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.

A conserved domain of Drosophila RNA-binding protein Pumilio interacts with multiple CCR4-NOT deadenylase complex subunits to repress target mRNAs.

Pumilio is a sequence-specific RNA-binding protein that controls development, stem cell fate, and neurological functions in Drosophila. Pumilio represses protein expression by destabilizing target mRNAs in a manner dependent on the CCR4-NOT deadenylase complex. Three unique repression domains in the N-terminal region of Pumilio were previously shown to recruit CCR4-NOT, but how they do so was not well understood. In this study, we identified the motifs that are necessary and sufficient for the activity of the third repression domain of Pumilio, designated RD3, which is present in all isoforms and has conserved regulatory function. We identified multiple conserved regions of RD3 that are important for repression activity in cell-based reporter gene assays. Using yeast two-hybrid assays, we show that RD3 contacts specific regions of the Not1, Not2, and Not3 subunits of the CCR4-NOT complex. Our results indicate that RD3 makes multivalent interactions with CCR4-NOT mediated by conserved short linear interaction motifs. Specifically, two phenylalanine residues in RD3 make crucial contacts with Not1 that are essential for its repression activity. Using reporter gene assays, we also identify three new target mRNAs that are repressed by Pumilio and show that RD3 contributes to their regulation. Together, these results provide important insights into the mechanism by which Pumilio recruits CCR4-NOT to regulate the expression of target mRNAs.

Ablation of Sam68 in adult mice increases thermogenesis and energy expenditure.

Genetic deletion of Src associated in mitosis of 68kDa (Sam68), a pleiotropic adaptor protein prevents high-fat diet-induced weight gain and insulin resistance. To clarify the role of Sam68 in energy metabolism in the adult stage, we generated an inducible Sam68 knockout mice. Knockout of Sam68 was induced at the age of 7-10 weeks, and then we examined the metabolic profiles of the mice. Sam68 knockout mice gained less body weight over time and at 34 or 36 weeks old, had smaller fat mass without changes in food intake and absorption efficiency. Deletion of Sam68 in mice elevated thermogenesis, increased energy expenditure, and attenuated core-temperature drop during acute cold exposure. Furthermore, we examined younger Sam68 knockout mice at 11 weeks old before their body weights deviate, and confirmed increased energy expenditure and thermogenic gene program. Thus, Sam68 is essential for the control of adipose thermogenesis and energy homeostasis in the adult.

In vivo assessment of bisphenol A induced histopathological alterations and inflammatory gene expression in lungs of male Wistar rats.

Bisphenol A (BPA), an imperative environmental contaminant used in polycarbonate plastics. Due to limited information concerning the effect of BPA on lungs, this study design to assess whether BPA cause alterations in histopathology and trace metal content in lungs of rats. They were divided into five groups with five rats per group. Group I was named as control group. Group L6 and L12 were received BPA (10 mg/kg body weight/day) for 6 weeks and 12 weeks respectively. Group H6 and L12 were given BPA (25 mg/kg body weight/day) for 6 weeks and 12 weeks respectively. Considerable alteration in Cu, Zn and Fe was detected in experimental groups. BPA also caused significant increase in the expression of tumor necrosis factor α that mediate the pulmonary inflammatory response. Comparative study of resolved proteins i.e. 72 KDa (matrix metalloproteinase 2 fragment) and 109 KDa (nucleolin) on SDS-PAGE showed their altered expression in experimental groups. Histopathology of experimental groups revealed altered architecture of lungs. Special staining of BPA treated groups showed significant number of mast cells in alveoli and bronchioles. Prolonged administration of BPA causes deleterious aggravating lung damage even at extremely low dose, so the use of BPA should be prohibited in plastic synthesizing industries.

Quantitative STAU2 measurement in lymphocytes for breast cancer risk assessment.

Although mammograms play a key role in early breast cancer detection, the test is not applicable to all women, for example, women under the age of 40. The development of a noninvasive blood test with high sensitivity and accessibility will improve the effectiveness of breast cancer screening programmes. Secretory factors released from cancer cells can induce the expression of certain genes in a large number of white blood cells (WBCs). Therefore, cancer-dependent proteins in WBCs can be used as tumour markers with high sensitivity. Five proteins (LMAN1, AZI2, STAU2, MMP9 and PLOD1) from a systemic analysis of a variety of array data of breast cancer patients were subjected to immunofluorescence staining to evaluate the presence of fixed WBCs on 96-well plates from 363 healthy females and 358 female breast cancer patients. The results revealed that the average fluorescence intensity of anti-STAU2 and the percentage of STAU2-positive T and B lymphocytes in breast cancer patients (110.50 ± 23.38 and 61.87 ± 12.44, respectively) were significantly increased compared with those in healthy females (56.47 ± 32.03 and 33.02 ± 18.10, respectively) (p = 3.56 × 10-71, odds ratio = 24.59, 95% CI = 16.64-36.34). The effect of secreted molecules from breast cancer cells was proven by the increase in STAU2 intensity in PBMCs cocultured with MCF-7 and T47D cells at 48 h (p = 0.0289). The test demonstrated 98.32%, 82.96%, and 48.32% sensitivity and 56.47%, 83.47%, and 98.62% specificity in correlation with the percentage of STAU2-positive cells at 40, 53.34 and 63.38, respectively. We also demonstrated how to use the STAU2 test for the assessment of risk in women under the age of 40. STAU2 is a novel breast cancer marker that can be assessed by quantitative immunofluorescence staining of fixed WBCs that are transportable at room temperature via mail, representing a useful risk assessment tool for women without access to mammograms.

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 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.

Comparative Colocalization Single-Molecule Spectroscopy (CoSMoS) with Multiple RNA Species.

Colocalization single-molecule spectroscopy (CoSMoS) allows studying RNA-protein complexes in the full complexity of their cellular environment at single-molecule resolution. Conventionally, the interaction between a single RNA species and multiple proteins is monitored in real time. However, comparing interactions of the same proteins with different RNA species in the same cell extract promises unique insights into RNA biology. Here, we describe an approach to monitor multiple RNA species simultaneously to enable direct comparison. This approach represents a technological development to avoid conventional inter-experiment comparisons.

In Vivo RNA Chemical Footprinting Analysis in Archaea.

RNA structural conformation and dynamics govern the functional properties of all RNA/RNP. Accordingly, defining changes of RNA structure and dynamics in various conditions may provide detailed insight into how RNA structural properties regulate the function of RNA/RNP. Traditional chemical footprinting analysis using chemical modifiers allows to sample the dynamics and conformation landscape of diverse RNA/RNP. However, many chemical modifiers are limited in their capacity to provide unbiased information reflecting the in vivo RNA/RNP structural landscape. In the recent years, the development of selective-2'-hydroxyl acylation analyzed by primer extension (SHAPE) methodology that uses powerful new chemical modifiers has significantly improved in vitro and in vivo structural probing of secondary and tertiary interactions of diverse RNA species at the single nucleotide level.Although the original discovery of Archaea as an independent domain of life is intimately linked to the technological development of RNA analysis, our understanding of in vivo RNA structural conformation and dynamics in this domain of life remains scarce.This protocol describes the in vivo use of SHAPE chemistry in two evolutionary divergent model Archaea, Sulfolobus acidocaldarius and Haloferax volcanii.

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.

QM/MM Calculations on Protein-RNA Complexes: Understanding Limitations of Classical MD Simulations and Search for Reliable Cost-Effective QM Methods.

Although atomistic explicit-solvent Molecular Dynamics (MD) is a popular tool to study protein-RNA recognition, satisfactory MD description of protein-RNA complexes is not always achieved. Unfortunately, it is often difficult to separate MD simulation instabilities primarily caused by the simple point-charge molecular mechanics (MM) force fields from problems related to the notorious uncertainties in the starting structures. Herein, we report a series of large-scale QM/MM calculations on the U1A protein-RNA complex. This experimentally well-characterized system has an intricate protein-RNA interface, which is very unstable in MD simulations. The QM/MM calculations identify several H-bonds poorly described by the MM method and thus indicate the sources of instabilities of the U1A interface in MD simulations. The results suggest that advanced QM/MM computations could be used to indirectly rationalize problems seen in MM-based MD simulations of protein-RNA complexes. As the most accurate QM method, we employ the computationally demanding meta-GGA density functional TPSS-D3(BJ)/def2-TZVP level of theory. Because considerably faster methods would be needed to extend sampling and to study even larger protein-RNA interfaces, a set of low-cost QM/MM methods is compared to the TPSS-D3(BJ)/def2-TZVP data. The PBEh-3c and B97-3c density functional composite methods appear to be suitable for protein-RNA interfaces. In contrast, HF-3c and the tight-binding Hamiltonians DFTB3-D3 and GFN-xTB perform unsatisfactorily and do not provide any advantage over the MM description. These conclusions are supported also by similar analysis of a simple HutP protein-RNA interface, which is well-described by MD with the exception of just one H-bond. Some other methodological aspects of QM/MM calculations on protein-RNA interfaces are discussed.

PureCLIP: capturing target-specific protein-RNA interaction footprints from single-nucleotide CLIP-seq data.

The iCLIP and eCLIP techniques facilitate the detection of protein-RNA interaction sites at high resolution, based on diagnostic events at crosslink sites. However, previous methods do not explicitly model the specifics of iCLIP and eCLIP truncation patterns and possible biases. We developed PureCLIP ( https://github.com/skrakau/PureCLIP ), a hidden Markov model based approach, which simultaneously performs peak-calling and individual crosslink site detection. It explicitly incorporates a non-specific background signal and, for the first time, non-specific sequence biases. On both simulated and real data, PureCLIP is more accurate in calling crosslink sites than other state-of-the-art methods and has a higher agreement across replicates.

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.

A Genetic Tool to Track Protein Aggregates and Control Prion Inheritance.

Protein aggregation is a hallmark of many diseases but also underlies a wide range of positive cellular functions. This phenomenon has been difficult to study because of a lack of quantitative and high-throughput cellular tools. Here, we develop a synthetic genetic tool to sense and control protein aggregation. We apply the technology to yeast prions, developing sensors to track their aggregation states and employing prion fusions to encode synthetic memories in yeast cells. Utilizing high-throughput screens, we identify prion-curing mutants and engineer "anti-prion drives" that reverse the non-Mendelian inheritance pattern of prions and eliminate them from yeast populations. We extend our technology to yeast RNA-binding proteins (RBPs) by tracking their propensity to aggregate, searching for co-occurring aggregates, and uncovering a group of coalescing RBPs through screens enabled by our platform. Our work establishes a quantitative, high-throughput, and generalizable technology to study and control diverse protein aggregation processes in cells.

Finding RNA-Protein Interaction Sites Using HMMs.

RNA-binding proteins play important roles in the various stages of RNA maturation through binding to its target RNAs. Cross-linking immunoprecipitation coupled with high-throughput sequencing (CLIP-Seq) has made it possible to identify the targeting sites of RNA-binding proteins in various cell culture systems and tissue types on a genome-wide scale. Several Hidden Markov model-based (HMM) approaches have been suggested to identify protein-RNA binding sites from CLIP-Seq datasets. In this chapter, we describe how HMM can be applied to analyze CLIP-Seq datasets, including the bioinformatics preprocessing steps to extract count information from the sequencing data before HMM and the downstream analysis steps following peak-calling.

Variation in the Calculation of Allostatic Load Score: 21 Examples from NHANES.

After decades of resistance, there is now a genuine consensus that disease cannot be prevented or even successfully treated unless the role of stress is addressed alongside traditionally recognized factors such as genes and the environment. Measurement of allostatic load, which is quantified by the allostatic load score (ALS), is one of the most frequently used methods to assess the physiologic response to stress. Even though there is universal agreement that in the calculation of ALS, biomarkers from three categories should be included (cardiovascular, metabolic and immune), enormous variation exists in how ALS is calculated. Specifically, there is no consensus on which biomarkers to include or the method which should be used to determine whether the value of a biomarker represents high risk. In this perspective, we outline the approach taken in 21 different NHANES studies.

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.

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.