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Shape change is an important early event in platelet activation. In this study we show that the Ca2+ chelator BAPTA and the Rho-kinase inhibitor Y-27632 inhibit ADP-induced myosin light chain (MLC) phosphorylation and platelet shape change through distinct pathways and with distinct kinetics. Ca2+ is largely responsible for the initial onset of shape change, whilst Rho-kinase plays a major role in the maintenance of the response. The relative contribution of these two pathways to each stage of the response was dependent on the method of platelet preparation, but in all cases shape change was shown to be downstream of the P2Y1 receptor. Similar observations were made in murine platelets. The shape change response was modulated via changes in cAMP levels, possibly via the P2T [sub AC] receptor, but not by tyrosine phosphorylation. We conclude that ADP-induced A shape change occurs via the P2Y1 receptor, which can be differentially coupled to Rho-kinase and Ca2+-linked pathways dependent on the method of platelet preparation. [ABSTRACT FROM AUTHOR]

Searching for factors to improve knockin efficiency for therapeutic applications, biotechnology, and generation of non-human primate models of disease, we found that the strand exchange protein RAD51 can significantly increase Cas9-mediated homozygous knockin in mouse embryos through an interhomolog repair (IHR) mechanism. IHR is a hallmark of meiosis but only occurs at low frequencies in somatic cells, and its occurrence in zygotes is controversial. Using multiple approaches, we provide evidence for an endogenous IHR mechanism in the early embryo that can be enhanced by RAD51. This process can be harnessed to generate homozygotes from wild-type zygotes using exogenous donors and to convert heterozygous alleles into homozygous alleles without exogenous templates. Furthermore, we identify additional IHR-promoting factors and describe features of IHR events. Together, our findings show conclusive evidence for IHR in mouse embryos and describe an efficient method for enhanced gene conversion. [Display omitted] • Interhomolog repair (IHR) occurs throughout the genome in the early mouse embryo • RAD51, BCCIP, and USP1/WDR48 enhance IHR at sites of Cas9-induced DNA breaks • Embryonic IHR occurs via gene conversion without crossover • Enhanced IHR can be used for homozygous knockin and donor-free allelic conversion Co-injection of mouse zygotes with RAD51 and Cas9 can enhance interhomolog repair and facilitate conversion of heterozygous alleles to homozygous alleles. [ABSTRACT FROM AUTHOR]

Until the late 1960s, Northern Goshawks, Accipiter gentilis atricapillus, were considered rare breeders in Wisconsin. Research initiated in 1971 revealed a small but growing breeding population, which continued to increase until the mid 1990s when depredations by mammalian predators began to reduce numbers. Since 1985, reproduction has fallenbelow the estimated threshold of 1.7 young fledged/active nest needed to maintain a stable population. The reintroduced Fisher, Martes pennanti, is largely responsible for increased nest failure and adult female mortality, with the turnover rate of nesting females having doubled to over 40%. Twenty-two years of reproductive data reveal a correlation between population levels and the 10-year wildlife cycle. Thegoshawk has been listed as a 'sensitive species' since 1986 in the Nicolet National Forest, and guidelines have been established for nestsite protection. However. no such standards exist for state, county and private lands, where populations are declining. Legal and illegal'take' of nestling goshawks is a continuing concern, especially as nest sites under national forest management become more vulnerable to falconers, who have taken an estimated 5% of young from monitored nests. [ABSTRACT FROM AUTHOR]

Parkinson's disease (PD) is a debilitating neurodegenerative disorder. Its symptoms are typically treated with levodopa or dopamine receptor agonists, but its action lacks specificity due to the wide distribution of dopamine receptors in the central nervous system and periphery. Here, we report the development of a gene therapy strategy to selectively manipulate PD-affected circuitry. Targeting striatal D1 medium spiny neurons (MSNs), whose activity is chronically suppressed in PD, we engineered a therapeutic strategy comprised of a highly efficient retrograde adeno-associated virus (AAV), promoter elements with strong D1-MSN activity, and a chemogenetic effector to enable precise D1-MSN activation after systemic ligand administration. Application of this therapeutic approach rescues locomotion, tremor, and motor skill defects in both mouse and primate models of PD, supporting the feasibility of targeted circuit modulation tools for the treatment of PD in humans. [Display omitted] • AAV8R12 efficiently transduces striatal D1-, but not D2-, MSNs after nigral delivery • G88P2/3/7 promoters derived from GPR88 gene induce robust gene expression in MSNs • Systemic ligand infusion selectively activates D1-MSNs after nigral AAV8R12/ rM3Ds delivery • Single-dose DCZ rescues PD symptoms in primates for at least 24 h without dyskinesia An AAV-based circuit modulation approach that consists of a designer retrograde AAV capsid, a medium spiny neuron-enriched promoter, and a selected chemogenetic effector specifically modulates direct pathway neurons and rescues parkinsonian symptoms in mouse and macaque Parkinson's disease models. [ABSTRACT FROM AUTHOR]

Abstract: During lung development, proper epithelial cell arrangements are critical for the formation of an arborized network of tubes. Each tube requires a lumen, the diameter of which must be tightly regulated to enable optimal lung function. Lung branching and lumen morphogenesis require close epithelial cell–cell contacts that are maintained as a result of adherens junctions, tight junctions and by intact apical–basal (A/B) polarity. However, the molecular mechanisms that maintain epithelial cohesion and lumen diameter in the mammalian lung are unknown. Here we show that Scribble, a protein implicated in planar cell polarity (PCP) signalling, is necessary for normal lung morphogenesis. Lungs of the Scrib mouse mutant Circletail (Crc) are abnormally shaped with fewer airways, and these airways often lack a visible, ‘open’ lumen. Mechanistically we show that Scrib genetically interacts with the core PCP gene Vangl2 in the developing lung and that the distribution of PCP pathway proteins and Rho mediated cytoskeletal modification is perturbed in Scrib Crc/Crc lungs. However A/B polarity, which is disrupted in Drosophila Scrib mutants, is largely unaffected. Notably, we find that Scrib mediates functions not attributed to other PCP proteins in the lung. Specifically, Scrib localises to both adherens and tight junctions of lung epithelia and knockdown of Scrib in lung explants and organotypic cultures leads to reduced cohesion of lung epithelial cells. Live imaging of Scrib knockdown lungs shows that Scrib does not affect bud bifurcation, as previously shown for the PCP protein Celsr1, but is required to maintain epithelial cohesion. To understand the mechanism leading to reduced cell–cell association, we show that Scrib associates with β-catenin in embryonic lung and the sub-cellular distribution of adherens and tight junction proteins is perturbed in mutant lung epithelia. Our data reveal that Scrib is required for normal lung epithelial organisation and lumen morphogenesis by maintaining cell–cell contacts. Thus we reveal novel and important roles for Scrib in lung development operating via the PCP pathway, and in regulating junctional complexes and cell cohesion. [Copyright &y& Elsevier]

Light transmission aggregometry (LTA) is used worldwide for the investigation of heritable platelet function disorders (PFDs), but interpretation of results is complicated by the feedback effects of ADP and thromboxane A2 (TxA2) and by the overlap with the response of healthy volunteers. Over 5 years, we have performed lumi-aggregometry on 9 platelet agonists in 111 unrelated research participants with suspected PFDs and in 70 healthy volunteers. Abnormal LTA or ATP secretion test results were identified in 58% of participants. In 84% of these, the patterns of response were consistent with defects in Gi receptor signaling, the TxA2 pathway, and dense granule secretion. Participants with defects in signaling to Gq-coupled receptor agonists and to collagen were also identified. Targeted genotyping identified 3 participants with function-disrupting mutations in the P2Y12 ADP and TxA2 receptors. The results of the present study illustrate that detailed phenotypic analysis using LTA and ATP secretion is a powerful tool for the diagnosis of PFDs. Our data also enable subdivision at the level of platelet-signaling pathways and in some cases to individual receptors. We further demonstrate that most PFDs can be reliably diagnosed using a streamlined panel of key platelet agonists and specified concentrations suitable for testing in most clinical diagnostic laboratories. [ABSTRACT FROM AUTHOR]

Reactive changes of glial cells during neuroinflammation impact brain disorders and disease progression. Elucidating the mechanisms that control reactive gliosis may help us to understand brain pathophysiology and improve outcomes. Here, we report that adult ablation of autism spectrum disorder (ASD)-associated CHD8 in astrocytes attenuates reactive gliosis via remodeling chromatin accessibility, changing gene expression. Conditional Chd8 deletion in astrocytes, but not microglia, suppresses reactive gliosis by impeding astrocyte proliferation and morphological elaboration. Astrocyte Chd8 ablation alleviates lipopolysaccharide-induced neuroinflammation and septic-associated hypothermia in mice. Astrocytic CHD8 plays an important role in neuroinflammation by altering the chromatin landscape, regulating metabolic and lipid-associated pathways, and astrocyte-microglia crosstalk. Moreover, we show that reactive gliosis can be directly mitigated in vivo using an adeno-associated virus (AAV)-mediated Chd8 gene editing strategy. These findings uncover a role of ASD-associated CHD8 in the adult brain, which may warrant future exploration of targeting chromatin remodelers in reactive gliosis and neuroinflammation in injury and neurological diseases., Competing Interests: Declaration of interests T.I. is a co-founder, member of the advisory board, and has an equity interest in Data4Cure and Serinus Biosciences. He is a consultant for and has an equity interest in Ideaya BioSciences and Light Horse Therapeutics. The terms of these arrangements have been reviewed and approved by University of California, San Diego in accordance with its conflict of interest policies. N.E.S. is an adviser to Qiagen and a co-founder of and adviser to TruEdit Bio and OverT Bio., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Introduction: The unique red blood cell (RBC) properties that characterize the rare neuroacanthocytosis syndromes (NAS) have prompted the exploration of osmotic gradient ektacytometry (Osmoscan) as a diagnostic tool for these disorders. In this exploratory study, we assessed if Osmoscans can discriminate NAS from other neurodegenerative diseases., Methods: A comprehensive assessment was conducted using Osmoscan on a diverse group of patients, including healthy controls ( n  = 9), neuroacanthocytosis syndrome patients ( n  = 6, 2 VPS13A and 4 XK disease), Parkinson's disease patients ( n  = 6), Huntington's disease patients (n = 5), and amyotrophic lateral sclerosis patients ( n  = 4). Concurrently, we collected and analyzed RBC indices and patients' characteristics., Results: Statistically significant changes were observed in NAS patients compared to healthy controls and other conditions, specifically in osmolality at minimal elongation index (O min ), maximal elongation index (EI max ), the osmolality at half maximal elongation index in the hyperosmotic part of the curve (O hyper ), and the width of the curve close to the osmolality at maximal elongation index (O max -width)., Discussion: This study represents an initial exploration of RBC properties from NAS patients using osmotic gradient ektacytometry. While specific parameters exhibited differences, only O hyper and O max -width yielded 100% specificity for other neurodegenerative diseases. Moreover, unique correlations between Osmoscan parameters and RBC indices in NAS versus controls were identified, such as osmolality at maximal elongation index (O max ) vs. mean cellular hemoglobin content (MCH) and minimal elongation index (EI min ) vs. red blood cell distribution width (RDW). Given the limited sample size, further studies are essential to establish diagnostic guidelines based on these findings., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Hernández, Peikert, Qiao, Darras, de Wilde, Bos, Leibowitz, Galea, Wagner, Rab, Walker, Hermann, van Beers, van Wijk and Kaestner.)

Optimizing behavioral strategy requires belief updating based on new evidence, a process that engages higher cognition. In schizophrenia, aberrant belief dynamics may lead to psychosis, but the mechanisms underlying this process are unknown, in part, due to lack of appropriate animal models and behavior readouts. Here, we address this challenge by taking two synergistic approaches. First, we generate a mouse model bearing patient-derived point mutation in Grin2a ( Grin2a Y700X+/- ), a gene that confers high-risk for schizophrenia and recently identified by large-scale exome sequencing. Second, we develop a computationally trackable foraging task, in which mice form and update belief-driven strategies in a dynamic environment. We found that Grin2a Y700X+/- mice perform less optimally than their wild-type (WT) littermates, showing unstable behavioral states and a slower belief update rate. Using functional ultrasound imaging, we identified the mediodorsal (MD) thalamus as hypofunctional in Grin2a Y700X+/- mice, and in vivo task recordings showed that MD neurons encoded dynamic values and behavioral states in WT mice. Optogenetic inhibition of MD neurons in WT mice phenocopied Grin2a Y700X+/- mice, and enhancing MD activity rescued task deficits in Grin2a Y700X+/- mice. Together, our study identifies the MD thalamus as a key node for schizophrenia-relevant cognitive dysfunction, and a potential target for future therapeutics.

The most common forms of sickle cell disease (SCD) are sickle cell anemia (SCA; HbSS) and HbSC disease. In both, especially the more dense, dehydrated and adherent red blood cells (RBCs) with reduced deformability are prone to hemolysis and sickling, and thereby vaso-occlusion. Based on plasma amino acid profiling in SCD, a composition of 10 amino acids and derivatives (RCitNacQCarLKHVS; Axcella Therapeutics, USA), referred to as endogenous metabolic modulators (EMMs), was designed to target RBC metabolism. The effects of ex vivo treatment with the EMM composition on different RBC properties were studied in SCD ( n  = 9 SCA, n  = 5 HbSC disease). Dose-dependent improvements were observed in RBC hydration assessed by hemocytometry (MCV, MCHC, dense RBCs) and osmotic gradient ektacytometry (Ohyper). Median (interquartile range [IQR]) increase in Ohyper compared to vehicle was 4.9% (4.0%-5.5%), 7.5% (6.9%-9.4%), and 12.8% (11.5%-14.0%) with increasing 20×, 40×, and 80X concentrations, respectively (all p  < 0.0001). RBC deformability (EImax using oxygen gradient ektacytometry) increased by 8.1% (2.2%-12.1%; p  = 0.0012), 9.6% (2.9%-15.1%; p  = 0.0013), and 13.3% (5.7%-25.5%; p  = 0.0007), respectively. Besides, RBC adhesion to subendothelial laminin decreased by 43% (6%-68%; p  = 0.4324), 58% (48%-72%; p  = 0.0185), and 71% (49%-82%; p  = 0.0016), respectively. Together, these results provide a rationale for further studies with the EMM composition targeting multiple RBC properties in SCD., Competing Interests: R.v.W. and M.R. received research funding from Axcella Therapeutics and Pfizer. E.v.B. and R.v.W. are consultants for Pfizer. E.v.B., R.v.W., and M.R. receive research funding and are consultants for Agios Pharmaceuticals Inc. M.J.K. and R.W. are employees of Axcella Therapeutics. The remaining authors declare no competing financial interests., (© 2024 The Authors. eJHaem published by British Society for Haematology and John Wiley & Sons Ltd.)

Novel developments in therapies for various hereditary hemolytic anemias reflect the pivotal role of pyruvate kinase (PK), a key enzyme of glycolysis, in red blood cell (RBC) health. Without PK catalyzing one of the final steps of the Embden-Meyerhof pathway, there is no net yield of adenosine triphosphate (ATP) during glycolysis, the sole source of energy production required for proper RBC function and survival. In hereditary hemolytic anemias, RBC health is compromised and therefore lifespan is shortened. Although our knowledge on glycolysis in general and PK function in particular is solid, recent advances in genetic, molecular, biochemical, and metabolic aspects of hereditary anemias have improved our understanding of these diseases. These advances provide a rationale for targeting PK as therapeutic option in hereditary hemolytic anemias other than PK deficiency. This review summarizes the knowledge, rationale, (pre)clinical trials, and future advances of PK activators for this important group of rare diseases., Competing Interests: Declaration of Competing Interest M.v.D., J.d.W., K.K., A.G., M.R., E.v.B. and R.v.W. receive research funding from Agios Pharmaceuticals Inc. K.K., E.v.B. and R.v.W. are consultants for Agios Pharmaceuticals Inc. K.K., M.R. and R.v.W. receive research funding from Pfizer. K.K. and R.v.W. are consultants for Pfizer. K.K. is also consultant for Alexion Pharmaceuticals, Celgene/Bristol Myers Squibb, Forma Therapeutics, Novo Nordisk, and chair of the Data Safety Monitoring Board for Bioverativ/Sanofi and Sangamo. A.G. receives research funding from Bristol Myers Squibb, Saniona and Sanofi and has done consulting for Agios, Bluebird Bio, Bristol Myers Squibb, Novartis, Novo Nordisk, Pharmacosmos. M.R. and R.v.W. receive research funding from Axcella Health Inc. The remaining author declares no conflict of interest., (Crown Copyright © 2023. Published by Elsevier Ltd. All rights reserved.)

Cornelia de Lange Syndrome (CdLS) is a rare, dominantly inherited multisystem developmental disorder characterized by highly variable manifestations of growth and developmental delays, upper limb involvement, hypertrichosis, cardiac, gastrointestinal, craniofacial, and other systemic features. Pathogenic variants in genes encoding cohesin complex structural subunits and regulatory proteins (NIPBL, SMC1A, SMC3, HDAC8, and RAD21) are the major pathogenic contributors to CdLS. Heterozygous or hemizygous variants in the genes encoding these five proteins have been found to be contributory to CdLS, with variants in NIPBL accounting for the majority (>60%) of cases, and the only gene identified to date that results in the severe or classic form of CdLS when mutated. Pathogenic variants in cohesin genes other than NIPBL tend to result in a less severe phenotype. Causative variants in additional genes, such as ANKRD11, EP300, AFF4, TAF1, and BRD4, can cause a CdLS-like phenotype. The common role that these genes, and others, play as critical regulators of developmental transcriptional control has led to the conditions they cause being referred to as disorders of transcriptional regulation (or "DTRs"). Here, we report the results of a comprehensive molecular analysis in a cohort of 716 probands with typical and atypical CdLS in order to delineate the genetic contribution of causative variants in cohesin complex genes as well as novel candidate genes, genotype-phenotype correlations, and the utility of genome sequencing in understanding the mutational landscape in this population., (© 2023 Wiley Periodicals LLC.)

Common polygenic diseases result from compounded risk contributed by multiple genetic variants, meaning that simultaneous correction or introduction of single nucleotide variants is required for disease modeling and gene therapy. Here, we show precise, efficient, and simultaneous multiplex base editing of up to three target sites across 11 genes/loci in cynomolgus monkey embryos using CRISPR-based cytidine- and adenine-base editors. Unbiased whole genome sequencing demonstrates high specificity of base editing in monkey embryos. Our data demonstrate feasibility of multiplex base editing for polygenic disease modeling in primate zygotes.

Background: Inherited thrombocytopenias (ITs) are a heterogeneous group of disorders characterized by low platelet counts and often disproportionate bleeding with over 30 genes currently implicated. Previously the UK-GAPP study using whole exome sequencing (WES) identified a pathogenic variant in 19 of 47 (40%) patients of which 71% had variants in genes known to cause IT., Aims: To employ a targeted next-generation sequencing platform to improve efficiency of diagnostic testing and reduce overall costs., Methods: We have developed an IT-specific gene panel as a pre-screen for patients prior to WES using the Agilent SureSelect QXT transposon-based enrichment system., Results: Thirty-one patients were analyzed using the panel-based sequencing, of which; 10% (3/31) were identified with a classified pathogenic variant, 16% (5/31) were identified with a likely pathogenic variant, 51% (16/31) were identified with variants of unknown significance, and 23% (7/31) were identified with either no variant or a benign variant., Discussion and Conclusion: Although requiring further clarification of the impact of the genetic variations, the application of an IT-specific next generation sequencing panel is an viable method of pre-screening patients for variants in known IT-causing genes prior to WES. With an added benefit of distinguishing IT from idiopathic thrombocytopenic purpura (ITP) and the potential to identify variants in genes known to have a predisposition to hematological malignancies, it could become a critical step in improving patient clinical management.

Inherited thrombocytopenias are a heterogeneous group of disorders characterized by abnormally low platelet counts which can be associated with abnormal bleeding. Next-generation sequencing has previously been employed in these disorders for the confirmation of suspected genetic abnormalities, and more recently in the discovery of novel disease-causing genes. However its full potential has not yet been exploited. Over the past 6 years we have sequenced the exomes from 55 patients, including 37 index cases and 18 additional family members, all of whom were recruited to the UK Genotyping and Phenotyping of Platelets study. All patients had inherited or sustained thrombocytopenia of unknown etiology with platelet counts varying from 11×10 9 /L to 186×10 9 /L. Of the 51 patients phenotypically tested, 37 (73%), had an additional secondary qualitative platelet defect. Using whole exome sequencing analysis we have identified "pathogenic" or "likely pathogenic" variants in 46% (17/37) of our index patients with thrombocytopenia. In addition, we report variants of uncertain significance in 12 index cases, including novel candidate genetic variants in previously unreported genes in four index cases. These results demonstrate that whole exome sequencing is an efficient method for elucidating potential pathogenic genetic variants in inherited thrombocytopenia. Whole exome sequencing also has the added benefit of discovering potentially pathogenic genetic variants for further study in novel genes not previously implicated in inherited thrombocytopenia., (Copyright© Ferrata Storti Foundation.)