Establishments of G3BP1-GFP stress granule monitoring system for real-time stress assessment in human neuroblastoma cells.

Acute stress caused by short-term exposure to deleterious chemicals can induce the aggregation of RNA-binding proteins (RBPs) in the cytosol and the formation of stress granules (SGs). The cytoplasmic RBP, Ras GTPase-activating protein-binding protein 1 (G3BP1) is a critical organizer of SG, and its aggregation is considered a hallmark of cellular stress. However, assembly of SG is a highly dynamic process that involves RBPs; hence, existing methods based on fixation processes or overexpression of RBPs exhibit limited efficacy in detecting the assembly of SG under stress conditions. In this study, we established a G3BP1- Green fluorescent protein (GFP) reporter protein in a human neuroblastoma cell line to overcome these limitations. GFP was introduced into the G3BP1 genomic sequence via homologous recombination to generate a G3BP1-GFP fusion protein and further analyze the aggregation processes. We validated the assembly of SG under stress conditions using the G3BP1-GFP reporter system. Additionally, this system supported the evaluation of bisphenol A-induced SG response in the established human neuroblastoma cell line. In conclusion, the established G3BP1-GFP reporter system enables us to monitor the assembly of the SG complex in a human neuroblastoma cell line in real time and can serve as an efficient tool for assessing potential neurotoxicity associated with short-term exposure to chemicals.

Assessment of CD8+ T-cell mediated immunity in an influenza A(H3N2) human challenge model in Belgium: a single centre, randomised, double-blind phase 2 study.

BACKGROUND: Protection afforded by inactivated influenza vaccines can theoretically be improved by inducing T-cell responses to conserved internal influenza A antigens. We assessed whether, in an influenza controlled human infection challenge, susceptible individuals receiving a vaccine boosting T-cell responses would exhibit lower viral load and decreased symptoms compared with placebo recipients. METHODS: In this single centre, randomised, double-blind phase 2 study, healthy adult (aged 18-55 years) volunteers with microneutralisation titres of less than 20 to the influenza A(H3N2) challenge strain were enrolled at an SGS quarantine facility in Antwerp, Belgium. Participants were randomly assigned double-blind using a permuted-block list with a 3:2 allocation ratio to receive 0·5 mL intramuscular injections of modified vaccinia Ankara (MVA) expressing H3N2 nucleoprotein (NP) and matrix protein 1 (M1) at 1·5 × 108 plaque forming units (4·3 × 108 50% tissue culture infectious dose [TCID50]; MVA-NP+M1 group) or saline placebo (placebo group). At least 6 weeks later, participants were challenged intranasally with 0·5 mL of a 1 × 106 TCID50/mL dose of influenza A/Belgium/4217/2015 (H3N2). Nasal swabs were collected twice daily from day 2 until day 11 for viral PCR, and symptoms of influenza were recorded from day 2 until day 11. The primary outcome was to determine the efficacy of MVA-NP+M1 vaccine to reduce the degree of nasopharyngeal viral shedding as measured by the cumulative viral area under the curve using a log-transformed quantitative PCR. This study is registered with ClinicalTrials.gov, NCT03883113. FINDINGS: Between May 2 and Oct 24, 2019, 145 volunteers were enrolled and randomly assigned to the MVA-NP+M1 group (n=87) or the placebo group (n=58). Of these, 118 volunteers entered the challenge period (71 in the MVA-NP+M1 group and 47 in the placebo group) and 117 participants completed the study (71 in the MVA-NP+M1 group and 46 in the placebo group). 78 (54%) of the 145 volunteers were female and 67 (46%) were male. The primary outcome, overall viral load as determined by quantitative PCR, did not show a statistically significant difference between the MVA-NP+M1 (mean 649·7 [95% CI 552·7-746·7) and placebo groups (mean 726·1 [604·0-848·2]; p=0·17). All reported treatment emergent adverse events (TEAEs; 11 in the vaccination phase and 51 in the challenge phase) were grade 1 and 2, except for two grade 3 TEAEs in the placebo group in the challenge phase. A grade 4 second trimester fetal death, considered possibly related to the MVA-NP+M1 vaccination, and an acute psychosis reported in a placebo participant during the challenge phase were reported. INTERPRETATION: The use of an MVA vaccine to expand CD4+ or CD8+ T cells to conserved influenza A antigens in peripheral blood did not affect nasopharyngeal viral load in an influenza H3N2 challenge model in seronegative, healthy adults. FUNDING: Department of Health and Human Services; Administration for Strategic Preparedness and Response; Biomedical Advanced Research and Development Authority; and Barinthus Biotherapeutics.

RNA m6A Modification Alteration by Black Phosphorus Quantum Dots Regulates Cell Ferroptosis: Implications for Nanotoxicological Assessment.

Nanosafety is a major concern for nanotechnology development. Evaluation of the transcriptome and the DNA methylome is proposed for nanosafety assessments. RNA m6A modification plays a crucial role in development, disease, and cell fate determination through regulating RNA stability and decay. Here, since black phosphorus quantum dots (BPQDs), among many other types of QDs, increase the global m6A level and decrease the demethylase ALKBH5 level in lung cells, the epitranscriptome is taken into consideration for the first time to evaluate nanosafety. Both the transcriptome and m6A epitranscriptome analyses show that BPQDs alter many biological processes, such as the response to selenium ions and the lipoxygenase pathway, indicating possible ferroptosis activation. The results further show that BPQDs cause lipid peroxidation, mitochondrial dysfunction, and iron overload. Recognition of these modified mRNAs by YTHDF2 leads to mRNAs' decay and eventually ferroptosis. This study shows that RNA m6A modification not only is a more sophisticated indicator for nanosafety assessment but also provides novel insight into the role of RNA m6A in regulating BPQD-induced ferroptosis, which may be broadly applicable to understanding the functions of RNA m6A under stress.

Assessment of MUSASHI 1 and MUSASHI 2 expression in spermatozoa and testicular tissue.

MUSASHI (MSI) family plays the main role in the spermatogenesis process. The purpose of this study was the assessment of sperm MSI1 and MSI2, and sperm functional tests in infertile men (n = 30) with varicocele and fertile men (n = 30). Furthermore, MSI1 and MSI2 proteins were assessed in testicular tissue of azoospermic men (n = 9) as well as epididymal spermatozoa and testis of mice. Expression of MSI1 and MSI2 was assessed at RNA and protein levels in human spermatozoa. Sperm concentration and motility were significantly lower, while abnormal sperm morphology, lipid peroxidation, DNA fragmentation and protamine deficiency were significantly higher in men with varicocele compared to fertile individuals. Any significant difference was not observed in the expression of MSI1 and MSI2 mRNA between the two groups. Unlike MSI1 protein that was not detectable in humans, the relative expression of MSI2 protein was similar in varicocele and fertile individuals. The expression level of both Msi1 and Msi2 proteins was also observable in mouse spermatozoa. No significant relationship was observed between sperm functional parameters with expression of these genes. The data of this study demonstrated that although MSI1 and MSI2 play important roles during spermatogenesis, their relative expression in spermatozoa was not affected by varicocele.

Assessment of LIN28A variants in Parkinson's disease in large European cohorts.

Parkinson's disease (PD) is a complex neurodegenerative disease with a strong genetic component. To date, several genes have been associated with monogenic forms of the disease, but these only explain a small fraction of the observed familial aggregation in PD. Recently, a heterozygous loss-of-function variant in LIN28A was associated with PD pathogenesis in the Asian population. Here, we comprehensively investigate the role of LIN28A variants in PD patients of European ancestry and assess susceptibility using individual-level genotyping data from 14,671 PD cases and 17,667 controls, as well as whole-genome sequencing data from 1647 patients with PD and 1050 controls. In addition, we further assess the summary statistics from the most recent genome-wide association studies meta-analyses to date for PD risk and age at onset. After evaluating these data, we did not find evidence to support a role for LIN28A as a major causal gene for PD. However, additional large-scale familial and case-control studies in non-European ancestry populations are necessary to further evaluate the role of LIN28A in PD etiology.

Comprehensive Survey and Comparative Assessment of RNA-Binding Residue Predictions with Analysis by RNA Type.

With close to 30 sequence-based predictors of RNA-binding residues (RBRs), this comparative survey aims to help with understanding and selection of the appropriate tools. We discuss past reviews on this topic, survey a comprehensive collection of predictors, and comparatively assess six representative methods. We provide a novel and well-designed benchmark dataset and we are the first to report and compare protein-level and datasets-level results, and to contextualize performance to specific types of RNAs. The methods considered here are well-cited and rely on machine learning algorithms on occasion combined with homology-based prediction. Empirical tests reveal that they provide relatively accurate predictions. Virtually all methods perform well for the proteins that interact with rRNAs, some generate accurate predictions for mRNAs, snRNA, SRP and IRES, while proteins that bind tRNAs are predicted poorly. Moreover, except for DRNApred, they confuse DNA and RNA-binding residues. None of the six methods consistently outperforms the others when tested on individual proteins. This variable and complementary protein-level performance suggests that users should not rely on applying just the single best dataset-level predictor. We recommend that future work should focus on the development of approaches that facilitate protein-level selection of accurate predictors and the consensus-based prediction of RBRs.

Assessment of PPARGC1A, PPARGC1B, and PON1 Genetic Polymorphisms in Esophageal Squamous Cell Carcinoma Susceptibility in the Eastern Chinese Han Population: A Case-Control Study Involving 2351 Subjects.

Previous studies suggested that alterations in the energy metabolism might be underlying cancer initiation and progression. Polymorphisms of genes involved in energy metabolism regulation, such as peroxisome proliferator-activated receptor gamma coactivator 1α (PPARGC1A), -ß (PPARGC1B), and paraoxonase 1 (PON1), might confer susceptibility to esophageal squamous cell carcinoma (ESCC) and partially explain its pathogenesis. We investigated the effects of several single nucleotide polymorphisms (SNPs) in three metabolic-related genes (e.g., PPARGC1A, PPARGC1B, and PON1) on ESCC susceptibility. In total, 829 patients with sporadic ESCC and 1522 nontumor controls were enrolled in the study. SNPs were genotyped using PCR-ligase detection reaction. Our study revealed that the PPARGC1A rs3736265 G/A SNP significantly increased the risk for ESCC (GA vs. GG: adjusted odds ratio [OR] = 1.25, 95% confidence interval [95% CI] = 1.02-1.54, p = 0.034; GA+AA vs. GG: adjusted OR = 1.25, 95% CI = 1.03-1.52, p = 0.027]. In addition, a stratified analysis revealed that the PPARGC1A rs3736265 SNP was correlated with the development of ESCC in male and nondrinking subgroups. We also confirmed that the PPARGC1B rs17572019 G/A SNP promoted the risk of ESCC in subgroup with high alcohol intake. The PPARGC1A rs8192678 C/T polymorphism decreased the susceptibility of ESCC in men. These findings highlight that polymorphisms in PPARGC1A and PPARGC1B may contribute to ESCC susceptibility. In the future, further well-designed epidemiological studies are needed to confirm our findings.

Assessment of Robustness to Temperature in a Negative Feedback Loop and a Feedforward Loop.

Robustness to temperature variation is an important specification in biomolecular circuit design. While the cancellation of parametric temperature dependencies has been shown to improve the temperature robustness of the period in a synthetic oscillator design, the performance of other biomolecular circuit designs in different temperature conditions is relatively unclear. Using a combination of experimental measurements and mathematical models, we assessed the temperature robustness of two biomolecular circuit motifs-a negative feedback loop and a feedforward loop. We found that the measured responses of both the circuits changed with temperature, both in the amplitude and in the transient response. We also found that, in addition to the cancellation of parametric temperature dependencies, certain parameter regimes could facilitate the temperature robustness of the negative feedback loop, although at a performance cost. We discuss these parameter regimes in the context of the measured data for the negative feedback loop. These results should help develop a framework for assessing and designing temperature robustness in biomolecular circuits.

Accurate inference of the full base-pairing structure of RNA by deep mutational scanning and covariation-induced deviation of activity.

Despite the large number of noncoding RNAs in human genome and their roles in many diseases include cancer, we know very little about them due to lack of structural clues. The centerpiece of the structural clues is the full RNA base-pairing structure of secondary and tertiary contacts that can be precisely obtained only from costly and time-consuming 3D structure determination. Here, we performed deep mutational scanning of self-cleaving CPEB3 ribozyme by error-prone PCR and showed that a library of <5 × 104 single-to-triple mutants is sufficient to infer 25 of 26 base pairs including non-nested, nonhelical, and noncanonical base pairs with both sensitivity and precision at 96%. Such accurate inference was further confirmed by a twister ribozyme at 100% precision with only noncanonical base pairs as false negatives. The performance was resulted from analyzing covariation-induced deviation of activity by utilizing both functional and nonfunctional variants for unsupervised classification, followed by Monte Carlo (MC) simulated annealing with mutation-derived scores. Highly accurate inference can also be obtained by combining MC with evolution/direct coupling analysis, R-scape or epistasis analysis. The results highlight the usefulness of deep mutational scanning for high-accuracy structural inference of self-cleaving ribozymes with implications for other structured RNAs that permit high-throughput functional selections.

Assessment of fear and anxiety associated behaviors, physiology and neural circuits in rats with reduced serotonin transporter (SERT) levels.

Genetic variation in serotonin transporter (SERT) that reduces transcriptional efficiency is associated with higher anxiety and fear traits and a greater incidence of post traumatic stress disorder (PTSD). Although previous studies have shown that rats with no expression of SERT (SERT-/-) have increased baseline anxiety behaviors, SERT+/- rats with low SERT expression (and more relevant to the clinical condition with low SERT expression) do not. Yet, no systematic studies of fear acquisition/extinction or their underlying neural mechanisms have been conducted in this preclinical genetic SERT+/- model. Here we sought to determine if SERT+/- or SERT-/-, compared to wildtype, rats would show exacerbated panic responses and/or persistent conditioned fear responses that may be associated with PTSD or phobia vulnerability. Results: Only SERT-/- rats showed increased baseline anxiety-like behaviors with heightened panic respiratory responses. However SERT+/- (also SERT-/-) rats showed enhanced acquisition of fear and delayed extinction of fear that was associated with changes in serotonergic-related genes (e.g., reduced 5-HT1A receptor) and disrupted inhibition within the basolateral amygdala (BLA). Furthermore, the disrupted fear responses in SERT+/- rats were normalized with 5HT1A antagonist infusions into the BLA. Enhanced acquisition and failure to extinguish fear memories displayed by both SERT-/- and SERT+/- rats are cardinal symptoms of disabling anxiety disorders such as phobias and PTSD. The data here support the hypothesis that reduced SERT function is a genetic risk that disrupts select gene expression and network properties in the amygdala that could result in vulnerability to these syndromes.

Partner-specific prediction of RNA-binding residues in proteins: A critical assessment.

RNA-protein interactions play essential roles in regulating gene expression. While some RNA-protein interactions are "specific", that is, the RNA-binding proteins preferentially bind to particular RNA sequence or structural motifs, others are "non-RNA specific." Deciphering the protein-RNA recognition code is essential for comprehending the functional implications of these interactions and for developing new therapies for many diseases. Because of the high cost of experimental determination of protein-RNA interfaces, there is a need for computational methods to identify RNA-binding residues in proteins. While most of the existing computational methods for predicting RNA-binding residues in RNA-binding proteins are oblivious to the characteristics of the partner RNA, there is growing interest in methods for partner-specific prediction of RNA binding sites in proteins. In this work, we assess the performance of two recently published partner-specific protein-RNA interface prediction tools, PS-PRIP, and PRIdictor, along with our own new tools. Specifically, we introduce a novel metric, RNA-specificity metric (RSM), for quantifying the RNA-specificity of the RNA binding residues predicted by such tools. Our results show that the RNA-binding residues predicted by previously published methods are oblivious to the characteristics of the putative RNA binding partner. Moreover, when evaluated using partner-agnostic metrics, RNA partner-specific methods are outperformed by the state-of-the-art partner-agnostic methods. We conjecture that either (a) the protein-RNA complexes in PDB are not representative of the protein-RNA interactions in nature, or (b) the current methods for partner-specific prediction of RNA-binding residues in proteins fail to account for the differences in RNA partner-specific versus partner-agnostic protein-RNA interactions, or both.

Allele-specific SHAPE-MaP assessment of the effects of somatic variation and protein binding on mRNA structure.

The impact of inherited and somatic mutations on messenger RNA (mRNA) structure remains poorly understood. Recent technological advances that leverage next-generation sequencing to obtain experimental structure data, such as SHAPE-MaP, can reveal structural effects of mutations, especially when these data are incorporated into structure modeling. Here, we analyze the ability of SHAPE-MaP to detect the relatively subtle structural changes caused by single-nucleotide mutations. We find that allele-specific sorting greatly improved our detection ability. Thus, we used SHAPE-MaP with a novel combination of clone-free robotic mutagenesis and allele-specific sorting to perform a rapid, comprehensive survey of noncoding somatic and inherited riboSNitches in two cancer-associated mRNAs, TPT1 and LCP1 Using rigorous thermodynamic modeling of the Boltzmann suboptimal ensemble, we identified a subset of mutations that change TPT1 and LCP1 RNA structure, with approximately 14% of all variants identified as riboSNitches. To confirm that these in vitro structures were biologically relevant, we tested how dependent TPT1 and LCP1 mRNA structures were on their environments. We performed SHAPE-MaP on TPT1 and LCP1 mRNAs in the presence or absence of cellular proteins and found that both mRNAs have similar overall folds in all conditions. RiboSNitches identified within these mRNAs in vitro likely exist under biological conditions. Overall, these data reveal a robust mRNA structural landscape where differences in environmental conditions and most sequence variants do not significantly alter RNA structural ensembles. Finally, predicting riboSNitches in mRNAs from sequence alone remains particularly challenging; these data will provide the community with benchmarks for further algorithmic development.

Assessment of BicC family RNA binding protein 1 and Ras protein specific guanine nucleotide releasing factor 1 as candidate genes for high myopia: A case-control study.

PURPOSE: The aim is to evaluate the association between high myopia and genetic variant in the BicC family RNA binding protein 1 (BICC1) as well as its association with Ras protein specific guanine nucleotide releasing factor 1 (RASGRF1) genes in a Chinese Han population with a case-control study. METHODS: Five TagSNPs in BICC1 and RASGRF1 genes were selected and genotyped in 821 unrelated subjects, which composed of 419 controls (spherical equivalent within ±1.00 D in both eyes and axial length ≦24.0 mm) and 402 cases (spherical equivalent ≤-6.0D in at least one eye and axial length ≥26.0 mm). Statistical analysis was performed with SNPstats. RESULTS: After an analysis adjusted by age and sex, rs4245599 in BICC1 was found to be significantly associated with high myopia under the codominant, dominant, recessive and log-additive model (P = 0.001, 0.0015, 0.0045 and 2e-04, odds ratio [OR] = 2.15, 1.59, 1.73 and 1.46, respectively), and rs10763559 in BICC1 was associated with high myopia and under the dominant and log-additive model (P = 0.032 and 0.036, OR = 0.72 and 0.78, respectively). Rs4778879 in RASGRF1 was found to be significantly associated with high myopia under codominant, dominant, recessive, and log-additive model (P = 0.0088, 0.0065, 0.026, and 0.0021, OR = 1.87, 1.48, 1.56, and 1.37, respectively). However, no significant association was found between rs745030 in RASGRF1 and high myopia, neither was there any association of rs745029 in RASGRF1. CONCLUSION: The present study indicated that genetic variants in BICC1 and RASGRF1 are closely associated with high myopia, which appears to be a potential candidate for high myopia in Chinese Han population. Considering the small sample size of this study, further work is needed to validate our results. The function of BICC1 and RASGRF1 in the process of developing high myopia needs to be explored in the future.

aPPRove: An HMM-Based Method for Accurate Prediction of RNA-Pentatricopeptide Repeat Protein Binding Events.

Pentatricopeptide repeat containing proteins (PPRs) bind to RNA transcripts originating from mitochondria and plastids. There are two classes of PPR proteins. The [Formula: see text] class contains tandem [Formula: see text]-type motif sequences, and the [Formula: see text] class contains alternating [Formula: see text], [Formula: see text] and [Formula: see text] type sequences. In this paper, we describe a novel tool that predicts PPR-RNA interaction; specifically, our method, which we call aPPRove, determines where and how a [Formula: see text]-class PPR protein will bind to RNA when given a PPR and one or more RNA transcripts by using a combinatorial binding code for site specificity proposed by Barkan et al. Our results demonstrate that aPPRove successfully locates how and where a PPR protein belonging to the [Formula: see text] class can bind to RNA. For each binding event it outputs the binding site, the amino-acid-nucleotide interaction, and its statistical significance. Furthermore, we show that our method can be used to predict binding events for [Formula: see text]-class proteins using a known edit site and the statistical significance of aligning the PPR protein to that site. In particular, we use our method to make a conjecture regarding an interaction between CLB19 and the second intronic region of ycf3. The aPPRove web server can be found at www.cs.colostate.edu/~approve.

Structural and functional assessment of APOBEC3G macromolecular complexes.

There are eleven members in the human APOBEC family of proteins that are evolutionarily related through their zinc-dependent cytidine deaminase domains. The human APOBEC gene clusters arose on chromosome 6 and 22 through gene duplication and divergence to where current day APOBEC proteins are functionally diverse and broadly expressed in tissues. APOBEC serve enzymatic and non enzymatic functions in cells. In both cases, formation of higher-order structures driven by APOBEC protein-protein interactions and binding to RNA and/or single stranded DNA are integral to their function. In some circumstances, these interactions are regulatory and modulate APOBEC activities. We are just beginning to understand how macromolecular interactions drive processes such as APOBEC subcellular compartmentalization, formation of holoenzyme complexes, gene targeting, foreign DNA restriction, anti-retroviral activity, formation of ribonucleoprotein particles and APOBEC degradation. Protein-protein and protein-nucleic acid cross-linking methods coupled with mass spectrometry, electrophoretic mobility shift assays, glycerol gradient sedimentation, fluorescence anisotropy and APOBEC deaminase assays are enabling mapping of interacting surfaces that are essential for these functions. The goal of this methods review is through example of our research on APOBEC3G, describe the application of cross-linking methods to characterize and quantify macromolecular interactions and their functional implications. Given the homology in structure and function, it is proposed that these methods will be generally applicable to the discovery process for other APOBEC and RNA and DNA editing and modifying proteins.

Influenza A viruses escape from MxA restriction at the expense of efficient nuclear vRNP import.

To establish a new lineage in the human population, avian influenza A viruses (AIV) must overcome the intracellular restriction factor MxA. Partial escape from MxA restriction can be achieved when the viral nucleoprotein (NP) acquires the critical human-adaptive amino acid residues 100I/V, 283P, and 313Y. Here, we show that introduction of these three residues into the NP of an avian H5N1 virus renders it genetically unstable, resulting in viruses harboring additional single mutations, including G16D. These substitutions restored genetic stability yet again yielded viruses with varying degrees of attenuation in mammalian and avian cells. Additionally, most of the mutant viruses lost the capacity to escape MxA restriction, with the exception of the G16D virus. We show that MxA escape is linked to attenuation by demonstrating that the three substitutions promoting MxA escape disturbed intracellular trafficking of incoming viral ribonucleoprotein complexes (vRNPs), thereby resulting in impaired nuclear import, and that the additional acquired mutations only partially compensate for this import block. We conclude that for adaptation to the human host, AIV must not only overcome MxA restriction but also an associated block in nuclear vRNP import. This inherent difficulty may partially explain the frequent failure of AIV to become pandemic.

Simultaneous characterization of sense and antisense genomic processes by the double-stranded hidden Markov model.

Hidden Markov models (HMMs) have been extensively used to dissect the genome into functionally distinct regions using data such as RNA expression or DNA binding measurements. It is a challenge to disentangle processes occurring on complementary strands of the same genomic region. We present the double-stranded HMM (dsHMM), a model for the strand-specific analysis of genomic processes. We applied dsHMM to yeast using strand specific transcription data, nucleosome data, and protein binding data for a set of 11 factors associated with the regulation of transcription.The resulting annotation recovers the mRNA transcription cycle (initiation, elongation, termination) while correctly predicting strand-specificity and directionality of the transcription process. We find that pre-initiation complex formation is an essentially undirected process, giving rise to a large number of bidirectional promoters and to pervasive antisense transcription. Notably, 12% of all transcriptionally active positions showed simultaneous activity on both strands. Furthermore, dsHMM reveals that antisense transcription is specifically suppressed by Nrd1, a yeast termination factor.

El uso de placebo en ensayos clínicos de fase III en Brasil / The use of placebos in phase III clinical trials in Brazil

El Consejo Federal de Medicina de Brasil (CFM) -órgano normativo y fiscalizador del ejercicio ético de la medicina- prohibió, en 2008, la participación de médicos brasileños en investigaciones que utilizaran placebo para enfermedades con tratamiento eficaz y efectivo, en contraposición a la Declaración de Helsinki, que permite su uso en condiciones metodológicamente justificadas. Con el objetivo de verificar si la normativa ética del CFM modificó el uso de placebo en ensayos clínicos de fase III en Brasil, se analizaron varias características de sus registros en el ClinicalTrials.gov, en los períodos de 2003 a 2007 y de 2009 a 2013. Se concluye que: a) la normativa promulgada por el CFM en 2008 fue ineficaz y prevaleció la posición adoptada por la Declaración de Helsinki; b) el patrocinio de ensayos con placebo por parte de la industria farmacéutica multinacional fue significativo; c) predominaron las investigaciones de fármacos para enfermedades crónicas, y fueron poco significativas para las enfermedades postergadas, de importancia para Brasil.

In 2008, Brazil's Federal Council of Medicine [Conselho Federal de Medicina] (CFM) - regulatory and supervisory agency on the ethical practice of medicine - banned the participation of Brazilian doctors in studies using placebos for diseases with efficient and effective treatment. This position differs with the Helsinki Declaration, which allows the use of placebos in methodologically justified conditions. To ascertain whether the CMF's ethical regulation modified the use of placebos in phase III clinical trials in Brazil, characteristics of the records in ClinicalTrials.gov were researched in the periods from 2003 to 2007 and from 2009 to 2013. The conclusions reached were: a) the regulations issued by the CFM in 2008 were ineffective and the position adopted by the Helsinki Declaration prevails; b) there was significant sponsorship by the multinational pharmaceutical industry of trials with placebos; c) the research was predominantly on new drugs for chronic diseases, with little study done of the neglected diseases which are of great importance to Brazil.

A fine decision tree consisted of CK5/6, IMP3 and TTF1 for cytological diagnosis among reactive mesothelial cells, metastatic adenocarcinoma of lung and non-lung origin in pleural effusion.

The utility of combination with CK5/6, IMP3 and TTF1 to differentiate among reactive mesothelial cells (RMs), metastatic adenocarcinoma of lung (LAC) and non-lung (NLAC) origin was investigated by using immunocytochemistry (ICC) and conventional PCR (C-PCR) in pleural effusion. A total of 108 cell blocks (32 RMs, 51 LAC and 25 NLAC were evaluated by ICC for CK5/6, IMP3 and TTF1 protein expression. In addition, we further performed C-PCR for amplification of CK5/6, IMP3 and TTF1 DNA from 28 specimens (9 MAC and 7 RMs, 6 LAC and 6 NLAC) for molecular diagnosis. CK5/6 staining was observed in the majority of reactive specimens (78.1%) and was rare in adenocarcinoma cells (14.5%), whereas the opposite was true for IMP3 and TTF1. We found a high frequency of TTF1 positivity (76.5%) in LAC, but not in NLAC (4.0%); while there was no significant difference of IMP3 expression in LAC (88.2%) and NLAC (88.0%). The 487 bp DNA fragments of IMP3 was expected to be amplified in 6/9 of adenocarcinoma cases showed negative in ICC; and the 394 bp DNA fragments of CK5/6 was also expected to be amplified in 4/7 of RMs cases showed negative in ICC. Consistent with ICC results, there was significant difference of TTF1 expression in the LAC and NLAC compared with IMP3 expression. The combination with CK5/6, IMP3 and TTF1 immunostaining appears to be useful to improve the accuracy of cytological diagnoses between RMs, metastatic adenocarcinoma of lung and non-lung origin in pleural effusion. In addition, C-PCR may act as a useful supplemental approach for ICC, especially negative cases in ICC for differential cytological diagnosis.

Computational assessment of the cooperativity between RNA binding proteins and MicroRNAs in Transcript Decay.

Transcript degradation is a widespread and important mechanism for regulating protein abundance. Two major regulators of transcript degradation are RNA Binding Proteins (RBPs) and microRNAs (miRNAs). We computationally explored whether RBPs and miRNAs cooperate to promote transcript decay. We defined five RBP motifs based on the evolutionary conservation of their recognition sites in 3'UTRs as the binding motifs for Pumilio (PUM), U1A, Fox-1, Nova, and UAUUUAU. Recognition sites for some of these RBPs tended to localize at the end of long 3'UTRs. A specific group of miRNA recognition sites were enriched within 50 nts from the RBP recognition sites for PUM and UAUUUAU. The presence of both a PUM recognition site and a recognition site for preferentially co-occurring miRNAs was associated with faster decay of the associated transcripts. For PUM and its co-occurring miRNAs, binding of the RBP to its recognition sites was predicted to release nearby miRNA recognition sites from RNA secondary structures. The mammalian miRNAs that preferentially co-occur with PUM binding sites have recognition seeds that are reverse complements to the PUM recognition motif. Their binding sites have the potential to form hairpin secondary structures with proximal PUM binding sites that would normally limit RISC accessibility, but would be more accessible to miRNAs in response to the binding of PUM. In sum, our computational analyses suggest that a specific set of RBPs and miRNAs work together to affect transcript decay, with the rescue of miRNA recognition sites via RBP binding as one possible mechanism of cooperativity.