Genetic variation in the vitamin D receptor gene and vitamin D serum levels in Egyptian women with polycystic ovary syndrome

Obesity, insulin resistance, and hyperandrogenism are considered crucial parameters of polycystic ovary syndrome (PCOS) which might be related to vitamin D metabolism. The aim of this study was to investigate the associations between polymorphisms (TaqI and ApaI) in the vitamin D receptor gene (VDR) and PCOS among Egyptian women. We aimed also to elucidate the impact of these polymorphisms on vitamin D level, hormonal and metabolic parameters of PCOS. One hundred and fifty Egyptian women with PCOS and 150 unrelated controls were enrolled in this study. Polymorphisms of VDR Taq-I T/C (rs731236) and Apa-I A/C (rs7975232) gene were genotyped using polymerase chain reaction restriction fragment length polymorphism (PCR–RFLP). Serum 25 hydroxy vitamin D [25(OH) D] levels were measured by high-performance liquid chromatography. PCOS women had significantly lower levels of 25(OH) D compared to healthy women. Our results revealed that Taq-I CC genotype and C allele were associated with increased risk of PCOS, while the Apa-I polymorphism was not. Haplotype Taq-I C/ Apa-I C was associated with a higher PCOS risk more than controls. Moreover, there was a significant decrease of 25(OH) D levels in carriers of haplotype Taq-I C/ Apa-I C (with variant alleles) compared to the non-carriers. Results showed also that there was an obesity- VDR Taq-I genotypes interactions. These results suggested that, VDR Taq-I gene polymorphism is associated with increased risk of PCOS in Egyptian women.     

A Role for p38 Stress-Activated Protein Kinase in Regulation of Cell Growth via TORC1

The target of rapamycin (TOR) complex 1 (TORC1) signaling pathway is a critical regulator of translation and cell growth. To identify novel components of this pathway, we performed a kinome-wide RNA interference (RNAi) screen in Drosophila melanogaster S2 cells. RNAi targeting components of the p38 stress-activated kinase cascade prevented the cell size increase elicited by depletion of the TOR negative regulator TSC2. In mammalian and Drosophila tissue culture, as well as in Drosophila ovaries ex vivo, p38-activating stresses, such as H₂O₂ and anisomycin, were able to activate TORC1. This stress-induced TORC1 activation could be blocked by RNAi against mitogen-activated protein kinase kinase 3 and 6 (MKK3/6) or by the overexpression of dominant negative Rags. Interestingly, p38 was also required for the activation of TORC1 in response to amino acids and growth factors. Genetic ablation either of p38b or licorne, its upstream kinase, resulted in small flies consisting of small cells. Mutants with mutations in licorne or p38b are nutrition sensitive; low-nutrient food accentuates the small-organism phenotypes, as well as the partial lethality of the p38b null allele. These data suggest that p38 is an important positive regulator of TORC1 in both mammalian and Drosophila systems in response to certain stresses and growth factors.The target of rapamycin, TOR, is a highly conserved serine/threonine kinase that is a critical regulator of cell growth. It is a core component of two signaling complexes, TORC1 and TORC2 (60, 74). TORC1 is defined by the presence of Raptor in the complex, while TORC2 contains Rictor. Rictor and Raptor are mutually exclusive. Activation of the TORC1 pathway leads to increased protein translation, increased cell size, and increased proliferation, making this pathway an important target for emerging cancer therapies. Rapamycin is an inhibitor of TORC1 that is commonly used as an immunosuppressant following kidney transplantation (51). At least three analogs of rapamycin are currently being tested in solid and hematological tumors and have shown some promising results (21).The TORC1 pathway responds to numerous inputs, sensing both the desirability of and the capacity for growth. Many of these pathways control TORC1 signaling through phosphorylation of the tuberous sclerosis protein TSC2. TSC2 associates with TSC1 to form a heterodimeric GTPase-activating protein complex (GAP) that inactivates the small GTPase Rheb (24, 29, 67). While the exact molecular mechanism remains a topic of debate, activation of Rheb promotes the kinase activity of TORC1 (24, 29, 67). Rheb is required for the activation of TORC1 in response to both amino acids and growth factors (55, 62). In Drosophila melanogaster, mutation of either TOR or Rheb inhibits growth, leading to reduced body size and reduced cell size in mutant clones (42, 64). Mutation of either TSC1 or TSC2 has the predicted opposite effect, as tissue deficient for either of these proteins overgrows and contains large cells (49, 66).TORC1 is activated via the phosphatidylinositol 3′ kinase (PI3′K) pathway by growth-promoting mitogens, such as insulin and growth factors. Drosophila mutants with mutations of PI3′K pathway components have size phenotypes similar to those of the TOR and Rheb mutants (71). In mammalian cells, the PI3′K-mediated activation of TORC1 occurs at least in part through the phosphorylation of TSC2 by the PI3′K target AKT (30, 50). Interestingly, mutation of these residues in Drosophila has no impact on TSC2 function in vivo, suggesting that there may be other mechanisms through which PI3′K can activate Drosophila TOR (20). Recent work has suggested that the proline-rich AKT substrate PRAS40 may provide part of this link (23, 59, 69, 70). In addition, signaling through RAS activates extracellular signal-regulated kinase (ERK) and ribosomal S6 kinase (RSK), which can phosphorylate TSC2 and Raptor to activate TORC1 (13, 40, 56). There are also likely to be additional mechanisms through which growth factors activate Drosophila TOR that have not yet been identified.TORC1 activity is also controlled by the intracellular building blocks necessary to support cellular growth. The energy-sensing AMP-activated protein kinase (AMPK) pathway relays information about the energy status of the cell to TORC1 by phosphorylating TSC2. Unlike the inactivating phosphorylation of TSC2 by Akt, phosphorylation of TSC2 by AMPK promotes the GAP activity of the TSC complex (31). AMPK also phosphorylates Raptor, leading to decreased TORC1 activity (28). Thus, when energy levels are low, active AMPK inhibits TORC1.Amino acids also activate the TORC1 pathway, through a mechanism that requires Rheb, as well as the type III PI3′K VPS34 and the serine/threonine kinase mitogen-activated protein kinase kinase kinase kinase 3 (MAP4K3) (11, 22, 43). TORC1 thereby integrates information about the availability of amino acids and the amount of energy available for growth with growth factor signaling. Given its ancient function in adapting growth rates to environmental conditions, it is likely that TOR responds to a variety of stimuli, suggesting that many TOR control mechanisms remain to be uncovered. The Rag family of Ras-related small GTPases has recently been identified as a key component of the amino acid-sensing pathway, acting in parallel to Rheb (34, 58). Rag GTPases form heterodimers; RagA or RagB interacts with RagC or RagD. RagA and RagB are active when GTP bound, while RagC and RagD are active when bound to GDP (34, 58). Activation of the Rags by amino acids results in TOR relocalization to Rab7-containing vesicles (58). While the function of these vesicles in TORC1 signaling remains unclear, this relocalization is associated with increased TORC1 activity.TORC1 controls cell growth and translation through the phosphorylation and activation of components of the translational machinery, such as S6 kinase (S6K) and 4EBP1, an inhibitor of eukaryotic translation initiation factor 4E (eIF4E) activity (reviewed in reference 27). S6K phosphorylates the S6 ribosomal subunit, thereby increasing translation. Mice deficient for S6K1 are small and have small pancreatic beta cells and a correspondingly low level of circulating insulin (45). Mutation of the phosphorylation sites on S6 results in a similar phenotype, with small beta cells and fibroblasts (57). In Drosophila, mutation of S6K again reduces both cell and organism size (42), as does the overexpression of 4EBP (41).Interestingly, while mutation of the TORC1 pathway in mammalian cells reduces cell size by 10 to 15%, ablation of core TORC1 pathway components in Drosophila cells can affect cell size by up to 40% (73). In an attempt to identify novel components of the TORC1 pathway, we undertook an RNA interference (RNAi)-based screen of Drosophila S2 cells. We reasoned that the extreme size phenotypes observed in Drosophila cells upon TORC1 manipulations would facilitate the identification of modulators. In order to increase the likelihood of isolating novel regulators of TOR, we uncoupled TOR activity from many of its known nutritional controls by depleting TSC2 and screened for double-stranded RNAs (dsRNAs) that could reverse the cell size increase elicited by loss of TSC2. Depletion of multiple components of the p38 pathway was found to revert the TSC2 RNAi-induced cell size increase. Furthermore, activation of p38 is necessary and sufficient for the activation of TOR. Strikingly, mutation of components of the stress-activated p38 pathway in Drosophila has a similar phenotype to mutations in the TOR and insulin signaling pathway: a cell-autonomous cell size decrease, reduced body size, and a sensitization to the effects of nutritional stress.     

Uprooting and Burial of Invasive Alien Plants: A New Tool in Coastal Restoration?

Invasive alien plants are a problem for conservation management, and control of these species can be combined with habitat restoration. Subsoil burial of uprooted plants is a new method of mechanical control, which might be suitable in disturbed habitats. The method was tested in Rosa rugosa (Japanese Rose), an invasive shrub in north‐western Europe with negative effects on coastal biodiversity. Two months after uprooting and burial in dunes of north‐eastern Denmark, 89% of the 58 shrubs resprouted from roots and rhizomes; on average 41 resprouts per shrub. Resprout density was twice as high at former shrub margins compared with the center; resprouts were taller and originated from more superficial soil layers at the margin than in the center. Resprouting was negatively correlated with fragment depth, and no resprouts were observed from greater than 15 cm depth. The number of resprouts increased with fragment dry mass (0.5–168.5 g). After 18 months with harrowing the species was still resprouting, flowering, and fruiting, albeit with no difference between shrub margin and center. Resprouts were taller (26 cm) and coverage was higher (0–4%) after two compared with three times harrowing, whereas no difference was found in cover of native dune species (1–5%). The results show that even small fragments of R. rugosa resprout, and that resprouting persists despite repeated harrowing. Thus, careful subsoil burial of all fragments is necessary, special attention should be paid to the shrub margin, and follow‐up treatments are needed. The effectiveness of the burial method is discussed for restoration of coastal dunes.     

A functional interaction between the MAGUK protein hDlg and the gap junction protein connexin 43 in cervical tumour cells

Gap junctions, composed of Cxs (connexins), allow direct intercellular communication. Gap junctions are often lost during the development of malignancy, although the processes behind this are not fully understood. Cx43 is a widely expressed Cx with a long cytoplasmic C-terminal tail that contains several potential protein-interaction domains. Previously, in a model of cervical carcinogenesis, we showed that the loss of gap junctional communication correlated with relocalization of Cx43 to the cytoplasm late in tumorigenesis. In the present study, we demonstrate a similar pattern of altered expression for the hDlg (human discs large) MAGUK (membrane-associated guanylate kinase) family tumour suppressor protein in cervical tumour cells, with partial co-localization of Cx43 and hDlg in an endosomal/lysosomal compartment. Relocalization of these proteins is not due to a general disruption of cell membrane integrity or Cx targeting. Cx43 (via its C-terminus) and hDlg interact directly in vitro and can form a complex in cells. This novel interaction requires the N- and C-termini of hDlg. hDlg is not required for Cx43 internalization in W12GPXY cells. Instead, hDlg appears to have a role in maintaining a cytoplasmic pool of Cx43. These results demonstrate that hDlg is a physiologically relevant regulator of Cx43?in transformed epithelial cells.     

Exploring Differences in the Content of Job Interviews between Youth with and without a Physical Disability

Objective

Although people with disabilities have great potential to provide advantages to work environments, many encounter barriers in finding employment, especially youth who are looking for their first job. A job interview is an essential component of obtaining employment. The objective of this study is to explore the content of the answers given in job interviews among youth with disabilities compared to typically developing youth.

Methods

A purposive sample of 31 youth (16 with typical development and 15 with disability) completed a mock job interview as part of an employment readiness study. The interview questions focused on skills and experiences, areas for improvement, and actions taken during problem-based scenarios. Transcribed interviews were analyzed using a content analysis of themes that emerged from the interviews.

Results

We found several similarities and differences between youth with disabilities and typically developing youth. Similarities included giving examples from school, emphasizing their “soft skills” (i.e., people and communication skills) and giving examples of relevant experience for the position. Both groups of youth gave similar examples for something they were proud of but fewer youth with disabilities provided examples. Differences in the content of job interview answers between the two groups included youth with disabilities: (1) disclosing their condition; (2) giving fewer examples related to customer service and teamwork skills; (3) experiencing greater challenges in providing feedback to team members and responding to scenario-based problem solving questions; and (4) drawing on examples from past work, volunteer and extra curricular activities.

Conclusions

Clinicians and educators should help youth to understand what their marketable skills are and how to highlight them in an interview. Employers need to understand that the experiences of youth with disabilities may be different than typically developing youth. Our findings also help to inform employment readiness programs by highlighting the areas where youth with disabilities may need extra help as compared to typically developing youth.     

Interaction of the Human Papillomavirus E6 Oncoprotein with Sorting Nexin 27 Modulates Endocytic Cargo Transport Pathways

A subset of high-risk Human Papillomaviruses (HPVs) are the causative agents of a large number of human cancers, of which cervical is the most common. Two viral oncoproteins, E6 and E7, contribute directly towards the development and maintenance of malignancy. A characteristic feature of the E6 oncoproteins from cancer-causing HPV types is the presence of a PDZ binding motif (PBM) at its C-terminus, which confers interaction with cellular proteins harbouring PDZ domains. Here we show that this motif allows E6 interaction with Sorting Nexin 27 (SNX27), an essential component of endosomal recycling pathways. This interaction is highly conserved across E6 proteins from multiple high-risk HPV types and is mediated by a classical PBM-PDZ interaction but unlike many E6 targets, SNX27 is not targeted for degradation by E6. Rather, in HPV-18 positive cell lines the association of SNX27 with components of the retromer complex and the endocytic transport machinery is altered in an E6 PBM-dependent manner. Analysis of a SNX27 cargo, the glucose transporter GLUT1, reveals an E6-dependent maintenance of GLUT1 expression and alteration in its association with components of the endocytic transport machinery. Furthermore, knockdown of E6 in HPV-18 positive cervical cancer cells phenocopies the loss of SNX27, both in terms of GLUT1 expression levels and its vesicular localization, with a concomitant marked reduction in glucose uptake, whilst loss of SNX27 results in slower cell proliferation in low nutrient conditions. These results demonstrate that E6 interaction with SNX27 can alter the recycling of cargo molecules, one consequence of which is modulation of nutrient availability in HPV transformed tumour cells.     

Inferring copy number and genotype in tumour exome data

Background

Using whole exome sequencing to predict aberrations in tumours is a cost effective alternative to whole genome sequencing, however is predominantly used for variant detection and infrequently utilised for detection of somatic copy number variation.

Results

We propose a new method to infer copy number and genotypes using whole exome data from paired tumour/normal samples. Our algorithm uses two Hidden Markov Models to predict copy number and genotypes and computationally resolves polyploidy/aneuploidy, normal cell contamination and signal baseline shift. Our method makes explicit detection on chromosome arm level events, which are commonly found in tumour samples. The methods are combined into a package named ADTEx (Aberration Detection in Tumour Exome). We applied our algorithm to a cohort of 17 in-house generated and 18 TCGA paired ovarian cancer/normal exomes and evaluated the performance by comparing against the copy number variations and genotypes predicted using Affymetrix SNP 6.0 data of the same samples. Further, we carried out a comparison study to show that ADTEx outperformed its competitors in terms of precision and F-measure.

Conclusions

Our proposed method, ADTEx, uses both depth of coverage ratios and B allele frequencies calculated from whole exome sequencing data, to predict copy number variations along with their genotypes. ADTEx is implemented as a user friendly software package using Python and R statistical language. Source code and sample data are freely available under GNU license (GPLv3) at http://adtex.sourceforge.net/.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-732) contains supplementary material, which is available to authorized users.     

p53 and Translation Attenuation Regulate Distinct Cell Cycle Checkpoints during Endoplasmic Reticulum (ER) Stress

Cell cycle checkpoints ensure that proliferation occurs only under permissive conditions, but their role in linking nutrient availability to cell division is incompletely understood. Protein folding within the endoplasmic reticulum (ER) is exquisitely sensitive to energy supply and amino acid sources because deficiencies impair luminal protein folding and consequently trigger ER stress signaling. Following ER stress, many cell types arrest within the G₁ phase, although recent studies have identified a novel ER stress G₂ checkpoint. Here, we report that ER stress affects cell cycle progression via two classes of signal: an early inhibition of protein synthesis leading to G₂ delay involving CHK1 and a later induction of G₁ arrest associated both with the induction of p53 target genes and loss of cyclin D1. We show that substitution of p53/47 for p53 impairs the ER stress G₁ checkpoint, attenuates the recovery of protein translation, and impairs induction of NOXA, a mediator of cell death. We propose that cell cycle regulation in response to ER stress comprises redundant pathways invoked sequentially first to impair G₂ progression prior to ultimate G₁ arrest.     

Nobiletin protects against diabetes-induced testicular injury via hypophysis–gonadal axis upregulation and amelioration of oxidative stress

Background

Testicular injury is one of the most serious problems associated with diabetes mellitus. The present study aimed to compare the effects of two different doses of nobiletin and analyze its mechanisms of action against diabetes-induced testicular impairment in rats.

Methods and results

Streptozotocin injection was used to induce diabetes. Diabetic rats received nobiletin orally at 10 or 25 mg/kg daily for 30 days. Diabetic rats displayed significant elevations in glucose, glycosylated hemoglobin (HbA1c), Homeostatic Model of Insulin Resistance (HOMA-IR), and pro-inflammatory cytokines, while the serum levels of insulin, testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) were significantly reduced. Histological changes to positivity for caspase-3 and decreased androgen receptors (AR) immunoexpression were observed in diabetic rats. Both doses of nobiletin improved hyperglycemia, reduced pro-inflammatory cytokines, and augmented insulin, testosterone, LH, and FSH levels. LH and FSH receptors and cytochrome P450 17 α-hydroxylase (CYP17A1) were markedly downregulated in terms of both gene and protein expression in testicular tissues of the diabetic group, effects that were markedly ameliorated with both doses of nobiletin. In addition, both doses significantly reduced lipid peroxidation and caspase-3 immunoexpression and improved the activity of the antioxidant enzymes and AR in testicular tissues of the diabetic group.

Conclusion

Both nobiletin doses showed protective effects against diabetes-induced testicular injury by reducing oxidative stress, hyperglycemia, inflammation, and caspase-3 and upregulating the hypophysis–gonadal axis and AR. The high dose of nobiletin was more effective than the lower one.