The MRN complex is transcriptionally regulated by MYCN during neural cell proliferation to control replication stress

The MRE11/RAD50/NBS1 (MRN) complex is a major sensor of DNA double strand breaks, whose role in controlling faithful DNA replication and preventing replication stress is also emerging. Inactivation of the MRN complex invariably leads to developmental and/or degenerative neuronal defects, the pathogenesis of which still remains poorly understood. In particular, NBS1 gene mutations are associated with microcephaly and strongly impaired cerebellar development, both in humans and in the mouse model. These phenotypes strikingly overlap those induced by inactivation of MYCN, an essential promoter of the expansion of neuronal stem and progenitor cells, suggesting that MYCN and the MRN complex might be connected on a unique pathway essential for the safe expansion of neuronal cells. Here, we show that MYCN transcriptionally controls the expression of each component of the MRN complex. By genetic and pharmacological inhibition of the MRN complex in a MYCN overexpression model and in the more physiological context of the Hedgehog-dependent expansion of primary cerebellar granule progenitor cells, we also show that the MRN complex is required for MYCN-dependent proliferation. Indeed, its inhibition resulted in DNA damage, activation of a DNA damage response, and cell death in a MYCN- and replication-dependent manner. Our data indicate the MRN complex is essential to restrain MYCN-induced replication stress during neural cell proliferation and support the hypothesis that replication-born DNA damage is responsible for the neuronal defects associated with MRN dysfunctions.Cell Death and Differentiation advance online publication, 12 June 2015; doi:10.1038/cdd.2015.81

Tales of Emergence - Synthetic Biology as a Scientific Community in the Making

International audienceThis article locates the beginnings of a synthetic biology network and thereby probes the formation of a potential disciplinary community. We consider the ways that ideas of community are mobilized, both by scientists and policy-makers in building an agenda for new forms of knowledge work, and by social scientists as an analytical device to understand new formations for knowledge production. As participants in, and analysts of, a network in synthetic biology, we describe our current understanding of synthetic biology by telling four tales of community making. The first tale tells of the mobilization of synthetic biology within a European context. The second tale describes the approach to synthetic biology community formation in the UK. The third narrates the creation of an institutionally based, funded 'network in synthetic biology'. The final tale de-localizes community-making efforts by focussing on 'devices' that make communities. In tying together these tales, our analysis suggests that the potential community can be understood in terms of 'movements'--the (re)orientation and enrolment of people, stories, disciplines and policies; and of 'stickiness'--the objects and glues that begin to bind together the various constitutive elements of community

A case-control analysis of common variants in GIP with type 2 diabetes and related biochemical parameters in a South Indian population

<p>Abstract</p> <p>Background</p> <p>Glucose-dependent insulinotropic polypeptide (GIP) is one of the incretins, which plays a crucial role in the secretion of insulin upon food stimulus and in the regulation of postprandial glucose level. It also exerts an effect on the synthesis and secretion of lipoprotein lipase, from adipocytes, important for lipid metabolism. The aim of our study was to do a case-control association analysis of common variants in <it>GIP </it>in association with type 2 diabetes and related biochemical parameters.</p> <p>Method</p> <p>A total of 2000 subjects which includes 1000 (584M/416F) cases with type 2 diabetes and 1000 (470M/530F) normoglycemic control subjects belonging to Dravidian ethnicity from South India were recruited to assess the effect of single nucleotide polymorphisms (SNPs) in <it>GIP </it>(rs2291725, rs2291726, rs937301) on type 2 diabetes in a case-control manner. The SNPs were genotyped by using tetra primer amplification refractory mutation system-PCR (ARMS PCR). For statistical analysis, our study population was divided into sub-groups based on gender (male and female). Association analysis was carried out using chi-squared test and the comparison of biochemical parameters among the three genotypes were performed using analysis of covariance (ANCOVA).</p> <p>Result</p> <p>Initial analysis revealed that, out of the total three SNPs selected for the present study, two SNPs namely rs2291726 and rs937301 were in complete linkage disequilibrium (LD) with each other. Therefore, only two SNPs, rs2291725 and rs2291726, were genotyped for the association studies. No significant difference in the allele frequency and genotype distribution of any of the SNPs in <it>GIP </it>were observed between cases and controls (<it>P </it>> 0.05). Analysis of biochemical parameters among the three genotypes showed a significant association of total cholesterol (<it>P </it>= 0.042) and low density lipoprotein (LDL) with the G allele of the SNP rs2291726 in <it>GIP </it>(<it>P </it>= 0.004), but this was observed only in the case of female subjects. However this association does not remain significant after correction for multiple testing by Bonferroni's inequality method.</p> <p>Conclusion</p> <p>No statistically significant association was observed between any of the SNPs analysed and type 2 diabetes in our population. But the analysis of biochemical parameters indicates that the G allele in rs2291726 may be a putative risk allele for increased LDL cholesterol and further studies in other population needs to be carried out for ascertaining its role in cholesterol metabolism and subsequent cardiovascular risk.</p

Frailty in primary care: a review of its conceptualization and implications for practice

Frail, older patients pose a challenge to the primary care physician who may often feel overwhelmed by their complex presentation and tenuous health status. At the same time, family physicians are ideally suited to incorporate the concept of frailty into their practice. They have the propensity and skill set that lends itself to patient-centred care, taking into account the individual subtleties of the patient's health within their social context. Tools to identify frailty in the primary care setting are still in the preliminary stages of development. Even so, some practical measures can be taken to recognize frailty in clinical practice and begin to address how its recognition may impact clinical care. This review seeks to address how frailty is recognised and managed, especially in the realm of primary care

Light Plays an Essential Role in Intracellular Distribution of Auxin Efflux Carrier PIN2 in Arabidopsis thaliana

BACKGROUND: Light plays a key role in multiple plant developmental processes. It has been shown that root development is modulated by shoot-localized light signaling and requires shoot-derived transport of the plant hormone, auxin. However, the mechanism by which light regulates root development is not largely understood. In plants, the endogenous auxin, indole-3-acetic acid, is directionally transported by plasma-membrane (PM)-localized auxin influx and efflux carriers in transporting cells. Remarkably, the auxin efflux carrier PIN proteins exhibit asymmetric PM localization, determining the polarity of auxin transport. Similar to PM-resident receptors and transporters in animal and yeast cells, PIN proteins undergo constitutive cycling between the PM and endosomal compartments. Auxin plays multiple roles in PIN protein intracellular trafficking, inhibiting PIN2 endocytosis at some concentrations and promoting PIN2 degradation at others. However, how PIN proteins are turned over in plant cells is yet to be addressed. METHODOLOGY AND PRINCIPLE FINDINGS: Using laser confocal scanning microscopy, and physiological and molecular genetic approaches, here, we show that in dark-grown seedlings, the PM localization of auxin efflux carrier PIN2 was largely reduced, and, in addition, PIN2 signal was detected in vacuolar compartments. This is in contrast to light-grown seedlings where PIN2 was predominantly PM-localized. In light-grown plants after shift to dark or to continuous red or far-red light, PIN2 also accumulated in vacuolar compartments. We show that PIN2 vacuolar targeting was derived from the PM via endocytic trafficking and inhibited by HY5-dependent light signaling. In addition, the ubiquitin 26S proteasome is involved in the process, since its inhibition by mutations in COP9 and a proteasome inhibitor MG132 impaired the process. CONCLUSIONS AND SIGNIFICANCE: Collectively, our data indicate that light plays an essential role in PIN2 intracellular trafficking, promoting PM-localization in the presence of light and, on the other hand, vacuolar targeting for protein degradation in the absence of light. Based on these results, we postulate that light regulation of root development is mediated at least in part by changes in the intracellular distribution of auxin efflux carriers, PIN proteins, in response to the light environment

Critical Involvement of the ATM-Dependent DNA Damage Response in the Apoptotic Demise of HIV-1-Elicited Syncytia

DNA damage can activate the oncosuppressor protein ataxia telangiectasia mutated (ATM), which phosphorylates the histone H2AX within characteristic DNA damage foci. Here, we show that ATM undergoes an activating phosphorylation in syncytia elicited by the envelope glycoprotein complex (Env) of human immunodeficiency virus-1 (HIV-1) in vitro. This was accompanied by aggregation of ATM in discrete nuclear foci that also contained phospho-histone H2AX. DNA damage foci containing phosphorylated ATM and H2AX were detectable in syncytia present in the brain or lymph nodes from patients with HIV-1 infection, as well as in a fraction of blood leukocytes, correlating with viral status. Knockdown of ATM or of its obligate activating factor NBS1 (Nijmegen breakage syndrome 1 protein), as well as pharmacological inhibition of ATM with KU-55933, inhibited H2AX phosphorylation and prevented Env-elicited syncytia from undergoing apoptosis. ATM was found indispensable for the activation of MAP kinase p38, which catalyzes the activating phosphorylation of p53 on serine 46, thereby causing p53 dependent apoptosis. Both wild type HIV-1 and an HIV-1 mutant lacking integrase activity induced syncytial apoptosis, which could be suppressed by inhibiting ATM. HIV-1-infected T lymphoblasts from patients with inactivating ATM or NBS1 mutations also exhibited reduced syncytial apoptosis. Altogether these results indicate that apoptosis induced by a fusogenic HIV-1 Env follows a pro-apoptotic pathway involving the sequential activation of ATM, p38MAPK and p53

Regulation of Alr1 Mg Transporter Activity by Intracellular Magnesium

Mg homeostasis is critical to eukaryotic cells, but the contribution of Mg transporter activity to homeostasis is not fully understood. In yeast, Mg uptake is primarily mediated by the Alr1 transporter, which also allows low affinity uptake of other divalent cations such as Ni2+, Mn2+, Zn2+ and Co2+. Using Ni2+ uptake to assay Alr1 activity, we observed approximately nine-fold more activity under Mg-deficient conditions. The mnr2 mutation, which is thought to block release of vacuolar Mg stores, was associated with increased Alr1 activity, suggesting Alr1 was regulated by intracellular Mg supply. Consistent with a previous report of the regulation of Alr1 expression by Mg supply, Mg deficiency and the mnr2 mutation both increased the accumulation of a carboxy-terminal epitope-tagged version of the Alr1 protein (Alr1-HA). However, Mg supply had little effect on ALR1 promoter activity or mRNA levels. In addition, while Mg deficiency caused a seven-fold increase in Alr1-HA accumulation, the N-terminally tagged and untagged Alr1 proteins increased less than two-fold. These observations argue that the Mg-dependent accumulation of the C-terminal epitope-tagged protein was primarily an artifact of its modification. Plasma membrane localization of YFP-tagged Alr1 was also unaffected by Mg supply, indicating that a change in Alr1 location did not explain the increased activity we observed. We conclude that variation in Alr1 protein accumulation or location does not make a substantial contribution to its regulation by Mg supply, suggesting Alr1 activity is directly regulated via as yet unknown mechanisms