CNNM2, encoding a basolateral protein required for renal Mg2+ handling, is mutated in dominant hypomagnesemia

Familial hypomagnesemia is a rare human disorder caused by renal or intestinal magnesium (Mg(2+)) wasting, which may lead to symptoms of Mg(2+) depletion such as tetany, seizures, and cardiac arrhythmias. Our knowledge of the physiology of Mg(2+) (re)absorption, particularly the luminal uptake of Mg(2+) along the nephron, has benefitted from positional cloning approaches in families with Mg(2+) reabsorption disorders; however, basolateral Mg(2+) transport and its regulation are still poorly understood. Here, by using a candidate screening approach, we identified CNNM2 as a gene involved in renal Mg(2+) handling in patients of two unrelated families with unexplained dominant hypomagnesemia. In the kidney, CNNM2 was predominantly found along the basolateral membrane of distal tubular segments involved in Mg(2+) reabsorption. The basolateral localization of endogenous and recombinant CNNM2 was confirmed in epithelial kidney cell lines. Electrophysiological analysis showed that CNNM2 mediated Mg(2+)-sensitive Na(+) currents that were significantly diminished in mutant protein and were blocked by increased extracellular Mg(2+) concentrations. Our data support the findings of a recent genome-wide association study showing the CNNM2 locus to be associated with serum Mg(2+) concentrations. The mutations found in CNNM2, its observed sensitivity to extracellular Mg(2+), and its basolateral localization signify a critical role for CNNM2 in epithelial Mg(2+) transport.     

The C‐terminal regions of YidC from Rhodopirellula baltica and Oceanicaulis alexandrii bind to ribosomes and partially substitute for SRP receptor function in Escherichia coli

The marine Gram‐negative bacteria Rhodopirellula baltica and Oceanicaulis alexandrii have, in contrast to Escherichia coli, membrane insertases with extended positively charged C‐terminal regions similar to the YidC homologues in mitochondria and Gram‐positive bacteria. We have found that chimeric forms of E. coli YidC fused to the C‐terminal YidC regions from the marine bacteria mediate binding of YidC to ribosomes and therefore may have a functional role for targeting a nascent protein to the membrane. Here, we show in E. coli that an extended C‐terminal region of YidC can compensate for a loss of SRP‐receptor function in vivo. Furthermore, the enhanced affinity of the ribosome to the chimeric YidC allows the isolation of a ribosome nascent chain complex together with the C‐terminally elongated YidC chimera. This complex was visualized at 8.6 Å by cryo‐electron microscopy and shows a close contact of the ribosome and a YidC monomer.     

Gastrointestinal absorption and metabolism of apple polyphenols ex vivo by the pig intestinal mucosa in the Ussing chamber

Polyphenols contained in food have various positive effects on human health. The absorption and metabolism of polyphenols in the intestinal tract needs to be studied to estimate these effects. The Ussing chamber technique was used to investigate the transport behavior of apple polyphenols through pig small intestinal mucosa, which served as a model for human gastrointestinal mucosa. The identities and concentrations of polyphenols and their metabolites in the half-chambers (luminal and basolateral) within an incubation period of 4 h were determined by HPLC–MS/MS and HPLC–DAD (DAD = diode-array detection). Flux values were also measured. It was found that 5-caffeoylquinic acid and caffeic acid were absorbed and translocated to the basolateral side (1.9 and 3.7%, respectively), but other compounds, including glycosides of phloretin and quercetin, were observed without translocation. A Ussing chamber utilizing pig small intestinal mucosa is a suitable model for assessing the effect of apple polyphenols on mucosal integrity and nutrition absorption across porcine mucosa.     

Lower Prevalence of Carotid Plaque Hemorrhage in Women,and Its Mediator Effect on Sex Differences in Recurrent Cerebrovascular Events

Background and Purpose

Women are at lower risk of stroke, and appear to benefit less from carotid endarterectomy (CEA) than men. We hypothesised that this is due to more benign carotid disease in women mediating a lower risk of recurrent cerebrovascular events. To test this, we investigated sex differences in the prevalence of MRI detectable plaque hemorrhage (MRI PH) as an index of plaque instability, and secondly whether MRI PH mediates sex differences in the rate of cerebrovascular recurrence.

Methods

Prevalence of PH between sexes was analysed in a single centre pooled cohort of 176 patients with recently symptomatic, significant carotid stenosis (106 severe [≥70%], 70 moderate [50–69%]) who underwent prospective carotid MRI scanning for identification of MRI PH. Further, a meta-analysis of published evidence was undertaken. Recurrent events were noted during clinical follow up for survival analysis.

Results

Women with symptomatic carotid stenosis (50%≥) were less likely to have plaque hemorrhage (PH) than men (46% vs. 70%) with an adjusted OR of 0.23 [95% CI 0.10–0.50, P<0.0001] controlling for other known vascular risk factors. This negative association was only significant for the severe stenosis subgroup (adjusted OR 0.18, 95% CI 0.067–0.50) not the moderate degree stenosis. Female sex in this subgroup also predicted a longer time to recurrent cerebral ischemic events (HR 0.38 95% CI 0.15–0.98, P = 0.045). Further addition of MRI PH or smoking abolished the sex effects with only MRI PH exerting a direct effect.Meta-analysis confirmed a protective effect of female sex on development of PH: unadjusted OR for presence of PH = 0.54 (95% CI 0.45–0.67, p<0.00001).

Conclusions

MRI PH is significantly less prevalent in women. Women with MRI PH and severe stenosis have a similar risk as men for recurrent cerebrovascular events. MRI PH thus allows overcoming the sex bias in selection for CEA.     

Comparative transcriptome profiling of amyloid precursor protein family members in the adult cortex

Background

SOX2 is a key gene implicated in maintaining the stemness of embryonic and adult stem cells. SOX2 appears to re-activate in several human cancers including glioblastoma multiforme (GBM), however, the detailed response program of SOX2 in GBM has not yet been defined.

Results

We show that knockdown of the SOX2 gene in LN229 GBM cells reduces cell proliferation and colony formation. We then comprehensively characterize the SOX2 response program by an integrated analysis using several advanced genomic technologies including ChIP-seq, microarray profiling, and microRNA sequencing. Using ChIP-seq technology, we identified 4883 SOX2 binding regions in the GBM cancer genome. SOX2 binding regions contain the consensus sequence wwTGnwTw that occurred 3931 instances in 2312 SOX2 binding regions. Microarray analysis identified 489 genes whose expression altered in response to SOX2 knockdown. Interesting findings include that SOX2 regulates the expression of SOX family proteins SOX1 and SOX18, and that SOX2 down regulates BEX1 (brain expressed X-linked 1) and BEX2 (brain expressed X-linked 2), two genes with tumor suppressor activity in GBM. Using next generation sequencing, we identified 105 precursor microRNAs (corresponding to 95 mature miRNAs) regulated by SOX2, including down regulation of miR-143, -145, -253-5p and miR-452. We also show that miR-145 and SOX2 form a double negative feedback loop in GBM cells, potentially creating a bistable system in GBM cells.

Conclusions

We present an integrated dataset of ChIP-seq, expression microarrays and microRNA sequencing representing the SOX2 response program in LN229 GBM cells. The insights gained from our integrated analysis further our understanding of the potential actions of SOX2 in carcinogenesis and serves as a useful resource for the research community.     

Ku70 and Rad51 vary in their importance for the repair of doxorubicin- versus etoposide-induced DNA damage

For DNA targeting anticancer drugs, cellular DNA repair mechanisms may cause resistance and hamper the therapeutic outcome. DNA damage induced by topoisomerase IIα inhibitors like etoposide and anthracyclines, which are a mainstay of cancer therapy, is also repaired in many cell types, but the impact and precise mechanisms of this repair are still obscure. To investigate the DNA damage response of human adenocarcinoma HT29-cells to doxorubicin and to compare the involvement of Ku70 and Rad51 in the repair of doxorubicin- versus etoposide-induced DNA damage, we assessed cell cycle distribution and cell death, DNA damage, proteins relevant for repair by homologous recombination and non-homologous end-joining, and clonogenicity following exposure to doxorubicin at clinically achievable concentrations. Also, we assessed changes in the repair kinetics after siRNA-mediated attenuation of Ku70 or Rad51 expression. We found that exposure to doxorubicin for 24 h induced a substantial amount of DNA damage that was largely repaired when doxorubicin was removed and the cells were maintained in drug-free medium. Nevertheless, a pronounced G₂/M arrest occurred at times when repair was maximal. This was followed by a distinct increase in cell death and loss of clonogenicity. In this regard, responses to doxorubicin and etoposide were similar. However, distinct differences in the repair process following doxorubicin versus etoposide were seen in concentration dependency, time-course and requirement of Ku70 and Rad51 proteins. In spite of the shared molecular target of doxorubicin and etoposide, DNA lesions induced by these compounds are repaired differently.