2% (95% CI = 1.7–2.3%), with the highest prevalence observed in sub-Saharan Africa. The relative risk (RR) of severe COVID-19 in PLWH was significant only in Africa (RR = 1.14, 95% CI = 1.05–1.24), while the relative risk of mortality was 1.5 (95% CI = 1.45–2.03) globally. The calculated global risk showed that HIV infection may be linked with increased COVID-19 death. The between-study heterogeneity was significantly high, while the risk of publication bias was not significant. Conclusions: Although there is a low prevalence of PLWH among COVID-19 cases, HIV infection may increase the severity of COVID-19 in Africa and increase the risk of death globally.

Summary: Regulatory T cells (Tregs) can impair anti-tumor immune responses and are associated with poor prognosis in multiple cancer types. Tregs in human tumors span diverse transcriptional states distinct from those of peripheral Tregs, but their contribution to tumor development remains unknown. Here, we use single-cell RNA sequencing (RNA-seq) to longitudinally profile dynamic shifts in the distribution of Tregs in a genetically engineered mouse model of lung adenocarcinoma. In this model, interferon-responsive Tregs are more prevalent early in tumor development, whereas a specialized effector phenotype characterized by enhanced expression of the interleukin-33 receptor ST2 is predominant in advanced disease. Treg-specific deletion of ST2 alters the evolution of effector Treg diversity, increases infiltration of CD8+ T cells into tumors, and decreases tumor burden. Our study shows that ST2 plays a critical role in Treg-mediated immunosuppression in cancer, highlighting potential paths for therapeutic intervention. : Li et al. show in a genetic mouse model of lung adenocarcinoma that during tumor development regulatory T cell (Treg) diversity shifts from an interferon-responsive to a ST2-positive, Klrg1+Areg+ effector-like phenotype. Treg-specific deletion of ST2 alters Treg heterogeneity, increases tumor infiltration by CD8+ T cells, and decreases tumor burden. Keywords: regulatory T cell, autochthonous mouse model of cancer, lung adenocarcinoma, tumor immunosuppression, single cell RNA sequencing, Treg heterogeneity, interleukin-33, ST2, Il1rl1

CRISPR-Cas9 screens are powerful high-throughput tools but can be confounded by nuclease toxicity. Here the authors design a library of variable length gRNAs with thousands of negative controls, including the targeting of ‘safe’ loci to account for on-target site DNA damage toxicity.

Plant-soil feedback (PSF) can provide a driving force during ecological succession by altering soil properties in ways that benefit or disadvantage other species in the successional sequence. Succession may be inevitable in disturbed sites remediated by planting early successional species, but information on PSF in such settings is lacking. We investigated whether gray birch (Betula populifolia), a native species but strong invader, alters succession from grassland to deciduous forest at a site contaminated with zinc, lead, and cadmium. We investigated PSF within the context of competition, herbivory, and soil contaminants, and evaluated whether gray birch, as a high metal accumulator, engages in elemental allelopathy, poisoning other species through its metal-contaminated leaf litter. We assessed the effects of gray birch on neighboring plant community structure, soil chemistry, fungal root symbionts, and the germination, growth, and herbivory of seedlings of black oak (Quercus velutina) and sugar maple (Acer saccharum), two tree species expected to follow gray birch in succession. Gray birch was associated with increased diversity in its neighborhood grassland community, increased herbivory on black oak seedlings, and influenced colonization by fungal root symbionts in both species. Seedling biomass was correlated with colonization by ectomycorrhizal fungi in black oak, but not with arbuscular mycorrhizal or dark septate fungal colonization in sugar maple. Gray birch had no effect on maple seedling performance or soil chemistry, and a small effect on black oak performance in the absence of aboveground competition. We found little evidence consistent with elemental allelopathy. Black oak and sugar maple seedlings responded more strongly to variation in soil nutrients than soil heavy metals, and they maintained leaf metal concentration profiles markedly different from those in their soils. We conclude that gray birch alters its environment in ways that could promote establishment and succession of woody species, potentially favoring those that are ectomycorrhizal, but most effects of PSF are overpowered by aboveground competition, herbivory, and/or existing abiotic soil factors. This study well illustrates why the potential for PSF to affect plant performance and community structure must be examined within the context of other ecological processes. Such a broad understanding can inform decisions made in the remediation and management of disturbed sites, and our understanding of plant succession and coexistence in general.

The small molecule Retro-2 prevents ricin toxicity through a poorly-defined mechanism of action (MOA), which involves halting retrograde vesicle transport to the endoplasmic reticulum (ER). CRISPRi genetic interaction analysis revealed Retro-2 activity resembles disruption of the transmembrane domain recognition complex (TRC) pathway, which mediates post-translational ER-targeting and insertion of tail-anchored (TA) proteins, including SNAREs required for retrograde transport. Cell-based and in vitro assays show that Retro-2 blocks delivery of newly-synthesized TA-proteins to the ER-targeting factor ASNA1 (TRC40). An ASNA1 point mutant identified using CRISPR-mediated mutagenesis abolishes both the cytoprotective effect of Retro-2 against ricin and its inhibitory effect on ASNA1-mediated ER-targeting. Together, our work explains how Retro-2 prevents retrograde trafficking of toxins by inhibiting TA-protein targeting, describes a general CRISPR strategy for predicting the MOA of small molecules, and paves the way for drugging the TRC pathway to treat broad classes of viruses known to be inhibited by Retro-2.

The "super-relaxed state" (SRX) of myosin represents a 'reserve' of motors in the heart. Myosin heads in the SRX are bound to the thick filament and have a very low ATPase rate. Changes in the SRX are likely to modulate cardiac contractility. We previously demonstrated that the SRX is significantly reduced in mouse cardiomyocytes lacking cardiac myosin binding protein-C (cMyBP-C). Here, we report the effect of mutations in the cMyBP-C gene (MYBPC3) using samples from human patients with hypertrophic cardiomyopathy (HCM). Left ventricular (LV) samples from 11 HCM patients were obtained following myectomy surgery to relieve LV outflow tract obstruction. HCM samples were genotyped as either MYBPC3 mutation positive (MYBPC3mut) or negative (HCMsmn) and were compared to eight non-failing donor hearts. Compared to donors, only MYBPC3mut samples display a significantly diminished SRX, characterised by a decrease in both the number of myosin heads in the SRX and the lifetime of ATP turnover. These changes were not observed in HCMsmn samples. There was a positive correlation (p < 0.01) between the expression of cMyBP-C and the proportion of myosin heads in the SRX state, suggesting cMyBP-C modulates and maintains the SRX. Phosphorylation of the myosin regulatory light chain in MYBPC3mut samples was significantly decreased compared to the other groups, suggesting a potential mechanism to compensate for the diminished SRX. We conclude that by altering both contractility and sarcomeric energy requirements, a reduced SRX may be an important disease mechanism in patients with MYBPC3 mutations.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Sensory integration difficulties have been reported in autism, but their underlying brain-circuit mechanisms are underexplored. Using five autism-related mouse models, Shank3+/ΔC, Mecp2R308/Y, Cntnap2−/−, L7-Tsc1 (L7/Pcp2Cre::Tsc1flox/+), and patDp(15q11-13)/+, we report specific perturbations in delay eyeblink conditioning, a form of associative sensory learning requiring cerebellar plasticity. By distinguishing perturbations in the probability and characteristics of learned responses, we found that probability was reduced in Cntnap2−/−, patDp(15q11-13)/+, and L7/Pcp2Cre::Tsc1flox/+, which are associated with Purkinje-cell/deep-nuclear gene expression, along with Shank3+/ΔC. Amplitudes were smaller in L7/Pcp2Cre::Tsc1flox/+ as well as Shank3+/ΔC and Mecp2R308/Y, which are associated with granule cell pathway expression. Shank3+/ΔC and Mecp2R308/Y also showed aberrant response timing and reduced Purkinje-cell dendritic spine density. Overall, our observations are potentially accounted for by defects in instructed learning in the olivocerebellar loop and response representation in the granule cell pathway. Our findings indicate that defects in associative temporal binding of sensory events are widespread in autism mouse models.