Collybistin and gephyrin are novel components of the eukaryotic translation initiation factor 3 complex

Abstract Background Collybistin (CB), a neuron-specific guanine nucleotide exchange factor, has been implicated in targeting gephyrin-GABAA receptors clusters to inhibitory postsynaptic sites. However, little is known about additional CB partners and functions. Findings Here, we identified the p40 subunit of the eukaryotic translation initiation factor 3 (eIF3H) as a novel binding partner of CB, documenting the interaction in yeast, non-neuronal cell lines, and the brain. In addition, we demonstrated that gephyrin also interacts with eIF3H in non-neuronal cells and forms a complex with eIF3 in the brain. Conclusions Together, our results suggest, for the first time, that CB and gephyrin associate with the translation initiation machinery, and lend further support to the previous evidence that gephyrin may act as a regulator of synaptic protein synthesis.

Potential Contribution of Translational Factors to Triiodo-L-Thyronine-Induced Insulin Synthesis by Pancreatic Beta Cells

Background: Thyroid hormones (THs) are known to regulate protein synthesis by acting at the transcriptional level and inducing the expression of many genes. However, little is known about their role in protein expression at the post-transcriptional level, even though studies have shown enhancement of protein synthesis associated with mTOR/p70S6K activation after triiodo-l-thyronine (T3) administration. On the other hand, the effects of TH on translation initiation and polypeptidic chain elongation factors, being essential for activating protein synthesis, have been poorly explored. Therefore, considering that preliminary studies from our laboratory have demonstrated an increase in insulin content in INS-1E cells in response to T3 treatment, the aim of the present study was to investigate if proteins of translational nature might be involved in this effect. Methods: INS-1E cells were maintained in the presence or absence of T3 (10(-6) or 10(-8) M) for 12 hours. Thereafter, insulin concentration in the culture medium was determined by radioimmunoassay, and the cells were processed for Western blot detection of insulin, eukaryotic initiation factor 2 (eIF2), p-eIF2, eIF5A, EF1A, eIF4E binding protein (4E-BP), p-4E-BP, p70S6K, and p-p70S6K. Results: It was found that, in parallel with increased insulin generation, T3 induced p70S6K phosphorylation and the expression of the translational factors eIF2, eIF5A, and eukaryotic elongation factor 1 alpha (eEF1A). In contrast, total and phosphorylated 4E-BP, as well as total p70S6K and p-eIF2 content, remained unchanged after T3 treatment. Conclusions: Considering that (i) p70S6K induces S6 phosphorylation of the 40S ribosomal subunit, an essential condition for protein synthesis; (ii) eIF2 is essential for the initiation of messenger RNA translation process; and (iii) eIF5A and eEF1A play a central role in the elongation of the polypeptidic chain during the transcripts decoding, the data presented here lead us to suppose that a part of T3-induced insulin expression in INS-1E cells depends on the protein synthesis activation at the post-transcriptional level, as these proteins of the translational machinery were shown to be regulated by T3.

Leucine Is Essential for Attenuating Fetal Growth Restriction Caused by a Protein-Restricted Diet in Rats

Certain amino acids, such as leucine (Leu) are not only substrates for protein synthesis but also are important regulators of protein metabolism. Moreover, it is known that alterations in intrauterine growth favor the development of chronic diseases in adulthood. Therefore, we investigated the role of Leu in combination with other BCAA on effects that are induced by maternal protein restriction on fetal growth. Wistar rats were divided into 4 groups according to the diet provided during pregnancy: control (C; 20% casein); V+I [5% casein + 2% L-valine (Val) + 2% L-isoleucine (Ile)1; KYT 15% casein + 1.8% L-lysine (Lys) + 1.2% L-tyrosine (Tyr) + 1% L-threonine (Thr)1; and BCAA (5% casein + 1.8% L-Leu + 1.2% L-Val + 1% L-Ile). Maternal protein restriction reduced the growth and organ weight of the offspring of dams receiving the V+I and KYT diets compared with the C group. Supplementation with BCAA reversed this growth deficit, minimizing the difference or restoring the mass of organs and carcass fat, the liver and muscle protein, and the RNA concentrations compared with newborns in the C group (P < 0.05). These effects could be explained by the activation of the mTOR signaling pathway, because phosphorylation of 4E-BP1 in the liver of offspring of the BCAA group was greater than that in the C, V+I, and KYT groups. The present results identify a critical role for Leu in association with other BCAA in the activation of the mTOR signaling pathway for the control of altered intrauterine growth induced by a maternal low-protein diet. J. Nutr. 142: 924-930, 2012.

Influence of HAART on Alternative Reading Frame Immune Responses over the Course of HIV-1 Infection

Background: Translational errors can result in bypassing of the main viral protein reading frames and the production of alternate reading frame (ARF) or cryptic peptides. Within HIV, there are many such ARFs in both sense and the antisense directions of transcription. These ARFs have the potential to generate immunogenic peptides called cryptic epitopes (CE). Both antiretroviral drug therapy and the immune system exert a mutational pressure on HIV-1. Immune pressure exerted by ARF CD8(+) T cells on the virus has already been observed in vitro. HAART has also been described to select HIV-1 variants for drug escape mutations. Since the mutational pressure exerted on one location of the HIV-1 genome can potentially affect the 3 reading frames, we hypothesized that ARF responses would be affected by this drug pressure in vivo. Methodology/Principal findings: In this study we identified new ARFs derived from sense and antisense transcription of HIV-1. Many of these ARFs are detectable in circulating viral proteins. They are predominantly found in the HIV-1 env nucleotide region. We measured T cell responses to 199 HIV-1 CE encoded within 13 sense and 34 antisense HIV-1 ARFs. We were able to observe that these ARF responses are more frequent and of greater magnitude in chronically infected individuals compared to acutely infected patients, and in patients on HAART, the breadth of ARF responses increased. Conclusions/Significance: These results have implications for vaccine design and unveil the existence of potential new epitopes that could be included as vaccine targets.

Cold Shock Genes cspA and cspB from Caulobacter crescentus Are Posttranscriptionally Regulated and Important for Cold Adaptation

Cold shock proteins (CSPs) are nucleic acid binding chaperones, first described as being induced to solve the problem of mRNA stabilization after temperature downshift. Caulobacter crescentus has four CSPs: CspA and CspB, which are cold induced, and CspC and CspD, which are induced only in stationary phase. In this work we have determined that the synthesis of both CspA and CspB reaches the maximum levels early in the acclimation phase. The deletion of cspA causes a decrease in growth at low temperature, whereas the strain with a deletion of cspB has a very subtle and transient cold-related growth phenotype. The cspA cspB double mutant has a slightly more severe phenotype than that of the cspA mutant, suggesting that although CspA may be more important to cold adaptation than CspB, both proteins have a role in this process. Gene expression analyses were carried out using cspA and cspB regulatory fusions to the lacZ reporter gene and showed that both genes are regulated at the transcriptional and posttranscriptional levels. Deletion mapping of the long 5'-untranslated region (5'-UTR) of each gene identified a common region important for cold induction, probably via translation enhancement. In contrast to what was reported for other bacteria, these cold shock genes have no regulatory regions downstream from ATG that are important for cold induction. This work shows that the importance of CspA and CspB to C. crescentus cold adaptation, mechanisms of regulation, and pattern of expression during the acclimation phase apparently differs in many aspects from what has been described so far for other bacteria.

Genetic analyses of smoking initiation, persistence, quantity, and age-at-onset of regular cigarette use in Brazilian families: the Baependi Heart Study

Background: The purpose of this study was to estimate the genetic influences on the initiation of cigarette smoking, the persistence, quantity and age-at-onset of regular cigarette use in Brazilian families. Methods: The data set consisted of 1,694 individuals enrolled in the Baependi Heart Study. The heritability and the heterogeneity in genetic and environmental variance components by gender were estimated from variance components approaches, using the SOLAR (Sequential Oligogenic Linkage Analysis Routines) computer package. The mixed-effects Cox model was used for the genetic analysis of the age-at onset of regular cigarette use. Results: The heritability estimates were high (> 50%) for smoking initiation and were intermediate, ranging from 23.4 to 31.9%, for smoking persistence and quantity. Significant evidence for heterogeneity in variance components by gender was observed for smoking initiation and age-at-onset of regular cigarette use. Genetic factors play an important role in the interindividual variation of these phenotypes in females, while in males there is a predominant environmental component, which could be explained by greater social influences in the initiation of tobacco use. Conclusions: Significant heritabilities were observed in smoking phenotypes for both males and females from the Brazilian population. These data add to the literature and are concordant with the notion of significant biological determination in smoking behavior. Samples from the Baependi Heart Study may be valuable for the mapping of genetic loci that modulate this complex biological trait.

TRANSLATION AND CULTURAL ADAPTATION OF THE GLOBAL APPRAISAL OF INDIVIDUAL NEEDS - INITIAL

The objective of this study was to perform the translation on and cultural adaptation of the Global Appraisal of Individual Needs - Initial instrument, and calculate its content validity index. This is a methodological study designed for the cultural adaptation of the instrument. The instrument was translated into Portuguese in two versions that originated the synthesis of the translations, which were then submitted to the evaluation of four judges, experts in the field of alcohol and other drugs. After the suggested changes were made, the instrument was back-translated and resubmitted to the judges and authors of the original instrument, resulting in the final version of the instrument, Avaliacao Global das Necessidades Individuais - Inicial. The content validity index of the instrument was 0.91, considered valid according to the literature. The instrument Avaliacao Global das Necessidades Individuais - Inicial was culturally adapted to the Portuguese language spoken in Brazil; however, it was not submitted to tests with the target population, which suggests further studies should be performed to test its reliability and validity.

TRANSLATION AND VALIDATION OF THE KNEE SOCIETY SCORE - KSS FOR BRAZILIAN PORTUGUESE

Objective: To translate, culturally adapt and validate the "Knee Society Score"(KSS) for the Portuguese language and determine its measurement properties, reproducibility and validity. Method: We analyzed 70 patients of both sexes, aged between 55 and 85 years, in a cross-sectional clinical trial, with diagnosis of primary osteoarthritis,undergoing total knee arthroplasty surgery. We assessed the patients with the English version of the KSS questionnaire and after 30 minutes with the Portuguese version of the KSS questionnaire, done by a different evaluator. All the patients were assessed preoperatively, and again at three, and six months postoperatively. Results: There was no statistical difference, using Cronbach's alpha index and the Bland-Altman graphical analysis, for the knees core during the preoperative period (p=1), and at three months (p=0.991) and six months postoperatively (p=0.985). There was no statistical difference for knee function score for all three periods (p=1.0). Conclusion: The Brazilian version of the Knee Society Score is easy to apply, as well providing as a valid and reliable instrument for measuring the knee score and function of Brazilian patients undergoing TKA. Level of Evidence: Level I - Diagnostic Studies Investigating a Diagnostic Test- Testing of previously developed diagnostic criteria on consecutive patients (with universally applied 'gold' reference standard).

Translation and Cross-cultural Adaptation of the Lower Extremity Functional Scale Into a Brazilian Portuguese Version and Validation on Patients With Knee Injuries

STUDY DESIGN: Clinical measurement. OBJECTIVE: To translate and culturally adapt the Lower Extremity Functional Scale (LEFS) into a Brazilian Portuguese version, and to test the construct and content validity and reliability of this version in patients with knee injuries. BACKGROUND: There is no Brazilian Portuguese version of an instrument to assess the function of the lower extremity after orthopaedic injury. METHODS: The translation of the original English version of the LEFS into a Brazilian Portuguese version was accomplished using standard guidelines and tested in 31 patients with knee injuries. Subsequently, 87 patients with a variety of knee disorders completed the Brazilian Portuguese LEES, the Medical Outcomes Study 36-Item Short-Form Health Survey, the Western Ontario and McMaster Universities Osteoarthritis Index, and the International Knee Documentation Committee Subjective Knee Evaluation Form and a visual analog scale for pain. All patients were retested within 2 days to determine reliability of these measures. Validation was assessed by determining the level of association between the Brazilian Portuguese LEFS and the other outcome measures. Reliability was documented by calculating internal consistency, test-retest reliability, and standard error of measurement. RESULTS: The Brazilian Portuguese LEES had a high level of association with the physical component of the Medical Outcomes Study 36-Item Short-Form Health Survey (r = 0.82), the Western Ontario and McMaster Universities Osteoarthritis Index (r = 0.87), the International Knee Documentation Committee Subjective Knee Evaluation Form (r = 0.82), and the pain visual analog scale (r = -0.60) (all, P<.05). The Brazilian Portuguese LEES had a low level of association with the mental component of the Medical Outcomes Study 36-Item Short-Form Health Survey (r = 0.38, P<.05). The internal consistency (Cronbach alpha = .952) and test-retest reliability (intraclass correlation coefficient = 0.957) of the Brazilian Portuguese version of the LEES were high. The standard error of measurement was low (3.6) and the agreement was considered high, demonstrated by the small differences between test and retest and the narrow limit of agreement, as observed in Bland-Altman and survival-agreement plots. CONCLUSION: The translation of the LEFS into a Brazilian Portuguese version was successful in preserving the semantic and measurement properties of the original version and was shown to be valid and reliable in a Brazilian population with knee injuries. J Ort hop Sports Phys Ther 2012;42(11):932-939, Epub 9 October 2012. doi:10.2519/jospt.2012.4101

Infantile Encephaloneuromyopathy and Defective Mitochondrial Translation Are Due to a Homozygous RMND1 Mutation

Defects of mitochondrial protein synthesis are clinically and genetically heterogeneous. We previously described a male infant who was born to consanguineous parents and who presented with severe congenital encephalopathy, peripheral neuropathy, myopathy, and lactic acidosis associated with deficiencies of multiple mitochondrial respiratory-chain enzymes and defective mitochondrial translation. In this work, we have characterized four additional affected family members, performed homozygosity mapping, and identified a homozygous splicing mutation in the splice donor site of exon 2 (c.504+1G>A) of RMND1 (required for meiotic nuclear division-1) in the affected individuals. Fibroblasts from affected individuals expressed two aberrant transcripts and had decreased wild-type mRNA and deficiencies of mitochondrial respiratory-chain enzymes. The RMND1 mutation caused haploinsufficiency that was rescued by overexpression of the wild-type transcript in mutant fibroblasts; this overexpression increased the levels and activities of mitochondrial respiratory-chain proteins. Knockdown of RMND1 via shRNA recapitulated the biochemical defect of the mutant fibroblasts, further supporting a loss-of-function pathomechanism in this disease. RMND1 belongs to the sif2 family, an evolutionary conserved group of proteins that share the DUF155 domain, have unknown function, and have never been associated with human disease. We documented that the protein localizes to mitochondria in mammalian and yeast cells. Further studies are necessary for understanding the function of this protein in mitochondrial protein translation.

Prodromal Questionnaire: translation, adaptation to Portuguese and preliminary results in ultra-high risk individuals and first episode psychosis

OBJECTIVE: The Prodromal Questionnaire (PQ) is a 92-item self-report screening tool for individuals at ultra-high risk (UHR) to develop psychosis. This study aims to present the translation to Portuguese and preliminary results in UHR and first episode (FE) psychosis in a Portuguese sample. METHODS: The PQ was translated from English to Portuguese by two bilingual researchers from the research program on early psychosis of the Instituto de Psiquiatria HCFMUSP, São Paulo, Brazil (ASAS - "Evaluation and Follow up of Adolescents and Young Adults in São Paulo") and back translated by two other researchers. The study participants (n = 11-) were evaluated through the Portuguese version of the Prodromal Questionnaire (PQ) and SIPS. RESULTS: The individuals at UHR (n = 7) presented a lower score than first episode patients (n = 4). The UHR mean scores and standard deviation on Portuguese version of the PQ were: 13.0 ± 10.0 points on positive symptoms subscale, and FE patients: 33.0 ± 10.0. CONCLUSION: The UHR and FE patients' of this study presented PQ scores similar to the ones found in the literature; what suggests that it is possible to use the PQ in Brazilian help-seeking individuals as a screening tool.

Personal and Social Performance (PSP) scale for patients with schizophrenia: translation to Portuguese, cross-cultural adaptation and interrater reliability

INTRODUCTION: Schizophrenia is a chronic mental disorder associated with impairment in social functioning. The most widely used scale to measure social functioning is the GAF (Global Assessment of Functioning), but it has the disadvantage of measuring at the same time symptoms and functioning, as described in its anchors. OBJECTIVES:Translation and cultural adaptation of the PSP, proposing a final version in Portuguese for use in Brazil. METHODS: We performed five steps: 1) translation; 2) back translation; 3) formal assessment of semantic equivalence; 4) debriefing; 5) analysis by experts. Interrater reliability (Intraclass correlation, ICC) between two raters was also measured. RESULTS: The final version was applied by two independent investigators in 18 adults with schizophrenia (DSM-IV-TR). The interrater reliability (ICC) was 0.812 (p < 0.001). CONCLUSION: The translation and adaptation of the PSP had an adequate level of semantic equivalence between the Portuguese version and the original English version. There were no difficulties related to understanding the content expressed in the translated texts and terms. Its application was easy and it showed a good interrater reliability. The PSP is a valid instrument for the measurement of personal and social functioning in schizophrenia.

Mitochondrial translation in health and disease

Mitochondrial disorders have become the most common cause of inborn errors of metabolism. Impairments in mitochondrial protein synthesis are one of the causes of these diseases, which are clinically and genetically heterogeneous. The mitochondrial translation machinery decodes 13 polypeptides essential for the oxidative phosphorylation process. Mitochondria protein synthesis depends on the integrity of mitochondrial rRNAs and tRNAs genes, and at least one hundred of nuclear encoded products. Diseases caused by mutations in mitochondrial genes as well as in ribosomal proteins, translational factors, RNA modifying enzymes, and all other constituents of the translational machinery have been described in patients with combine respiratory chain deficiency, and are the object of this review.

Testing the influence of far-field topographic forcing on subduction initiation at a passive margin

Despite favourable gravitational instability and ridge-push, elastic and frictional forces prevent subduction initiation fromarising spontaneously at passive margins. Here,we argue that forces arising fromlarge continental topographic gradients are required to initiate subduction at passivemargins. In order to test this hypothesis,we use 2Dnumerical models to assess the influence of the Andean Plateau on stressmagnitudes and deformation patterns at the Brazilian passive margin. The numerical results indicate that “plateau-push” in this region is a necessary additional force to initiate subduction. As the SE Brazilianmargin currently shows no signs of self-sustained subduction, we examined geological and geophysical data to determine if themargin is in the preliminary stages of subduction initiation. The compiled data indicate that the margin is presently undergoing tectonic inversion, which we infer as part of the continental–oceanic overthrusting stage of subduction initiation. We refer to this early subduction stage as the “Brazilian Stage”, which is characterized by N10 kmdeep reverse fault seismicity at themargin, recent topographic uplift on the continental side, thick continental crust at themargin, and bulging on the oceanic side due to loading by the overthrusting continent. The combined results of the numerical simulations and passivemargin analysis indicate that the SE Brazilian margin is a prototype candidate for subduction initiation.