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Src family kinases (SFKs) coordinate the initiating and propagating activation signals in platelets, but it remains unclear how they are regulated. Here, we show that ablation of C-terminal Src kinase (Csk) and receptor-like protein tyrosine-phosphatase CD148 in mice results in a dramatic increase in platelet SFK activity, demonstrating that these proteins are essential regulators of platelet reactivity. Paradoxically, Csk/CD148-deficient mice exhibit reduced in vivo and ex vivo thrombus formation and increased bleeding following injury rather than a prothrombotic phenotype. This is a consequence of multiple negative feedback mechanisms, including downregulation of the immunoreceptor tyrosine-based activation motif (ITAM)- and hemi-ITAM-containing receptors glycoprotein VI (GPVI)-Fc receptor (FcR) gamma-chain and CLEC-2, respectively and upregulation of the immunoreceptor tyrosine-based inhibition motif (ITIM)-containing receptor G6b-B and its interaction with the tyrosine phosphatases Shp1 and Shp2. Results from an analog-sensitive Csk mouse model demonstrate the unconventional role of SFKs in activating ITIM signaling. This study establishes Csk and CD148 as critical molecular switches controlling the thrombotic and hemostatic capacity of platelets and reveals cell-intrinsic mechanisms that prevent pathological thrombosis from occurring.

The eukaryotic helicase is an 11-subunit machine containing an Mcm2-7 motor ring that encircles DNA, Cdc45 and the GINS tetramer, referred to as CMG (Cdc45, Mcm2-7, GINS). CMG is "built" on DNA at origins in two steps. First, two Mcm2-7 rings are assembled around duplex DNA at origins in G1 phase, forming the Mcm2-7 "double hexamer." In a second step, in S phase Cdc45 and GINS are assembled onto each Mcm2-7 ring, hence producing two CMGs that ultimately form two replication forks that travel in opposite directions. Here, we review recent findings about CMG structure and function. The CMG unwinds the parental duplex and is also the organizing center of the replisome: it binds DNA polymerases and other factors. EM studies reveal a 20-subunit core replisome with the leading Pol epsilon and lagging Pol alpha-primase on opposite faces of CMG, forming a fundamentally asymmetric architecture. Structural studies of CMG at a replication fork reveal unexpected details of how CMG engages the DNA fork. The structures of CMG and the Mcm2-7 double hexamer on DNA suggest a completely unanticipated process for formation of bidirectional replication forks at origins.

Background: Autosomal dominant anhidrotic ectodermal dysplasia with immune deficiency (AD EDA-ID) is caused by heterozygous point mutations at or close to serine 32 and serine 36 or N-terminal truncations in I kappa B alpha that impair its phosphorylation and degradation and thus activation of the canonical nuclear factor kappa light chain enhancer of activated B cells (NF-kappa B) pathway. The outcome of hematopoietic stem cell transplantation is poor in patients with AD EDA-ID despite achievement of chimerism. Mice heterozygous for the serine 32I mutation in I kappa B alpha have impaired noncanonical NF-kappa B activity and defective lymphorganogenesis. Objective: We sought to establish genotype-phenotype correlation in patients with AD EDA-ID. Methods: A disease severity scoring system was devised. Stability of I kappa B alpha mutants was examined in transfected cells. Immunologic, biochemical, and gene expression analyses were performed to evaluate canonical and noncanonical NF-kappa B signaling in skin-derived fibroblasts. Results: Disease severity was greater in patients with IkBa point mutations than in those with truncation mutations. IkBa point mutants were expressed at significantly higher levels in transfectants compared with truncation mutants. Canonical NF-kappa B-dependent IL-6 secretion and upregulation of the NF-kappa B subunit 2/p100 and RELB proto-oncogene, NF-kappa B subunit (RelB) components of the noncanonical NF-kappa B pathway were diminished significantly more in patients with point mutations compared with those with truncations. Noncanonical NF-kappa B-driven generation of the transcriptionally active p100 cleavage product p52 and upregulation of CCL20, intercellular adhesion molecule 1 (ICAM1), and vascular cell adhesion molecule 1 (VCAM1), which are important for lymphorganogenesis, were diminished significantly more in LPS plus alpha-lymphotoxin beta receptor-stimulated fibroblasts from patients with point mutations compared with those with truncations. Conclusions: I kappa B alpha point mutants accumulate at higher levels compared with truncation mutants and are associated with more severe disease and greater impairment of canonical and noncanonical NF-kappa B activity in patients with AD EDA-ID.

Purpose: The purpose of this study was to compare the maternal and neonatal outcomes among laboring women permitted ad lib oral intake with those permitted nothing by mouth except for ice chips. Design: This was a quantitative retrospective observational cross-sectional study. Sample: The initial data set consisted of all closed medical records for 2,817 women who were admitted to a suburban community hospital in the northeastern United States between January 2008 and December 2012. Some subjects' records were missing either covariate data or outcomes data, resulting in final sample sizes of 2,797 women (for comparison across covariates) and 2,784 women (for comparison across outcomes). Methods: A deidentified limited data set was extracted from the electronic health record for descriptive and inferential comparisons between groups. Demographics and maternal comorbidities present on admission were compared between groups before data analysis. Outcome comparisons were obtained with traditional between-groups analysis and propensity score matching. Results: The groups were found to be sufficiently equivalent for comparison. The group permitted nothing by mouth was significantly more likely to have unplanned cesarean section births than the group permitted ad lib oral intake. There were no significant differences in unplanned maternal ICU admissions postpartum, in neonate condition as determined by Apgar scores, or in the need for a higher level of care. Allowing women ad lib oral intake during labor caused no increase in morbidity, and there were no mortalities in either group. Conclusion: Allowing women ad lib oral intake during labor does not increase adverse maternal or neonatal outcomes. It stands to reason that allowing such intake could increase patient satisfaction. Further study is needed to determine what types of food and drink are most beneficial as well as what types are preferred.

BACKGROUND Taylor's law (TL) states a linear relationship on logarithmic scales between the variance and the mean of a nonnegative quantity. TL has been observed in spatiotemporal contexts for the population density of hundreds of species including humans. TL also describes temporal variation in human mortality in developed countries, but no explanation has been proposed. OBJECTIVE To understand why and to what extent TL describes temporal variation in human mortality, we examine whether the mortality models of Gompertz, Makeham, and Siler are consistent with TL. We also examine how strongly TL differs between observed and modeled mortality, between women and men, and among countries. METHOD We analyze how well each mortality model explains TL fitted to observed occurrence-exposure death rates by comparing three features: the log-log linearity of the temporal variance as a function of the temporal mean, the age profile, and the slope of TL. We support some empirical findings from the Human Mortality Database with mathematical proofs. RESULTS TL describes modeled mortality better than observed mortality and describes Gompertz mortality best. The age profile of TL is closest between observed and Siler mortality. The slope of TL is closest between observed and Makeham mortality. The Gompertz model predicts TL with a slope of exactly 2 if the modal age at death increases linearly with time and the parameter that specifies the growth rate of mortality with age is constant in time. Observed mortality obeys TL with a slope generally less than 2. An explanation is that, when the parameters of the Gompertz model are estimated from observed mortality year by year, both the modal age at death and the growth rate of mortality with age change over time. CONCLUSIONS TL describes human mortality well in developed countries because their mortality schedules are approximated well by classical mortality models, which we have shown to obey TL. CONTRIBUTION We provide the first theoretical linkage between three classical demographic models of mortality and TL.

Demodex mites are microscopic, cigar-shaped, follicular mites often regarded as commensal microfauna in mammals. Although Demodex spp. can cause dermatologic disease in any immunocompromised mammal, they are rarely reported in laboratory mice. Recent identification of Demodex musculi in a colony of immunodeficient mice with dermatitis afforded us the opportunity to investigate the comparative sensitivity of 4 antemortem diagnostic techniques to detect D. musculi-superficial skin scrape (SSS), tape impression (TI), fur pluck (FP), and deep skin scrape (DSS)-which we performed on 4 anatomic sites (face, interscapular region [IS], caudal ventrum [CV], and caudal dorsum [CD]) in 46 mice. DSS had an overall detection rate of 91.1% (n = 112 tests), with the highest detection rates in IS (93.5%), CV (89.1%), and CD (90.0%). The detection rates for SSS (62.5%; n = 112 tests), TI (57.5%; n = 138 tests), and FP (62.7%; n = 158 tests) were all lower than for DSS. IS was the most reliable site. Results from combined FP and DSS samples collected from IS and CV yielded 100% detection, whereas the face was not a desirable sampling site due to inadequate sample quality and low detection rate. Demodex eggs and larvae were observed from FP more often than DSS (19.0% of 158 tests compared with 14.3% of 112 tests). In a subset of samples, an 18S rRNA PCR assay was equivalent to DSS for detection of mites (both 100%, n = 8). We recommend collecting samples from both IS and CV by both FP and DSS to assess for the presence of D. musculi and performing further studies to assess whether PCR analysis can be used as a diagnostic tool for the detection of Demodex mites in laboratory mice.

Virtually the entire surface of the HIV-1-envelope trimer is recognized by neutralizing antibodies, except for a highly glycosylated region at the center of the "silent face" on the gp120 subunit. From an HIV-1-infected donor, #74, we identified antibody VRC-PG05, which neutralized 27% of HIV-1 strains. The crystal structure of the antigen-binding fragment of VRC-PG05 in complex with gp120 revealed an epitope comprised primarily of N-linked glycans from N262, N295, and N448 at the silent face center. Somatic hypermutation occurred preferentially at antibody residues that interacted with these glycans, suggesting somatic development of glycan recognition. Resistance to VRC-PG05 in donor #74 involved shifting of glycan-N448 to N446 or mutation of glycan-proximal residue E293. HIV-1 neutralization can thus be achieved at the silent face center by glycan-recognizing antibody; along with other known epitopes, the VRC-PG05 epitope completes coverage by neutralizing antibody of all major exposed regions of the prefusion closed trimer.

Somatosensory neurons mediate responses to diverse mechanical stimuli, from innocuous touch to noxious pain. While recent studies have identified distinct populations of A mechanonociceptors (AMs) that are required for mechanical pain, the molecular underpinnings of mechanonociception remain unknown. Here, we show that the bioactive lipid sphingosine 1-phosphate (S1P) and S1P Receptor 3 (S1PR3) are critical regulators of acute mechanonociception. Genetic or pharmacological ablation of S1PR3, or blockade of S1P production, significantly impaired the behavioral response to noxious mechanical stimuli, with no effect on responses to innocuous touch or thermal stimuli. These effects are mediated by fast-conducting A mechanonociceptors, which displayed a significant decrease in mechanosensitivity in S1PR3 mutant mice. We show that S1PR3 signaling tunes mechanonociceptor excitability via modulation of KCNQ2/3 channels. Our findings define a new role for S1PR3 in regulating neuronal excitability and establish the importance of S1P/S1PR3 signaling in the setting of mechanical pain thresholds.

By using a cell fraction technique that separates chromatin-associated nascent RNA, newly completed nucleoplasmic mRNA and cytoplasmic mRNA, we have shown in a previous study that residues in exons are methylated (m(6)A) in nascent pre-mRNA and remain methylated in the same exonic residues in nucleoplasmic and cytoplasmic mRNA. Thus, there is no evidence of a substantial degree of demethylation in mRNA exons that would correspond to so-called "epigenetic" demethylation. The turnover rate of mRNA molecules is faster, depending on m(6)A content in HeLa cell mRNA, suggesting that specification of mRNA stability may be the major role of m(6)A exon modification. In mouse embryonic stem cells (mESCs) lacking Mettl3, the major mRNA methylase, the cells continue to grow, making the same mRNAs with unchanged splicing profiles in the absence (>90%) of m(6)A in mRNA, suggesting no common obligatory role of m(6)A in splicing. All these data argue strongly against a commonly used "reversible dynamic methylation/demethylation" of mRNA, calling into question the concept of "RNA epigenetics" that parallels the well-established role of dynamic DNA epigenetics.

Elevated body mass index (BMI) is associated with increased multi-morbidity and mortality. The investigation of the relationship between BMI and brain organization has the potential to provide new insights relevant to clinical and policy strategies for weight control. Here, we quantified the association between increasing BMI and the functional organization of resting-state brain networks in a sample of 496 healthy individuals that were studied as part of the Human Connectome Project. We demonstrated that higher BMI was associated with changes in the functional connectivity of the default-mode network (DMN), central executive network (CEN), sensorimotor network (SMN), visual network (VN), and their constituent modules. In siblings discordant for obesity, we showed that person-specific factors contributing to obesity are linked to reduced cohesiveness of the sensory networks (SMN and VN). We conclude that higher BMI is associated with widespread alterations in brain networks that balance sensory-driven (SMN, VN) and internally guided (DMN, CEN) states which may augment sensory-driven behavior leading to overeating and subsequent weight gain. Our results provide a neurobiological context for understanding the association between BMI and brain functional organization while accounting for familial and person-specific influences.