Circulating serum exosomes i-tRF-AspGTC and tRF-1-SerCGA as diagnostic indicators for non-small cell lung cancer.

BACKGROUND: tRF-RNA-a representative of non-coding RNA (ncRNA)-is a precursor or fragment of mature tRNA and plays a crucial regulatory role in the occurrence and development of cancer. There is currently little research on tRF-RNA as a diagnostic marker in cancer, especially for NSCLC from serum exosomes. METHOD: Serum exosomes were successfully extracted from serum; their physical morphology was captured by transmission electron microscopy (TEM); appropriate particle size detection was performed using qNano; surface labeling was verified through western blotting. Serum exosomes i-tRF-AspGTC and tRF-1-SerCGA were selected through gene microarray, and qPCR was used to validate their significance in 242 patients and 201 healthy individuals. The area under the curve (AUC) was used to evaluate the diagnostic indicators of non-small cell lung cancer (NSCLC). RESULT: Compared with 201 healthy individuals, i-tRF-AspGTC and tRF-1-SerCGA were significantly downregulated in 242 NSCLC patients and 95 early-stage patients. For tRF-AspGTC and tRF-1-SerCGA, the predictive diagnostic efficiency rates of AUC were 0.690 and 0.680, respectively, whereas the early diagnostic efficiency rates were 0.656 and 0.688, respectively. The result of combined diagnosis with CEA and CYFRA21-1 was 0.928, and the early diagnostic efficiency was 0.843, which is a very high biological predictive factor for NSCLC. CONCLUSION: The expression of serum exosomes i-tRF-AspGTC and tRF-1-SerCGA was significantly downregulated in NSCLC patients. These exosomes could be used as predictive indicators for diagnosis or early diagnosis of NSCLC.

Nicked tRNAs are stable reservoirs of tRNA halves in cells and biofluids.

Nonvesicular extracellular RNAs (nv-exRNAs) constitute the majority of the extracellular RNAome, but little is known about their stability, function, and potential use as disease biomarkers. Herein, we measured the stability of several naked RNAs when incubated in human serum, urine, and cerebrospinal fluid (CSF). We identified extracellularly produced tRNA-derived small RNAs (tDRs) with half-lives of several hours in CSF. Contrary to widespread assumptions, these intrinsically stable small RNAs are full-length tRNAs containing broken phosphodiester bonds (i.e., nicked tRNAs). Standard molecular biology protocols, including phenol-based RNA extraction and heat, induce the artifactual denaturation of nicked tRNAs and the consequent in vitro production of tDRs. Broken bonds are roadblocks for reverse transcriptases, preventing amplification and/or sequencing of nicked tRNAs in their native state. To solve this, we performed enzymatic repair of nicked tRNAs purified under native conditions, harnessing the intrinsic activity of phage and bacterial tRNA repair systems. Enzymatic repair regenerated an RNase R-resistant tRNA-sized band in northern blot and enabled RT-PCR amplification of full-length tRNAs. We also separated nicked tRNAs from tDRs by chromatographic methods under native conditions, identifying nicked tRNAs inside stressed cells and in vesicle-depleted human biofluids. Dissociation of nicked tRNAs produces single-stranded tDRs that can be spontaneously taken up by human epithelial cells, positioning stable nv-exRNAs as potentially relevant players in intercellular communication pathways.

Role of the TATA-box binding protein (TBP) and associated family members in transcription regulation.

The assembly of transcription complexes on eukaryotic promoters involves a series of steps, including chromatin remodeling, recruitment of TATA-binding protein (TBP)-containing complexes, the RNA polymerase II holoenzyme, and additional basal transcription factors. This review describes the transcriptional regulation by TBP and its corresponding homologs that constitute the TBP family and their interactions with promoter DNA. The C-terminal core domain of TBP is highly conserved and contains two structural repeats that fold into a saddle-like structure, essential for the interaction with the TATA-box on DNA. Based on the TBP C-terminal core domain similarity, three TBP-related factors (TRFs) or TBP-like factors (TBPLs) have been discovered in metazoans, TRF1, TBPL1, and TBPL2. TBP is autoregulated, and once bound to DNA, repressors such as Mot1 induce TBP to dissociate, while other factors such as NC2 and the NOT complex convert the active TBP/DNA complex into inactive, negatively regulating TBP. TFIIA antagonizes the TBP repressors but may be effective only in conjunction with the RNA polymerase II holoenzyme recruitment to the promoter by promoter-bound activators. TRF1 has been discovered inDrosophila melanogasterandAnophelesbut found absent in vertebrates and yeast. TBPL1 cannot bind to the TATA-box; instead, TBPL1 prefers binding to TATA-less promoters. However, TBPL1 shows a stronger association with TFIIA than TBP. The TCT core promoter element is present in most ribosomal protein genes inDrosophilaand humans, and TBPL1 is required for the transcription of these genes. TBP directly participates in the DNA repair mechanism, and TBPL1 mediates cell cycle arrest and apoptosis. TBPL2 is closely related to its TBP paralog, showing 95% sequence similarity with the TBP core domain. Like TBP, TBPL2 also binds to the TATA-box and shows interactions with TFIIA, TFIIB, and other basal transcription factors. Despite these advances, much remains to be explored in this family of transcription factors.

Time-restricted feeding combined with aerobic exercise training can prevent weight gain and improve metabolic disorders in mice fed a high-fat diet.

KEY POINTS: Time-restricted feeding (TRF, in which energy intake is restricted to 8 h/day during the dark phase) alone or combined with aerobic exercise (AE) training can prevent weight gain and metabolic disorders in Swiss mice fed a high-fat diet. The benefits of TRF combined with AE are associated with improved hepatic metabolism and decreased hepatic lipid accumulation. TRF combined with AE training increased fatty acid oxidation and decreased expression of lipogenic and gluconeogenic genes in the liver of young male Swiss mice. TRF combined with AE training attenuated the detrimental effects of high-fat diet feeding on the insulin signalling pathway in the liver. ABSTRACT: Time-restricted feeding (TRF) or physical exercise have been shown to be efficient in the prevention and treatment of metabolic disorders; however, the additive effects of TRF combined with aerobic exercise (AE) training on liver metabolism have not been widely explored. In this study TRF (8 h in the active phase) and TRF combined with AE (TRF+Exe) were compared in male Swiss mice fed a high-fat diet, with evaluation of the effects on insulin sensitivity and expression of hepatic genes involved in fatty acid oxidation, lipogenesis and gluconeogenesis. As in previous reports, we show that TRF alone (eating only between zeitgeber time 16 and 0) was sufficient to reduce weight and adiposity gain, increase fatty acid oxidation and decrease lipogenesis genes in the liver. In addition, we show that mice of the TRF+Exe group showed additional adaptations such as increased oxygen consumption ( V̇O2${\dot V_{{{\rm{O}}_{\rm{2}}}}}$ ), carbon dioxide production ( V̇CO2${\dot V_{{\rm{C}}{{\rm{O}}_{\rm{2}}}}}$ ) and production of ketone bodies (ß-hydroxybutyrate). Also, TRF+Exe attenuated the negative effects of high-fat diet feeding on the insulin signalling pathway (insulin receptor, insulin receptor substrate, Akt), and led to increased fatty acid oxidation (Ppara, Cpt1a) and decreased gluconeogenic (Fbp1, Pck1, Pgc1a) and lipogenic (Srebp1c, Cd36) gene expression in the liver. These molecular results were accompanied by increased glucose metabolism, lower serum triglycerides and reduced hepatic lipid content in the TRF+Exe group. The data presented in this study show that TRF alone has benefits but TRF+Exe has additive benefits and can mitigate the harmful effects of consuming a high-fat diet on body adiposity, liver metabolism and glycaemic homeostasis in young male Swiss mice.

Suggestive diagnosis of attention-deficit/hyperactivity disorder in indigenous children and adolescents from the Brazilian Amazon.

The prevalence of attention-deficit/hyperactivity disorder (ADHD) symptoms has been scarcely studied in indigenous cultures that preserve ancestral cultural characteristics. The objective of the study is to estimate the prevalence of suggestive diagnosis of ADHD among indigenous children and adolescents from villages in the Amazon. This is an analytical cross-sectional study using instruments to track ADHD symptoms (the Child Behaviour Checklist for ages 6-18: CBCL/6-18 and the teacher report form for ages 6-18: TRF/6-18) and to investigate their negative impact on the patients (using the Strengths and Difficulties Questionnaire-SDQ). The prevalence of a suggestive ADHD diagnosis according to the CBCL/TRF DSM-IV ADHD subscale without and with negative impact as assessed by the SDQ was 4.3% and 1.1%, respectively. Comorbid oppositional-defiant, conduct problems and anxious symptoms were present in all cases screening positive for ADHD. We also presented a case report as an illustration of the observed clinical presentation. ADHD is a recognizable disorder even in a culture that preserves millennial characteristics. Furthermore, the presence of ADHD symptoms was associated with significant impairment.

Production and evaluation of leukocyte- and thrombocyte-rich fibrin membranes in birds.

The aim of this study was the preparation and histological evaluation of Leukocyte- and Thrombocyte-Rich Fibrin (L-TRF) membranes obtained from the blood of four bird species. Forty adult healthy birds were divided into four groups of equal size: G1 - macaws, G2 - domestic chickens, G3 - parrots, G4 - toco toucans. A total of 0.5 mL of blood was collected from each bird, put into a glass tube without anticoagulant and centrifuged at 3000 rpm for 10 min. L-TRF membranes produced after compression of the clot were processed for histological analysis. The ratio of thrombocytes/area was not significantly different among Groups G2, G3 and G4, but a significant difference was found between Groups G1 and G2 with the highest thrombocyte concentration/area in G1. The groups did not differ statistically in the number of leukocytes/area. The fibrin-to-cells ratio did not vary statistically among Groups G1, G2 and G3, but this ratio was significantly higher in Group G4 than in the other groups. The thrombocyte-to-leukocyte ratio was the highest in Group G1, but it did not differ among Groups G2, G3 and G4. In conclusion, the centrifugation protocol allowed the production of L-TRF membranes in the four bird species studied. Histologically, cell ratios were analogous in domestic chickens and parrots, and macaws had the highest ratio of thrombocytes.

Percepção de professores dos problemas de comportamento em duas amostras clínicas com TDAH: Brasil e Espanha / Teachers' perception of behavioral problems in two clinical samples with ADHD: Brazil and Spain

Considerando a díade professor-aluno no contexto escolar, a figura desse profissional tem sido estimada como uma fonte de informação válida na avaliação comportamental do aluno. Trata-se de uma investigação documental do tipo exploratória-descritiva, retrospectiva e transversal, em que se recorreu a análise de prontuários de crianças e adolescentes entre os anos de 2013 e 2014 diagnosticados com TDAH em duas unidades clínicas de psicologia situadas no Brasil e Espanha. Encontraram-se diferenças significativas apenas em duas síndromes: Problemas de Sociabilidade e Problemas de Atenção. A contribuição deste estudo foi comparar a percepção de professores residentes em diferentes países, e constatar que, independentemente das diferenças culturais, foram encontrados comportamentos similares nos indivíduos com o diagnóstico de TDAH.

Considering teacher-student dyadic interactions, the figure of this professional has been estimated as a valid source of information in the behavioral evaluation of the student. This is an exploratory-descriptive, retrospective and cross-sectional documentary investigation, in which the analysis of medical records of children and adolescents between the years of 2013 and 2014 diagnosed with ADHD in two clinical psychology units located in Brazil and Spain . In the total analysis of TRF scores, significant differences were found only in two syndromes: Sociability Problems and Attention Problems. The contribution of this study was to compare the perception of teachers from different countries and verify that regardless of cultural differences, some behaviors were similarly in individuals with the diagnosis of ADHD.

Telomeric Repeat-Binding Factor Homologs in Entamoeba histolytica: New Clues for Telomeric Research.

Telomeric Repeat Binding Factors (TRFs) are architectural nuclear proteins with critical roles in telomere-length regulation, chromosome end protection and, fusion prevention, DNA damage detection, and senescence regulation. Entamoeba histolytica, the parasite responsible of human amoebiasis, harbors three homologs of human TRFs, based on sequence similarities to their Myb DNA binding domain. These proteins were dubbed EhTRF-like I, II and III. In this work, we revealed that EhTRF-like I and II share similarity with human TRF1, while EhTRF-like III shares similarity with human TRF2 by in silico approach. The analysis of ehtrf-like genes showed they are expressed differentially under basal culture conditions. We also studied the cellular localization of EhTRF-like I and III proteins using subcellular fractionation and western blot assays. EhTRF-like I and III proteins were enriched in the nuclear fraction, but they were also present in the cytoplasm. Indirect immunofluorescence showed that these proteins were located at the nuclear periphery co-localizing with Lamin B1 and trimethylated H4K20, which is a characteristic mark of heterochromatic regions and telomeres. We found by transmission electron microscopy that EhTRF-like III was located in regions of more condensed chromatin. Finally, EMSA assays showed that EhTRF-like III forms specific DNA-protein complexes with telomeric related sequences. Our data suggested that EhTRF-like proteins play a role in the maintenance of the chromosome ends in this parasite.

Development and characterization of polyclonal antibodies against the linker region of the telomere-binding protein TRF2

Background: TRF2 (telomeric repeat binding factor 2) is an essential component of the telomere-binding protein complex shelterin. TRF2 induces the formation of a special structure of telomeric DNA and counteracts activation of DNA damage-response pathways telomeres. TRF2 has a poorly characterized linker region (udTRF2) between its homodimerization and DNA-binding domains. Some lines of evidence have shown that this region could be involved in TRF2 interaction with nuclear lamina. Results: In this study, the fragment of the TERF2 gene encoding udTRF2 domain of telomere-binding protein TRF2 was produced by PCR and cloned into the pET32a vector. The resulting plasmid pET32a-udTRF2 was used for the expression of the recombinant udTRF2 in E. coli RosettaBlue (DE3). The protein was isolated and purified using ammonium sulfate precipitation followed by ion-exchange chromatography. The purified recombinant protein udTRF2 was injected into guinea pigs to generate polyclonal antibodies. The ability of anti-udTRF2 antibodies to bind endogenous TRF2 in human skin fibroblasts was tested by western blotting and immunofluorescent staining. Conclusions: In this study, the recombinant protein udTRF2 and antibodies to it were generated. Both protein and antibodies will provide a useful tool for investigation of the functions of the udTRF2 domain and its role in the interaction between TRF2 and nuclear lamina.

Parenting program versus telephone support for Mexican parents of children with acquired brain injury: A blind randomized controlled trial.

INTRODUCTION: Acquired brain injury (ABI) during childhood typically causes behavior problems in the child and high levels of stress in the family. The aims of this study are: (1) to investigate the effectiveness and feasibility of a parenting intervention in improving behavior and self-regulation in Mexican children with ABI compared to telephone support; (2) to investigate the effectiveness and feasibility of a parenting intervention in improving parenting skills, parent self-efficacy and decreasing parental stress in parents of children with ABI compared to telephone support. Our secondary aims are (1) to explore the impact that parent characteristics have on the intervention outcomes; (2) to investigate if changes are maintained 3 months after the intervention. METHODS: The research design is a blind randomized controlled trial (RCT). Eligible participants include children with a diagnosis of ABI, between 6 and 12 years of age, and their parents. Sixty-six children and their parents will be randomly allocated to either a parenting program group or telephone support group. The parenting program involves six face-to-face weekly group sessions of 2.5 h each. Participants in the control group receive an information sheet with behavioral strategies, and six weekly phone calls, in which strategies to improve academic skills are provided. Children and their parents are evaluated by blind assessors before the intervention, immediately after the intervention and 3-months post-intervention. DISCUSSION: This study will be the first to evaluate the efficacy and feasibility of a parenting program for Mexican parents of children with ABI. TRIAL IDENTIFIER: ACTRN12617000360314.

Genome-wide identification and characterization of tRNA-derived RNA fragments in land plants.

KEY MESSAGE: The manuscript by Alves et al. entitled "Genome-wide identification and characterization of tRNA-derived RNA fragments in land plants" describes the identification and characterization of tRNAderived sRNA fragments in plants. By combining bioinformatic analysis and genetic and molecular approaches, we show that tRF biogenesis does not rely on canonical microRNA/siRNA processing machinery (i.e., independent of DICER-LIKE proteins). Moreover, we provide evidences that the Arabidopsis S-like Ribonuclease 1 (RNS1) might be involved in the biogenesis of tRFs. Detailed analyses showed that plant tRFs are sorted into different types of ARGONAUTE proteins and that they have potential target candidate genes. Our work advances the understanding of the tRF biology in plants by providing evidences that plant and animal tRFs shared common features and raising the hypothesis that an interplay between tRFs and other sRNAs might be important to fine-tune gene expression and protein biosynthesis in plant cells. Small RNA (sRNA) fragments derived from tRNAs (3'-loop, 5'-loop, anti-codon loop), named tRFs, have been reported in several organisms, including humans and plants. Although they may interfere with gene expression, their biogenesis and biological functions in plants remain poorly understood. Here, we capitalized on small RNA sequencing data from distinct species such as Arabidopsis thaliana, Oryza sativa, and Physcomitrella patens to examine the diversity of plant tRFs and provide insight into their properties. In silico analyzes of 19 to 25-nt tRFs derived from 5' (tRF-5s) and 3'CCA (tRF-3s) tRNA loops in these three evolutionary distant species showed that they are conserved and their abundance did not correlate with the number of genomic copies of the parental tRNAs. Moreover, tRF-5 is the most abundant variant in all three species. In silico and in vivo expression analyses unraveled differential accumulation of tRFs in Arabidopsis tissues/organs, suggesting that they are not byproducts of tRNA degradation. We also verified that the biogenesis of most Arabidopsis 19-25 nt tRF-5s and tRF-3s is not primarily dependent on DICER-LIKE proteins, though they seem to be associated with ARGONAUTE proteins and have few potential targets. Finally, we provide evidence that Arabidopsis ribonuclease RNS1 might be involved in the processing and/or degradation of tRFs. Our data support the notion that an interplay between tRFs and other sRNAs might be important to fine tune gene expression and protein biosynthesis in plant cells.

Low-level infrared laser modulates muscle repair and chromosome stabilization genes in myoblasts.

Infrared laser therapy is used for skeletal muscle repair based on its biostimulative effect on satellite cells. However, shortening of telomere length limits regenerative potential in satellite cells, which occurs after each cell division cycle. Also, laser therapy could be more effective on non-physiologic tissues. This study evaluated low-level infrared laser exposure effects on mRNA expression from muscle injury repair and telomere stabilization genes in myoblasts in normal and stressful conditions. Laser fluences were those used in clinical protocols. C2C12 myoblast cultures were exposed to low-level infrared laser (10, 35, and 70 J/cm(2)) in standard or normal (10 %) and reduced (2 %) fetal bovine serum concentrations; total RNA was extracted for mRNA expression evaluation from muscle injury repair (MyoD and Pax7) and chromosome stabilization (TRF1 and TRF2) genes by real time quantitative polymerization chain reaction. Data show that low-level infrared laser increases the expression of MyoD and Pax7 in 10 J/cm(2) fluence, TRF1 expression in all fluences, and TRF2 expression in 70 J/cm(2) fluence in both 10 and 2 % fetal bovine serum. Low-level infrared laser increases mRNA expression from genes related to muscle repair and telomere stabilization in myoblasts in standard or normal and stressful conditions.

A relação entre professores com sofrimento psíquico e crianças escolares com problemas de comportamento / The relation between teachers with psychological distress and schoolchildren with behavior problems

O presente estudo tem por objetivo analisar a relação entre professores com sofrimento psíquico e crianças escolares com problemas de comportamento. Um total de 151 professores avaliou a presença de problemas de comportamento em 372 alunos, através da escala Teacher´s Report Form (TRF). Os alunos foram selecionados através de amostragem por conglomerados e estavam matriculados na primeira série do ensino fundamental. Os professores também responderam a um questionário auto-aplicado a fim de avaliar algumas características pessoais e a presença de sofrimento psíquico (Self Reported Questionnaire SRQ-20). A prevalência de sofrimento psíquico encontrada entre os professores foi de 21,8 por cento. Os resultados mostram percentuais mais elevados na identificação de problemas internalizantes pelas professoras que apresentam sofrimento psíquico.

The present article aims to analyze the relation between teachers suffering from psychological distress and school children with behavioral problems. A total of 372 children were evaluated by teachers through utilizing the Teachers Report Form (TRF). The teachers also answered a questionnaire in order to evaluate some personal characteristics and the presence of psychological distress (Self Reported Questionnaire, SRQ-20). The prevalence of psychological distress found was of 21.8 percent. The results show that the teachers presenting psychological distress identified higher percentages of internalizing problems in the children.