eIF2B promotes eIF5 dissociation from eIF2*GDP to facilitate guanine nucleotide exchange for translation initiation.

Protein synthesis factor eIF2 delivers initiator tRNA to the ribosome. Two proteins regulate its G-protein cycle: eIF5 has both GTPase-accelerating protein (GAP) and GDP dissociation inhibitor (GDI) functions, and eIF2B is the guanine nucleotide exchange factor (GEF). In this study, we used protein-protein interaction and nucleotide exchange assays to monitor the kinetics of eIF2 release from the eIF2•GDP/eIF5 GDI complex and determine the effect of eIF2B on this release. We demonstrate that eIF2B has a second activity as a GDI displacement factor (GDF) that can recruit eIF2 from the eIF2•GDP/eIF5 GDI complex prior to GEF action. We found that GDF function is dependent on the eIF2Bε and eIF2Bγ subunits and identified a novel eIF2-eIF2Bγ interaction. Furthermore, GDF and GEF activities are shown to be independent. First, eIF2B GDF is insensitive to eIF2α phosphorylation, unlike GEF. Second, we found that eIF2Bγ mutations known to disrupt GCN4 translational control significantly impair GDF activity but not GEF function. Our data therefore define an additional step in the protein synthesis initiation pathway that is important for its proper control. We propose a new model to place eIF2B GDF function in the context of efficient eIF2 recycling and its regulation by eIF2 phosphorylation.

Adult anthropometry in Type 2 diabetic population: A case-control study.

OBJECTIVES: This study was aimed to compare the body mass index (BMI) and waist-to-hip ratio (WHR) in their ability to predict type 2 diabetes risk in a large prospective cohort of men and women in Pakistan. METHODS: This was a case-control study conducted at Diabetic and medical OPD of GTTH. Anthropometric measures including BMI and WHR were analyzed. Student's t-test, Chi-squared test along with Cramer's V value, was applied to evaluate association between variables. Receiver operating curve (ROC) was used to assess anthropometric measures. RESULTS: The study included 804 diabetics and 396 non-diabetics between 30-60 years of age. Comparing the BMI parameters it was found that 717 (89.2%) in diabetic group were overweight or obese (p-value < 0.001). On comparing the WHR, 97.9% diabetics had increased WHR (p-value <0.001). Both BMI & WHR were further compared using ROC curve which found out that WHR had an area under ROC of 0.720 & BMI has 0.680, suggesting that WHR is more better predictor of diabetes as compared to BMI. CONCLUSIONS: Both BMI and WHR were strong discriminators of T2DM but WHR was found superior according to ROC value. Family history is significantly associated in patients with diabetes.

The 4E-BP Caf20p Mediates Both eIF4E-Dependent and Independent Repression of Translation.

Translation initiation factor eIF4E mediates mRNA selection for protein synthesis via the mRNA 5'cap. A family of binding proteins, termed the 4E-BPs, interact with eIF4E to hinder ribosome recruitment. Mechanisms underlying mRNA specificity for 4E-BP control remain poorly understood. Saccharomyces cerevisiae 4E-BPs, Caf20p and Eap1p, each regulate an overlapping set of mRNAs. We undertook global approaches to identify protein and RNA partners of both 4E-BPs by immunoprecipitation of tagged proteins combined with mass spectrometry or next-generation sequencing. Unexpectedly, mass spectrometry indicated that the 4E-BPs associate with many ribosomal proteins. 80S ribosome and polysome association was independently confirmed and was not dependent upon interaction with eIF4E, as mutated forms of both Caf20p and Eap1p with disrupted eIF4E-binding motifs retain ribosome interaction. Whole-cell proteomics revealed Caf20p mutations cause both up and down-regulation of proteins and that many changes were independent of the 4E-binding motif. Investigations into Caf20p mRNA targets by immunoprecipitation followed by RNA sequencing revealed a strong association between Caf20p and mRNAs involved in transcription and cell cycle processes, consistent with observed cell cycle phenotypes of mutant strains. A core set of over 500 Caf20p-interacting mRNAs comprised of both eIF4E-dependent (75%) and eIF4E-independent targets (25%), which differ in sequence attributes. eIF4E-independent mRNAs share a 3' UTR motif. Caf20p binds all tested motif-containing 3' UTRs. Caf20p and the 3'UTR combine to influence ERS1 mRNA polysome association consistent with Caf20p contributing to translational control. Finally ERS1 3'UTR confers Caf20-dependent repression of expression to a heterologous reporter gene. Taken together, these data reveal conserved features of eIF4E-dependent Caf20p mRNA targets and uncover a novel eIF4E-independent mode of Caf20p binding to mRNAs that extends the regulatory role of Caf20p in the mRNA-specific repression of protein synthesis beyond its interaction with eIF4E.

Utilizing non-ablative fractional photothermolysis prior to ALA-photodynamic therapy in the treatment of acne vulgaris: a case series.

Aminolevulinic acid (ALA) photodynamic therapy (PDT) is an emerging modality in the treatment of acne. While ablative fractional lasers have been used to enhance drug delivery into the epidermis, recent evidence suggests that non-ablative fractional photothermolysis may also improve uptake of ALA. We explored the use of non-ablative 1550 nm laser as a safe alternative in the delivery of ALA prior to red-light PDT for refractory inflammatory and cystic acne. Subjects referred for treatment of acne refractory to several topical and oral regimens, including isotretinoin, were pre-treated with non-ablative fractional photothermolysis (NAFP). This was followed by 20 % ALA application with an incubation time of 1-3 h and then exposure to 50-100 J/cm2 red light. Follow-up was at 1, 3, and 6 months. In all three cases, patients demonstrated marked reduction in inflammatory lesions. Two subjects had remission of acne after a single combination treatment. Non-ablative fractional laser applied immediately prior to PDT may be used in the treatment of acne with minimal side effects and fewer sessions needed than PDT alone. This may be due to enhanced delivery of ALA from pre-treating the skin with non-ablative fractional photothermolysis.

Soil-Transmitted Helminth Infections Are Associated With an Increase in Human Papillomavirus Prevalence and a T-Helper Type 2 Cytokine Signature in Cervical Fluids.

BACKGROUND: An ecological correlation between invasive cervical cancer incidence and burden of soil-transmitted helminths (STH) is hypothesized to explain the excess in detectable human papillomavirus (HPV) infection in Latin America, via a global T-helper type 2 (Th2)-biased mucosal immune response secondary to STH infection. METHODS: The association between current STH infection and HPV prevalence was compared in regions of Peru where STH is or is not endemic. Adjusted prevalence ratios (PRs) with robust variance were estimated as an effect measure of STH infection on HPV prevalence in each study site. Soluble immune marker profiles in STH-infected and STH-uninfected women were compared using Spearman rank correlation with the Sidak correction. RESULTS: Among women in the helminth-endemic region of the Peruvian Amazon, those with STH infection women had a 60% higher prevalence of HPV, compared with those without STH infection (PR, 1.6; 95% confidence interval, 1.0-2.7). Non-STH parasitic/protozoal infections in the non-STH-endemic population of Peru were not associated with HPV prevalence. In Iquitos, A Th2 immune profile was observed in cervical fluid from helminth-infected women but not helminth-uninfected women. CONCLUSIONS: A proportion of the increased HPV prevalence at older ages observed in Latin America may be due to a population-level difference in the efficiency of immunological control of HPV across the lifespan due to endemic STH infection.

Exposure to teledermatology and resident preparedness for future practice: results of a national survey.

Teledermatology (TD) is an emerging modality for providing remote dermatologic care with high diagnostic and management 25 concordance compared to face-to-face clinic dermatology. TD training among dermatology residency programs in the United 26 States has not been characterized. We disseminated a survey to all dermatology residents at ACGME accredited programs in the 27 United States to explore the prevalence and distribution of TD training and trainee perceptions of TD. One hundred out of a 28 potential 1170 responses (RR 8.5%) were collected from residents in every geographic location from all years in training: 67/100 29 of residents reported that TD was practiced at their institutions, although at these sites only 21/100 residents participated in 30 clinical sessions. Residents with TD exposure were more likely to feel comfortable managing a TD consult after residency 31 (p<0.001), but were not more likely to incorporate teledermatology into their future plans. Results of this study provide insight 32 into the impact of TD exposure on resident perceptions of TD and demonstrate the need for expanding TD training across all 33 dermatology residency programs.

Identification of intersubunit domain interactions within eukaryotic initiation factor (eIF) 2B, the nucleotide exchange factor for translation initiation.

In eukaryotic translation initiation, eIF2B is the guanine nucleotide exchange factor (GEF) required for reactivation of the G protein eIF2 between rounds of protein synthesis initiation. eIF2B is unusually complex with five subunits (α-ε) necessary for GEF activity and its control by phosphorylation of eIF2α. In addition, inherited mutations in eIF2B cause a fatal leukoencephalopathy. Here we describe experiments examining domains of eIF2Bγ and ε that both share sequence and predicted tertiary structure similarity with a family of phospho-hexose sugar nucleotide pyrophosphorylases. Firstly, using a genetic approach, we find no evidence to support a significant role for a potential nucleotide-binding region within the pyrophosphorylase-like domain (PLD) of eIF2Bε for nucleotide exchange. These findings are at odds with one mechanism for nucleotide exchange proposed previously. By using a series of constructs and a co-expression and precipitation strategy, we find that the eIF2Bε and -γ PLDs and a shared second domain predicted to form a left-handed ß helix are all critical for interprotein interactions between eIF2B subunits necessary for eIF2B complex formation. We have identified extensive interactions between the PLDs and left-handed ß helix domains that form the eIF2Bγε subcomplex and propose a model for domain interactions between eIF2B subunits.

Critical contacts between the eukaryotic initiation factor 2B (eIF2B) catalytic domain and both eIF2beta and -2gamma mediate guanine nucleotide exchange.

Diverse guanine nucleotide exchange factors (GEFs) regulate the activity of GTP binding proteins. One of the most complicated pairs is eukaryotic initiation factor 2B (eIF2B) and eIF2, which function during protein synthesis initiation in eukaryotes. We have mutated conserved surface residues within the eIF2B GEF domain, located at the eIF2Bepsilon C terminus. Extensive genetic and biochemical characterization established how these residues contribute to GEF activity. We find that the universally conserved residue E569 is critical for activity and that even a conservative E569D substitution is lethal in vivo. Several mutations within residues close to E569 have no discernible effect on growth or GCN4 expression, but an alanine substitution at the adjacent L568 is cold sensitive and deregulates GCN4 activity at 15 degrees C. The mutation of W699, found on a separate surface approximately 40 A from E569, is also lethal. Binding studies show that W699 is critical for interaction with eIF2beta, while L568 and E569 are not. In contrast, all three residues are critical for interaction with eIF2gamma. These data show that multiple contacts between eIF2gamma and eIF2Bepsilon mediate nucleotide exchange.

Purification of FLAG-tagged eukaryotic initiation factor 2B complexes, subcomplexes, and fragments from Saccharomyces cerevisiae.

The eukaryotic initiation factor 2B (eIF2B) is a five-subunit guanine nucleotide exchange factor, that functions during translation initiation to catalyze the otherwise slow exchange of GDP for GTP on its substrate eIF2. Assays to measure substrate interaction and guanine nucleotide release ability of eIF2B require the complex to be purified free of interacting proteins. We have also found that a subcomplex of two subunits, gamma and epsilon or the largest one, epsilon alone, promotes this activity. Within eIF2Bepsilon, the catalytic center requires the C-terminal 200 residues only. Here, we describe our protocols for purifying the Saccharomyces cerevisiae eIF2B complexes and the catalytic subunit using FLAG-tagged proteins overexpressed in yeast cells. Using commercially available FLAG-affinity resin and high salt buffer, we are able to purify active eIF2B virtually free of contaminants.

rs3751812, a common variant in fat mass and obesity-associated (FTO) gene, is associated with serum high- and low-density lipoprotein cholesterol in Pakistani individuals.

OBJECTIVES: The aim of the present study was to investigate the effect of a common variant of the fat mass and obesity associated (FTO) gene, rs3751812 in obese Pakistani individuals, compare this effect with nonobese controls of the same ethnicity, and then correlate it with serum lipid profile and anthropometric parameters. METHODS: We genotyped 475 samples using TaqMan allelic discrimination assay. Serum total cholesterol, triacylglycerols, and high- (HDL-C) and low-density lipoprotein cholesterol (LDL-C) concentrations were measured. Statistical analysis was done by SPSS version 22 and the results were analyzed for any significant associations. RESULTS: Results demonstrated that the variant is significantly associated with obesity in Pakistan. The study found, for the first time, that the variant has a significant effect on lowering HDL-C and increasing LDL-C. Among anthropometric measures, the variant showed significant association with body mass index and weight. CONCLUSION: The study concludes that the FTO variant is consistently associated with obesity in the Pakistani population and its association with anthropometric and lipid parameters show that it may mediate its role by altering fat deposition and disturbing serum lipid profile. However, future studies with larger sample size are needed to validate the results of the present study.

Reduced incidence of skin cancer in patients with alopecia areata: A retrospective cohort study.

The risk of skin cancer in patients with alopecia areata (AA) is unknown. While the risk of skin cancer in chronic inflammatory alopecias may be elevated, AA shares many characteristics with vitiligo, an autoimmune illness associated with decreased risk of melanoma and non-melanoma skin cancers. In this retrospective cohort study, we determined the risk of developing skin cancer among patients with AA in a validated cohort relative to matched controls at two tertiary care hospitals in Massachusetts. There was a significantly decreased risk of NMSC in AA patients than controls (OR=0.63, 95% CI=0.48-0.81). There was a trend towards a protective effect of AA associated with melanoma (OR=0.65, 95% CI=0.39-1.09). There was no difference in anatomic distribution of skin cancer between patients with AA and controls. Our study demonstrates a decreased risk of nonmelanoma skin cancer and a trend towards reduced risk of melanoma in patients with AA.

Irritable Bowel Syndrome: A Global Challenge Among Medical Students.

Irritable bowel syndrome (IBS) has been identified as one of the more highly prevalent and costly gastrointestinal disorders. Despite its uncertain etiology, risk factors, such as stress and academic load, are well correlated with the prevalence of the disease. Being in one of the most stressful and challenging environments, medical students are predisposed to have high rates of IBS. The socioeconomic burden of the disease on its sufferers is devastating as their quality of life is reduced, mandating additional health care precautions. The aim of this article, therefore, is to review the current literature about IBS among medical students, its prevalence, associated risk factors, and diagnostic criteria. Additionally, different solutions and management options are recommended to control the disease.

Global mRNA selection mechanisms for translation initiation.

BACKGROUND: The selection and regulation of individual mRNAs for translation initiation from a competing pool of mRNA are poorly understood processes. The closed loop complex, comprising eIF4E, eIF4G and PABP, and its regulation by 4E-BPs are perceived to be key players. Using RIP-seq, we aimed to evaluate the role in gene regulation of the closed loop complex and 4E-BP regulation across the entire yeast transcriptome. RESULTS: We find that there are distinct populations of mRNAs with coherent properties: one mRNA pool contains many ribosomal protein mRNAs and is enriched specifically with all of the closed loop translation initiation components. This class likely represents mRNAs that rely heavily on the closed loop complex for protein synthesis. Other heavily translated mRNAs are apparently under-represented with most closed loop components except Pab1p. Combined with data showing a close correlation between Pab1p interaction and levels of translation, these data suggest that Pab1p is important for the translation of these mRNAs in a closed loop independent manner. We also identify a translational regulatory mechanism for the 4E-BPs; these appear to self-regulate by inhibiting translation initiation of their own mRNAs. CONCLUSIONS: Overall, we show that mRNA selection for translation initiation is not as uniformly regimented as previously anticipated. Components of the closed loop complex are highly relevant for many mRNAs, but some heavily translated mRNAs interact poorly with this machinery. Therefore, alternative, possibly Pab1p-dependent mechanisms likely exist to load ribosomes effectively onto mRNAs. Finally, these studies identify and characterize a complex self-regulatory circuit for the yeast 4E-BPs.

Prevalence of depression among community dwelling elderly in karachi, pakistan.

OBJECTIVE: The objectives of the study were to find out the prevalence of depression and to identify associated risk factors among community dwelling elderly in Karachi. METHODS: It was a cross-sectional, descriptive study involving 284 community-dwelling elderly residing in Karachi, Pakistan. A non-probability convenience sampling was done. The Geriatric Depression Scale (GDS-15) was used to assess depression. Descriptive statistics was performed using SPSS version 12. Cross tabulation for different variables was done and Chi-square was used as test of significance. The level of significance was set as p < 0.05. An informal (verbal) consent was taken. Anonymity and confidentiality was assured. RESULTS: Among 284 respondents, 74% were males while 26% were females. The mean age was 68.44 ±7.59 years. The study found that 16.5% respondents were depressed while 23.6% were suggestive of depression. Depression was more among men than in women. Depression was statistically significant among married respondents (p<0.05) and illiterate (p<0.001). Although a large proportion of the participants were satisfied with their income, this was statistically significant (p<0.001) for depression among those who were not satisfied with their income. Similarly, sleep was significantly disturbed (p<0.001) among the depressed respondents. CONCLUSION: A significant prevalence of geriatric depression was reported. In order to reduce its prevalence, general physicians and other health care professionals need to be sensitized about geriatric depression and its risk factors.

Clues to the mechanism of action of eIF2B, the guanine-nucleotide-exchange factor for translation initiation.

A variety of cellular processes rely on G-proteins, which cycle through active GTP-bound and inactive GDP-bound forms. The switch between these states is commonly regulated by GEFs (guanine-nucleotide-exchange factors) and GAPs (GTPase-activating proteins). Although G-proteins have structural similarity, GEFs are very diverse proteins. A complex example of this system is seen in eukaryotic translation initiation between eIF (eukaryotic initiation factor) 2, a G-protein, its five-subunit GEF, eIF2B, and its GAP, eIF5. eIF2 delivers Met-tRNA(i) (initiator methionyl-tRNA) to the 40S ribosomal subunit before mRNA binding. Upon AUG recognition, eIF2 hydrolyses GTP, aided by eIF5. eIF2B then re-activates eIF2 by removing GDP, thereby promoting association of GTP. In the present article, we review data from studies of representative G-protein-GEF pairs and compare these with observations from our research on eIF2 and eIF2B to propose a model for how interactions between eIF2B and eIF2 promote guanine nucleotide exchange.

An eIF5/eIF2 complex antagonizes guanine nucleotide exchange by eIF2B during translation initiation.

In eukaryotic translation initiation, the eIF2.GTP/Met-tRNA(i)(Met) ternary complex (TC) binds the eIF3/eIF1/eIF5 complex to form the multifactor complex (MFC), whereas eIF2.GDP binds the pentameric factor eIF2B for guanine nucleotide exchange. eIF5 and the eIF2Bvarepsilon catalytic subunit possess a conserved eIF2-binding site. Nearly half of cellular eIF2 forms a complex with eIF5 lacking Met-tRNA(i)(Met), and here we investigate its physiological significance. eIF5 overexpression increases the abundance of both eIF2/eIF5 and TC/eIF5 complexes, thereby impeding eIF2B reaction and MFC formation, respectively. eIF2Bvarepsilon mutations, but not other eIF2B mutations, enhance the ability of overexpressed eIF5 to compete for eIF2, indicating that interaction of eIF2Bvarepsilon with eIF2 normally disrupts eIF2/eIF5 interaction. Overexpression of the catalytic eIF2Bvarepsilon segment similarly exacerbates eIF5 mutant phenotypes, supporting the ability of eIF2Bvarepsilon to compete with MFC. Moreover, we show that eIF5 overexpression does not generate aberrant MFC lacking tRNA(i)(Met), suggesting that tRNA(i)(Met) is a vital component promoting MFC assembly. We propose that the eIF2/eIF5 complex represents a cytoplasmic reservoir for eIF2 that antagonizes eIF2B-promoted guanine nucleotide exchange, enabling coordinated regulation of translation initiation.