RGS10 shapes the hemostatic response to injury through its differential effects on intracellular signaling by platelet agonists.

Platelets express ≥2 members of the regulators of G protein signaling (RGS) family. Here, we have focused on the most abundant, RGS10, examining its impact on the hemostatic response in vivo and the mechanisms involved. We have previously shown that the hemostatic thrombi formed in response to penetrating injuries consist of a core of fully activated densely packed platelets overlaid by a shell of less-activated platelets responding to adenosine 5\u27-diphosphate (ADP) and thromboxane A2 (TxA2). Hemostatic thrombi formed in RGS10-/- mice were larger than in controls, with the increase due to expansion of the shell but not the core. Clot retraction was slower, and average packing density was reduced. Deleting RGS10 had agonist-specific effects on signaling. There was a leftward shift in the dose/response curve for the thrombin receptor (PAR4) agonist peptide AYPGKF but no increase in the maximum response. This contrasted with ADP and TxA2, both of which evoked considerably greater maximum responses in RGS10-/- platelets with enhanced Gq- and Gi-mediated signaling. Shape change, which is G13-mediated, was unaffected. Finally, we found that free RGS10 levels in platelets are actively regulated. In resting platelets, RGS10 was bound to 2 scaffold proteins: spinophilin and 14-3-3γ. Platelet activation caused an increase in free RGS10, as did the endothelium-derived platelet antagonist prostacyclin. Collectively, these observations show that RGS10 serves as an actively regulated node on the platelet signaling network, helping to produce smaller and more densely packed hemostatic thrombi with a greater proportion of fully activated platelets

GRK6 regulates the hemostatic response to injury through its rate-limiting effects on GPCR signaling in platelets.

G protein-coupled receptors (GPCRs) mediate the majority of platelet activation in response to agonists. However, questions remain regarding the mechanisms that provide negative feedback toward activated GPCRs to limit platelet activation and thrombus formation. Here we provide the first evidence that GPCR kinase 6 (GRK6) serves this role in platelets, using GRK6-/- mice generated by CRISPR-Cas9 genome editing to examine the consequences of GRK6 knockout on GPCR-dependent signaling. Hemostatic thrombi formed in GRK6-/- mice are larger than in wild-type (WT) controls during the early stages of thrombus formation, with a rapid increase in platelet accumulation at the site of injury. GRK6-/- platelets have increased platelet activation, but in an agonist-selective manner. Responses to PAR4 agonist or adenosine 5\u27-diphosphate stimulation in GRK6-/- platelets are increased compared with WT littermates, whereas the response to thromboxane A2 (TxA2) is normal. Underlying these changes in GRK6-/- platelets is an increase in Ca2+ mobilization, Akt activation, and granule secretion. Furthermore, deletion of GRK6 in human MEG-01 cells causes an increase in Ca2+ response and PAR1 surface expression in response to thrombin. Finally, we show that human platelet activation in response to thrombin causes an increase in binding of GRK6 to PAR1, as well as an increase in the phosphorylation of PAR1. Deletion of GRK6 in MEG-01 cells causes a decrease in PAR1 phosphorylation. Taken together, these data show that GRK6 regulates the hemostatic response to injury through PAR- and P2Y12-mediated effects, helping to limit the rate of platelet activation during thrombus growth and prevent inappropriate platelet activation

Molecular simulation of hydrogen storage and transport in cellulose

In this work we describe a computational workflow to model the sorption and transport of molecular hydrogen in cellulose frameworks. The work demonstrates the value of the molecular dynamics code, DL_POLY and Monte Carlo code, DL_MONTE sharing common input formats to enhance the compatibility of the codes, being supported by DL_FIELD. Structures generated using cellulose-builder were processed by DL_FIELD to generate input files for DL_POLY using the OPLS_2005 force field. After relaxation in molecular dynamics, structures were used for GCMC simulations in DL_MONTE before passing back to DL_POLY to evaluate transport properties at different levels of sorption. While no hydrogen sorption was seen in pure crystalline cellulose, increasing separation between layers did allow sorption. When slit-pores were sufficiently wide, interactions with the cellulose led to the volumetric density of adsorbed hydrogen exceeding vacuum density at accessible partial pressures as well as allowing diffusion through the system. These model systems can give useful insight into the behaviour of amorphous cellulose in future simulation and experiment

Individual phosphatidylinositol transfer proteins have distinct functions that do not involve lipid transfer activity

Platelets utilize signal transduction pathways facilitated by Class I phosphatidylinositol transfer proteins (PITPs). The two mammalian Class I PITPs, PITPα and PITPβ, are single PITP domain soluble proteins that are encoded by different genes and have 77% sequence identity, though their individual roles in mammalian biology remain uncharacterized. These proteins are believed to shuttle phosphatidylinositol and phosphatidylcholine between separate intracellular membrane compartments, thereby regulating phosphoinositide synthesis and second messenger formation. Previously, we observed that platelet-specific deletion of PITPα, the predominant expressed murine PITP isoform, had no effect on hemostasis, but had impaired tumor metastasis formation and disrupted phosphoinositide signaling. Here, we find that mice lacking the lesser expressed PITPβ in their platelets exhibit a similar phenotype. However, in contrast to PITPα-null platelet lysates that have impaired lipid transfer activity, PITPβ-null platelet lysates have essentially normal lipid transfer activity, although both isoforms contribute to phosphoinositide synthesis in vitro. Moreover, we found that platelet-specific deletion of both PITPs leads to ex vivo platelet aggregation/secretion and spreading defects, impaired tail bleeding, and profound tumor dissemination. Our studies also demonstrate that PITP isoforms are required for maintaining endogenous phosphoinositide PI(4,5)P2 levels and agonist stimulated second messenger formation. The data shown here demonstrate that both class I PITP isoforms contribute to phosphoinositide signaling in platelets, likely through distinct biochemical mechanisms or in different subcellular domains. They are functionally overlapping and either single isoform is able to maintain the homeostasis of platelets

A scalable, fully automated process for construction of sequence-ready human exome targeted capture libraries

Genome targeting methods enable cost-effective capture of specific subsets of the genome for sequencing. We present here an automated, highly scalable method for carrying out the Solution Hybrid Selection capture approach that provides a dramatic increase in scale and throughput of sequence-ready libraries produced. Significant process improvements and a series of in-process quality control checkpoints are also added. These process improvements can also be used in a manual version of the protocol

Peptides derived from MARCKS block coagulation complex assembly on phosphatidylserine.

Blood coagulation involves activation of platelets and coagulation factors. At the interface of these two processes resides the lipid phosphatidylserine. Activated platelets expose phosphatidylserine on their outer membrane leaflet and activated clotting factors assemble into enzymatically active complexes on the exposed lipid, ultimately leading to the formation of fibrin. Here, we describe how small peptide and peptidomimetic probes derived from the lipid binding domain of the protein myristoylated alanine-rich C-kinase substrate (MARCKS) bind to phosphatidylserine exposed on activated platelets and thereby inhibit fibrin formation. The MARCKS peptides antagonize the binding of factor Xa to phosphatidylserine and inhibit the enzymatic activity of prothrombinase. In whole blood under flow, the MARCKS peptides colocalize with, and inhibit fibrin cross-linking, of adherent platelets. In vivo, we find that the MARCKS peptides circulate to remote injuries and bind to activated platelets in the inner core of developing thrombi

Are Third World Emigration Forces Abating?

Most observers appear to believe that Third World emigration pressure is on the rise. But history suggests that migration typically follows a bell shape, in which case it might be entering on the downward phase. This paper estimates the economic and demographic fundamentals driving emigration from the developing world to the United States since 1970. The results suggest that emigration pressure, determined largely by source country demographics, education, poverty, and migrant stock dynamics, has recently been abating. Projections into the future suggest that it may even decline, and that its composition will become much more African and much less Hispanic

Bi-allelic loss-of-function CACNA1B mutations in progressive epilepsy-dyskinesia

The occurrence of non-epileptic hyperkinetic movements in the context of developmental epileptic encephalopathies is an increasingly recognized phenomenon. Identification of causative mutations provides an important insight into common pathogenic mechanisms that cause both seizures and abnormal motor control. We report bi-allelic loss-of-function CACNA1B variants in six children from three unrelated families whose affected members present with a complex and progressive neurological syndrome. All affected individuals presented with epileptic encephalopathy, severe neurodevelopmental delay (often with regression), and a hyperkinetic movement disorder. Additional neurological features included postnatal microcephaly and hypotonia. Five children died in childhood or adolescence (mean age of death: 9 years), mainly as a result of secondary respiratory complications. CACNA1B encodes the pore-forming subunit of the pre-synaptic neuronal voltage-gated calcium channel Cav2.2/N-type, crucial for SNARE-mediated neurotransmission, particularly in the early postnatal period. Bi-allelic loss-of-function variants in CACNA1B are predicted to cause disruption of Ca2+ influx, leading to impaired synaptic neurotransmission. The resultant effect on neuronal function is likely to be important in the development of involuntary movements and epilepsy. Overall, our findings provide further evidence for the key role of Cav2.2 in normal human neurodevelopment

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

The UK10K project identifies rare variants in health and disease.

The contribution of rare and low-frequency variants to human traits is largely unexplored. Here we describe insights from sequencing whole genomes (low read depth, 7×) or exomes (high read depth, 80×) of nearly 10,000 individuals from population-based and disease collections. In extensively phenotyped cohorts we characterize over 24 million novel sequence variants, generate a highly accurate imputation reference panel and identify novel alleles associated with levels of triglycerides (APOB), adiponectin (ADIPOQ) and low-density lipoprotein cholesterol (LDLR and RGAG1) from single-marker and rare variant aggregation tests. We describe population structure and functional annotation of rare and low-frequency variants, use the data to estimate the benefits of sequencing for association studies, and summarize lessons from disease-specific collections. Finally, we make available an extensive resource, including individual-level genetic and phenotypic data and web-based tools to facilitate the exploration of association results