To measure the strength of natural selection that acts upon single nucleotide variants (SNVs) in a set of human genes, we calculate the ratio between nonsynonymous SNVs (nsSNVs) per nonsynonymous site and synonymous SNVs (sSNVs) per synonymous site. We transform this ratio with a respective factor f that corrects for the bias of synonymous sites towards transitions in the genetic code and different mutation rates for transitions and transversions. This method approximates the relative density of nsSNVs (rdnsv) in comparison with the neutral expectation as inferred from the density of sSNVs. Using SNVs from a diploid genome and 200 exomes, we apply our method to immune system genes (ISGs), nervous system genes (NSGs), randomly sampled genes (RSGs), and gene ontology annotated genes. The estimate of rdnsv in an individual exome is around 20% for NSGs and 30-40% for ISGs and RSGs. This smaller rdnsv of NSGs indicates overall stronger purifying selection. To quantify the relative shift of nsSNVs towards rare variants, we next fit a linear regression model to the estimates of rdnsv over different SNV allele frequency bins. The obtained regression models show a negative slope for NSGs, ISGs and RSGs, supporting an influence of purifying selection on the frequency spectrum of segregating nsSNVs. The y-intercept of the model predicts rdnsv for an allele frequency close to 0. This parameter can be interpreted as the proportion of nonsynonymous sites where mutations are tolerated to segregate with an allele frequency notably greater than 0 in the population, given the performed normalization of the observed nsSNV to sSNV ratio. A smaller y-intercept is displayed by NSGs, indicating more nonsynonymous sites under strong negative selection. This predicts more monogenically inherited or de-novo mutation diseases that affect the nervous system.

Trauma to the spinal cord and brain can result in irreparable loss of function. This failure of recovery is in part due to inhibition of axon regeneration by myelin and chondroitin sulfate proteoglycans (CSPGs). Peripheral nervous system (PNS) neurons exhibit increased regenerative ability compared to central nervous system neurons, even in the presence of inhibitory environments. Previously, we identified over a thousand genes differentially expressed in PNS neurons relative to CNS neurons. These genes represent intrinsic differences that may account for the PNS's enhanced regenerative ability. Cerebellar neurons were transfected with cDNAs for each of these PNS genes to assess their ability to enhance neurite growth on inhibitory (CSPG) or permissive (laminin) substrates. Using high content analysis, we evaluated the phenotypic profile of each neuron to extract meaningful data for over 1100 genes. Several known growth associated proteins potentiated neurite growth on laminin. Most interestingly, novel genes were identified that promoted neurite growth on CSPGs (GPX3, EIF2B5, RBMX). Bioinformatic approaches also uncovered a number of novel gene families that altered neurite growth of CNS neurons.

Impairment of working memory (WM) performance in schizophrenia patients (SZ) is well-established. Compared to healthy controls (HC), SZ patients show aberrant blood oxygen level dependent (BOLD) activations and disrupted functional connectivity during WM performance. In this study, we examined the small-world network metrics computed from functional magnetic resonance imaging (fMRI) data collected as 35 HC and 35 SZ performed a Sternberg Item Recognition Paradigm (SIRP) at three WM load levels. Functional connectivity networks were built by calculating the partial correlation on preprocessed time courses of BOLD signal between task-related brain regions of interest (ROIs) defined by group independent component analysis (ICA). The networks were then thresholded within the small-world regime, resulting in undirected binarized small-world networks at different working memory loads. Our results showed: 1) at the medium WM load level, the networks in SZ showed a lower clustering coefficient and less local efficiency compared with HC; 2) in SZ, most network measures altered significantly as the WM load level increased from low to medium and from medium to high, while the network metrics were relatively stable in HC at different WM loads; and 3) the altered structure at medium WM load in SZ was related to their performance during the task, with longer reaction time related to lower clustering coefficient and lower local efficiency. These findings suggest brain connectivity in patients with SZ was more diffuse and less strongly linked locally in functional network at intermediate level of WM when compared to HC. SZ show distinctly inefficient and variable network structures in response to WM load increase, comparing to stable highly clustered network topologies in HC.

Marine sponge species are of significant interest to many scientific fields including marine ecology, conservation biology, genetics, host-microbe symbiosis and pharmacology. One of the most intriguing aspects of the sponge "holobiont" system is the unique physiology, interaction with microbes from the marine environment and the development of a complex commensal microbial community. However, intraspecific variability and temporal stability of sponge-associated bacterial symbionts remain relatively unknown.

To determine the maximum tolerated dose (MTD) and safety, and explore efficacy and biomarkers of vandetanib with cetuximab and irinotecan in second-line metastatic colorectal cancer.

Constitutive activation of FGFR1, through rearrangement with various dimerization domains, leads to atypical myeloproliferative disorders where, although T cell lymphoma are common, the BCR-FGFR1 chimeric kinase results in CML-like leukemia. As with the human disease, mouse bone marrow transduction/transplantation with BCR-FGFR1 leads to CML-like myeloproliferation as well as B-cell leukemia/lymphoma. The murine disease described in this report is virtually identical to the human disease in that both showed bi-lineage involvement of myeloid and B-cells, splenomegaly, leukocytosis and bone marrow hypercellularity. A CD19(+) IgM(-) CD43(+) immunophenotype was seen both in primary tumors and two cell lines derived from these tumors. In all primary tumors, subpopulations of these CD19(+) IgM(-) CD43(+) were also either B220(+) or B220(-), suggesting a block in differentiation at the pro-B cell stage. The B220(-) phenotype was retained in one of the cell lines while the other was B220(+). When the two cell lines were transplanted into syngeneic mice, all animals developed the same B-lymphoblastic leukemia within 2-weeks. Thus, the murine model described here closely mimics the human disease with bilineage myeloid and B-cell leukemia/lymphoma which provides a representative model to investigate therapeutic intervention and a better understanding of the etiology of the disease.

Hyperuricemia, hyperlipidemia and inflammation are associated with diabetic nephropathy. The NLRP3 inflammasome-mediated inflammation is recently recognized in the development of kidney injury. Urate and lipid are considered as danger signals in the NLRP3 inflammasome activation. Although dietary flavonoid quercetin and allopurinol alleviate hyperuricemia, dyslipidmia and inflammation, their nephroprotective effects are currently unknown. In this study, we used streptozotocin (STZ)-induced diabetic nephropathy model with hyperuricemia and dyslipidemia in rats, and found over-expression of renal inflammasome components NLRP3, apoptosis-associated speck-like protein and Caspase-1, resulting in elevation of IL-1β and IL-18, with subsequently deteriorated renal injury. These findings demonstrated the possible association between renal NLRP3 inflammasome activation and lipid accumulation to superimpose causes of nephrotoxicity in STZ-treated rats. The treatment of quercetin and allopurinol regulated renal urate transport-related proteins to reduce hyperuricemia, and lipid metabolism-related genes to alleviate kidney lipid accumulation in STZ-treated rats. Furthermore, quercetin and allopurinol were found to suppress renal NLRP3 inflammasome activation, at least partly, via their anti-hyperuricemic and anti-dyslipidemic effects, resulting in the amelioration of STZ-induced the superimposed nephrotoxicity in rats. These results may provide a basis for the prevention of diabetes-associated nephrotoxicity with urate-lowering agents such as quercetin and allopurinol.

Mechanotransduction has demonstrated potential for regulating tissue adaptation in vivo and cellular activities in vitro. It is well documented that ultrasound can produce a wide variety of biological effects in biological systems. For example, pulsed ultrasound can be used to noninvasively accelerate the rate of bone fracture healing. Although a wide range of studies has been performed, mechanism for this therapeutic effect on bone healing is currently unknown. To elucidate the mechanism of cellular response to mechanical stimuli induced by pulsed ultrasound radiation, we developed a method to apply focused acoustic radiation force (ARF) (duration, one minute) on osteoblastic MC3T3-E1 cells and observed cellular responses to ARF using a spinning disk confocal microscope. This study demonstrates that the focused ARF induced F-actin cytoskeletal rearrangement in MC3T3-E1 cells. In addition, these cells showed an increase in intracellular calcium concentration following the application of focused ARF. Furthermore, passive bending movement was noted in primary cilium that were treated with focused ARF. Cell viability was not affected. Application of pulsed ultrasound radiation generated only a minimal temperature rise of 0.1°C, and induced a streaming resulting fluid shear stress of 0.186 dyne/cm(2), suggesting that hyperthermia and acoustic streaming might not be the main causes of the observed cell responses. In conclusion, these data provide more insight in the interactions between acoustic mechanical stress and osteoblastic cells. This experimental system could serve as basis for further exploration of the mechanosensing mechanism of osteoblasts triggered by ultrasound.

To determine the capacity of dendritic cells (DCs) loaded with single or multiple-peptide mixtures of novel hepatitis C virus (HCV) epitopes to stimulate HCV-specific cytotoxic T lymphocyte (CTL) effector functions.

Sex differences in responses to myocardial ischemia have been described, but whether cardiomyocyte function is influenced by sex in the setting of ischemia and reperfusion has not been elucidated. This study compared contractions and intracellular Ca(2+) in isolated ventricular myocytes exposed to ischemia and reperfusion. Cells were isolated from anesthetized 3-month-old male and female Fischer 344 rats, paced at 4 Hz (37°C), exposed to simulated ischemia (20 mins) and reperfused. Cell shortening (edge detector) and intracellular Ca(2+) (fura-2) were measured simultaneously. Cell viability was assessed with Trypan blue. Ischemia reduced peak contractions and increased Ca(2+) levels equally in myocytes from both sexes. However, contraction amplitudes were reduced in reperfusion in male myocytes, while contractions recovered to exceed control levels in females (62.6±5.1 vs. 140.1±15.8%; p<0.05). Only 60% of male myocytes excluded trypan blue dye after ischemia and reperfusion, while all female cardiomyocytes excluded the dye (p<0.05). Parallel experiments were conducted in myocytes from ∼24-month-old female rats or 5-6-month-old rats that had an ovariectomy at 3-4 weeks of age. Beneficial effects of female sex on myocyte viability and contractile dysfunction in reperfusion were abolished in cells from 24-month-old females. Aged female myocytes also exhibited elevated intracellular Ca(2+) and alternans in ischemia. Cells from ovariectomized rats displayed increased Ca(2+) transients and spontaneous activity in ischemia compared to sham-operated controls. None of the myocytes from ovariectomized rats were viable after 15 minutes of ischemia, while 75% of sham cells remained viable at end of reperfusion (p<0.05). These findings demonstrate that cardiomyocytes from young adult females are more resistant to ischemia and reperfusion injury than cells from males. Age and OVX abolish these beneficial effects and induce Ca(2+) dysregulation at the level of the cardiomyocyte. Thus, beneficial effects of estrogen in ischemia and reperfusion are mediated, in part, by effects on cardiomyocytes.

Unfavorable maternal diet during pregnancy can predispose the offspring to diseases later in life, such as hypertension, metabolic syndrome, and obesity. However, the molecular basis for this phenomenon of "developmental programming" is poorly understood. We have recently shown that a diet nutritionally optimized for pregnancy can nevertheless be harmful in the context of diabetic pregnancy in the mouse, associated with a high incidence of neural tube defects and intrauterine growth restriction. We hypothesized that placental abnormalities may contribute to impaired fetal growth in these pregnancies, and therefore investigated the role of maternal diet in the placenta. LabDiet 5015 diet was associated with reduced placental growth, commencing at midgestation, when compared to pregnancies in which the diabetic dam was fed LabDiet 5001 maintenance chow. Furthermore, by quantitative RT-PCR we identify 34 genes whose expression in placenta at midgestation is modulated by diet, diabetes, or both, establishing biomarkers for gene-environment interactions in the placenta. These results implicate maternal diet as an important factor in pregnancy complications and suggest that the early phases of placenta development could be a critical time window for developmental origins of adult disease.

To identify the immediate economic impact of maternal death on rural Chinese households.

Recent evidence established a crucial role for mammalian oxygen sensing transcription factor hypoxia inducible factor-1 (HIF-1) in innate immunity against intracellular pathogens. In response to most of these pathogens host phagocytes increase transcription of HIF-1α, the regulatory component of HIF-1 to express various effector molecules against invaders. Leishmania donovani (LD), a protozoan parasite and the causative agent of fatal visceral leishmaniasis resides in macrophages within mammalian host. The mechanism of HIF-1 activation or its role in determining the fate of LD in infected macrophages is still not known. To determine that J774 macrophages were infected with LD and about four-fold increase in HIF-1 activity and HIF-1α expression were detected. A strong increase in HIF-1α expression and nuclear localization was also detected in LD-infected J774 cells, peritoneal macrophages and spleen derived macrophages of LD-infected BALB/c mice. A two-fold increase in HIF-1α mRNA was detected in LD-infected macrophages suggesting involvement of a transcriptional mechanism that was confirmed by promoter activity. We further revealed that LD also induced HIF-1α expression by depleting host cellular iron pool to affect prolyl hydroxylase activity resulting in to stabilization of HIF-1α. To determine the role of HIF-1 on intracellular LD, cells were transfected with HIF-1α siRNA to attenuate its expression and then infected with LD. Although, initial infection rate of LD in HIF-1α attenuated cells was not affected but intracellular growth of LD was significantly inhibited; while, over-expression of stabilized form of HIF-1α promoted intracellular growth of LD in host macrophage. Our results strongly suggest that LD activates HIF-1 by dual mechanism for its survival advantage within macrophage.

In order to describe the influence of Pleistocene glaciations on the genetic structure and demography of a highly mobile, but specialized, passerine, the Savi's Warbler (Locustella luscinioides), mitochondrial DNA sequences (ND2) and microsatellites were analysed in c.330 individuals of 17 breeding and two wintering populations. Phylogenetic, population genetics and coalescent methods were used to describe the genetic structure, determine the timing of the major splits and model the demography of populations. Savi's Warblers split from its sister species c.8 million years ago and have two major haplotype groups that diverged in the early/middle Pleistocene. One of these clades originated in the Balkans and is currently widespread, showing strong evidence for population expansion; whereas the other is restricted to Iberia and remained stable. Microsatellites agreed with a genetic break around the Pyrenees, but showed considerable introgression and a weaker genetic structure. Both genetic markers showed an isolation-by-distance pattern associated with the population expansion of the eastern clade. Breeding populations seem to be segregated at the wintering sites, but results on migratory connectivity are preliminary. Savi's Warbler is the only known migratory bird species in which Iberian birds did not expand beyond the Pyrenees after the last glaciation. Despite the long period of independent evolution of western and eastern populations, complete introgression occurred when these groups met in Iberia. Mitochondrial sequences indicated the existence of refugia-within-refugia in the Iberian Peninsula during the last glacial period, which is surprising given the high dispersal capacity of this species. Plumage differences of eastern subspecies seemed to have evolved recently through natural selection, in agreement with the glacial expansion hypothesis. This study supports the great importance of the Iberian Peninsula and its role for the conservation of genetic variation.

The desmosome anchors keratin filaments in epithelial cells leading to the formation of a tissue wide IF network. Loss of the desmosomal plaque protein plakophilin3 (PKP3) in HCT116 cells, leads to an increase in neoplastic progression and metastasis, which was accompanied by an increase in K8 levels. The increase in levels was due to an increase in the protein levels of the Phosphatase of Regenerating Liver 3 (PRL3), which results in a decrease in phosphorylation on K8. The increase in PRL3 and K8 protein levels could be reversed by introduction of an shRNA resistant PKP3 cDNA. Inhibition of K8 expression in the PKP3 knockdown clone S10, led to a decrease in cell migration and lamellipodia formation. Further, the K8 PKP3 double knockdown clones showed a decrease in colony formation in soft agar and decreased tumorigenesis and metastasis in nude mice. These results suggest that a stabilisation of K8 filaments leading to an increase in migration and transformation may be one mechanism by which PKP3 loss leads to tumor progression and metastasis.

MicroRNA (miRNA) function is required for normal animal development, in particular in differentiation pathways from stem cell and precursor populations. In neurogenesis, it is becoming increasingly appreciated that miRNAs act at many stages to ensure proper progression. In this study we examined the role of miR-34a in neural progenitor cells (NPC) derived from murine embryonic cortex. We found that over-expression of miR-34a in NPC significantly reduced the neuron yield upon in vitro induction of differentiation. MiR-34a has several predicted targets in the Notch pathway, which operates to balance progenitor self-renewal and differentiation during cortical neurogenesis. We tested several Notch pathway players for regulation by miR-34a in undifferentiated NPC, and found that mRNA and protein levels of Numbl, a negative regulator of Notch signaling, as well as two downstream pro-neural genes usually blocked by Notch signaling, NeuroD1 and Mash1, were diminished, while Notch1 and Cbf1 transcripts were enhanced by miR-34a over-expression. Using a luciferase reporter assay, we verified the Numbl 3'-UTR as a direct miR-34a target. Correspondingly, knock-down of endogenous miR-34a resulted in increased Numbl, NeuroD1 and Mash1, and reduced Notch1 transcript levels. Together these results implicate Numbl as a physiologically relevant target of miR-34a in NPC, allowing for enhanced Notch signaling and inhibition of neuronal differentiation. This work extends our understanding of miR-34a-mediated control of cell differentiation from cancer to mammalian nervous system development.

Adult community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) and methicillin-susceptible S. aureus (CA-MSSA) skin and soft tissue infection (SSTI) in China is not well described. A prospective cohort of adults with SSTI was established between January 2009 and August 2010 at 4 hospitals in Beijing. Susceptibility testing and molecular typing, including multilocus sequence typing, spa, agr typing, and toxin detection were assessed for all S. aureus isolates. Overall, 501 SSTI patients were enrolled. Cutaneous abscess (40.7%) was the most common infection, followed by impetigo (6.8%) and cellulitis (4.8%). S. aureus accounted for 32.7% (164/501) of SSTIs. Five isolates (5/164, 3.0%) were CA-MRSA. The most dominant ST in CA-MSSA was ST398 (17.6%). The prevalence of Panton-Valentine Leukocidin (pvl) gene was 41.5% (66/159) in MSSA. Female, younger patients and infections requiring incision or drainage were more commonly associated with pvl-positive S. aureus (P<0.03); sec gene was more often identified in CC5 (P<0.03); seh gene was more prevalent in CC1 (P = 0.001). Importantly, ST59 isolates showed more resistance to erythromycin, clindamycin and tetracycline, and needed more surgical intervention. In conclusion, CA-MRSA infections were rare among adult SSTI patients in Beijing. Six major MSSA clones were identified and associated with unique antimicrobial susceptibility, toxin profiles, and agr types. A high prevalence of livestock ST398 clone (17.1% of all S. aureus infections) was found with no apparent association to animal contact.

While the essential role of bone morphogenetic protein (BMP) signaling in nervous system development is well established, its function in the adult CNS is poorly understood. We investigated the role of BMP signaling in the adult mouse retina following damage in vivo. Intravitreal injection of N-methyl-D-aspartic acid (NMDA) induced extensive retinal ganglion cell death by 2 days. During this period, BMP2, -4 and -7 were upregulated, leading to phosphorylation of the downstream effector, Smad1/5/8 in the inner retina, including in retinal ganglion cells. Expression of Inhibitor of differentiation 1 (Id1; a known BMP-Smad1/5/8 target) was also upregulated in the retina. This activation of BMP-Smad1/5/8 signaling was also observed following light damage, suggesting that it is a general response to retinal injuries. Co-injection of BMP inhibitors with NMDA effectively blocked the damage-induced BMP-Smad1/5/8 activation and led to further cell death of retinal ganglion cells, when compared with NMDA injection alone. Moreover, treatment of the retina with exogenous BMP4 along with NMDA damage led to a significant rescue of retinal ganglion cells. These data demonstrate that BMP-Smad1/5/8 signaling is neuroprotective for retinal ganglion cells after damage, and suggest that stimulation of this pathway can serve as a potential target for neuroprotective therapies in retinal ganglion cell diseases, such as glaucoma.

The ability to synthesize and salvage purines is crucial for colonization by a variety of human bacterial pathogens. Helicobacter pylori colonizes the gastric epithelium of humans, yet its specific purine requirements are poorly understood, and the transport mechanisms underlying purine uptake remain unknown. Using a fully defined synthetic growth medium, we determined that H. pylori 26695 possesses a complete salvage pathway that allows for growth on any biological purine nucleobase or nucleoside with the exception of xanthosine. Doubling times in this medium varied between 7 and 14 hours depending on the purine source, with hypoxanthine, inosine and adenosine representing the purines utilized most efficiently for growth. The ability to grow on adenine or adenosine was studied using enzyme assays, revealing deamination of adenosine but not adenine by H. pylori 26695 cell lysates. Using mutant analysis we show that a strain lacking the gene encoding a NupC homolog (HP1180) was growth-retarded in a defined medium supplemented with certain purines. This strain was attenuated for uptake of radiolabeled adenosine, guanosine, and inosine, showing a role for this transporter in uptake of purine nucleosides. Deletion of the GMP biosynthesis gene guaA had no discernible effect on mouse stomach colonization, in contrast to findings in numerous bacterial pathogens. In this study we define a more comprehensive model for purine acquisition and salvage in H. pylori that includes purine uptake by a NupC homolog and catabolism of adenosine via adenosine deaminase.

Dispersal is an important nematode behavior. Upon crowding or food depletion, the free living bacteriovorus nematode Caenorhabditis elegans produces stress resistant dispersal larvae, called dauer, which are analogous to second stage juveniles (J2) of plant parasitic Meloidogyne spp. and infective juveniles (IJ)s of entomopathogenic nematodes (EPN), e.g., Steinernema feltiae. Regulation of dispersal behavior has not been thoroughly investigated for C. elegans or any other nematode species. Based on the fact that ascarosides regulate entry in dauer stage as well as multiple behaviors in C. elegans adults including mating, avoidance and aggregation, we hypothesized that ascarosides might also be involved in regulation of dispersal behavior in C. elegans and for other nematodes such as IJ of phylogenetically related EPNs.