Use of mRNA- and protein-destabilizing elements to develop a highly responsive reporter system

Reporter assays are widely used in applications that require measurement of changes in gene expression over time (e.g. drug screening). With standard reporter vectors, the measurable effect of a treatment or compound (altered reporter activity) is substantially diluted and delayed, compared with its true effect (altered transcriptional activity). This problem is caused by the relatively long half-lives of both the reporter protein and its mRNA. As a result, the activities of compounds, ligands or treatments that have a relatively minor effect, or a substantial but transient effect, often remain undetected. To circumvent this problem, we introduced modular protein- and mRNA-destabilizing elements into a range of commonly used reporters. Our data show that both elements are required for maximal responses to both increases and decreases in transcriptional activity. The double-destabilized reporter vectors showed markedly improved performance in drug screening, kinetic assays and dose–response titrations

Decrease of resistance to air flow with nasal strips as measured with the airflow perturbation device

BACKGROUND: Nasal strips are used by athletes, people who snore, and asthmatics to ease the burden of breathing. Although there are some published studies that demonstrate higher flow with nasal strips, none had directly measured the effect of the strips on nasal resistance using the airflow perturbation device (APD). The APD is an inexpensive instrument that can measure respiratory resistance based on changes in mouth pressure and rate of airflow. METHOD: This study tested forty-seven volunteers (14 men and 33 women), ranging in age from 17 to 51. Each volunteer was instructed to breathe normally into the APD using an oronasal mask with and without nasal strips. The APD measured respiratory resistance during inhalation, exhalation, and an average of the two. RESULTS: Results of a paired mean t-test comparing nasal strip against no nasal strip were statistically significant at the p = 0.05 level. The Breathe Right™ nasal dilator strips lowered nasal resistance by an average of 0.5 cm H(2)0/Lps from an average nasal resistance of 5.5 cm H(2)0/Lps. CONCLUSIONS: Nasal strips reduce nasal resistance when measured with the APD. The effect is equal during exhalation and during inhalation

Short-term tissue decomposition alters stable isotope values and C:N ratio, but does not change relationships between lipid content, C:N ratio, and Δδ13C in marine animals

Measures (e.g. δ15N, δ13C, %C, %N and C:N) derived from animal tissues are commonlyused to estimate diets and trophic interactions. Since tissue samples are often exposed toair or kept chilled in ice over a short-term during sample preparation, they may degrade.Herein, we hypothesize that tissue decomposition will cause changes in these measures. Inthis study, we kept marine fish, crustacean and mollusc tissues in air or ice over 120 h (5days). We found that tissue decomposition in air enriched δ15N (range 0.6½ to 1.3½) andδ13C (0.2½ to 0.4½), decreased %N (0.47 to 3.43 percentage points from staring values of~13%) and %C (4.53 to 8.29 percentage points from starting values of ~43%), and subsequentlyincreased C:N ratio (0.14 to 0.75). In air, while such changes to δ13C were relativelyminor and therefore likely tolerable, changes in δ15N, %N, %C and C:N ratio should be interpretedwith caution. Ice effectively reduced the extent to which decomposition enrichedδ15N ( 0.4½) and δ13C ( 0.2½), and eliminated decomposition in C:N ratio, %N and %C.In our second experiment, for fish tissues in either air or ice over 120 h, we observed noeffects of decomposition on relationships between lipid content, C:N ratio, and Δδ13C(change in δ13C after lipid removal), which are employed to correct δ13C for samples containinglipid. We also confirmed that lipid in tissues caused large errors when estimatingδ13C (mean ± standard error = -1.8½ ± 0.1½, range -0.6½ to -3.8½), and showed both lipidextraction and mathematical correction performed equally well to correct for lipids when estimatingδ13C. We, therefore, recommend that specimens of marine animals should be keptin ice during sample preparation for a short-term, as it is an effective means for minimizingchanges of the stable isotope measures in their tissue

Clinical Significance of BCL2, C-MYC, and BCL6 Genetic Abnormalities, Epstein-Barr Virus Infection, CD5 Protein Expression, Germinal Center B Cell/Non-Germinal Center B-Cell Subtypes, Co-expression of MYC/BCL2 Proteins and Co-expression of MYC/BCL2/BCL6 Proteins in Diffuse Large B-Cell Lymphoma : A Clinical and Pathological Correlation Study of 120 Patients

Background: Clinical significance of germinal center B-cell (GCB) and non-GCB sub-categorization, expression of MYC, BCL2, BCL6, CD5 proteins and Epstein Barr virus encoded RNA (EBER) positivity in diffuse large B-cell lymphoma (DLBCL) remain controversial. Could these biomarkers accurately identify high risk DLBCL patients? Are MYC, BCL2 and BCL6 proteins expression feasible as baseline testing to predict c-Myc, BCL2 or BCL6 gene rearrangements? Aims: To investigate prognostic values of GCB/non-GCB sub-categorization, Double Protein Expression Lymphoma (DPL), Triple Protein Expression Lymphoma (TPL), positivity of CD5 protein and EBER in patients with DLBCL disease. To evaluate correlation between BCL2 , c-Myc and BCL6 gene rearrangements with BCL2, MYC and BCL6 proteins expression. Methods: Diagnostic tissue samples of 120 DLBCL patients between January 2012 to December 2013 from four major hospitals in Malaysia were selected. Samples were subjected to immunohistochemical staining, fluorescent in-situ hybridization (FISH) testing, and central pathological review. Pathological data were correlated with clinical characteristics and treatment outcome. Results: A total of 120 cases were analysed. Mean age of diagnosis was 54.1 years ± 14.6, 64 were males, 56 were females, mean follow up period was 25 months (ranged from 1 to 36 months). Of the 120 cases, 74.2% were non-GCB whereas 25.8% were GCB, 6.7% were EBER positive, 6.7% expressed CD5 protein, 13.3% were DPL and 40% were TPL. The prevalence of c-Myc, BCL2, BCL6 gene rearrangements were 5.8%, 5.8%, and 14.2%, respectively; and 1.6% were Double Hit Lymphoma (DHL). EBER positivity, DPL, TPL, c-Myc gene rearrangement, BCL2 gene rearrangement, extra copies of BCL2 gene and BCL6 gene rearrangement were associated with shorter median overall survival (P0.05). Overall, c-Myc, BCL2 and BCL6 gene rearrangements showed weak correlation with expression of MYC, BCL2 and BCL6 proteins (P>0.05). Fluorescent in situ hybridization is the preferred technique for prediction of treatment outcome in DLBCL patients. Conclusion: c-Myc, BCL2, and BCL6 gene rearrangements, EBER expression, DHL, TPL and IPI score are reliable risk stratification tools. MYC, BCL2 and BCL6 proteins expression are not applicable as baseline biomarkers to predict c-Myc, BCL2, and BCL6 gene rearrangements

A Pre-mRNA–Associating Factor Links Endogenous siRNAs to Chromatin Regulation

In plants and fungi, small RNAs silence gene expression in the nucleus by establishing repressive chromatin states. The role of endogenous small RNAs in metazoan nuclei is largely unknown. Here we show that endogenous small interfering RNAs (endo-siRNAs) direct Histone H3 Lysine 9 methylation (H3K9me) in Caenorhabditis elegans. In addition, we report the identification and characterization of nuclear RNAi defective (nrde)-1 and nrde-4. Endo-siRNA–driven H3K9me requires the nuclear RNAi pathway including the Argonaute (Ago) NRDE-3, the conserved nuclear RNAi factor NRDE-2, as well as NRDE-1 and NRDE-4. Small RNAs direct NRDE-1 to associate with the pre-mRNA and chromatin of genes, which have been targeted by RNAi. NRDE-3 and NRDE-2 are required for the association of NRDE-1 with pre-mRNA and chromatin. NRDE-4 is required for NRDE-1/chromatin association, but not NRDE-1/pre-mRNA association. These data establish that NRDE-1 is a novel pre-mRNA and chromatin-associating factor that links small RNAs to H3K9 methylation. In addition, these results demonstrate that endo-siRNAs direct chromatin modifications via the Nrde pathway in C. elegans

Nanoceria Inhibit the Development and Promote the Regression of Pathologic Retinal Neovascularization in the Vldlr Knockout Mouse

Many neurodegenerative diseases are known to occur and progress because of oxidative stress, the presence of reactive oxygen species (ROS) in excess of the cellular defensive capabilities. Age related macular degeneration (AMD), diabetic retinopathy (DR) and inherited retinal degeneration share oxidative stress as a common node upstream of the blinding effects of these diseases. Knockout of the Vldlr gene results in a mouse that develops intraretinal and subretinal neovascular lesions within the first month of age and is an excellent model for a form of AMD called retinal angiomatous proliferation (RAP). Cerium oxide nanoparticles (nanoceria) catalytically scavenge ROS by mimicking the activities of superoxide dismutase and catalase. A single intravitreal injection of nanoceria into the Vldlr-/- eye was shown to inhibit: the rise in ROS in the Vldlr-/- retina, increases in vascular endothelial growth factor (VEGF) in the photoreceptor layer, and the formation of intraretinal and subretinal neovascular lesions. Of more therapeutic interest, injection of nanoceria into older mice (postnatal day 28) resulted in the regression of existing vascular lesions indicating that the pathologic neovessels require the continual production of excessive ROS. Our data demonstrate the unique ability of nanoceria to prevent downstream effects of oxidative stress in vivo and support their therapeutic potential for treatment of neurodegenerative diseases such as AMD and DR

Systematic, comprehensive, evidence-based approach to identify neuroprotective interventions for motor neuron disease: using systematic reviews to inform expert consensus

Objectives: Motor neuron disease (MND) is an incurable progressive neurodegenerative disease with limited treatment options. There is a pressing need for innovation in identifying therapies to take to clinical trial. Here, we detail a systematic and structured evidence-based approach to inform consensus decision making to select the first two drugs for evaluation in Motor Neuron Disease-Systematic Multi-arm Adaptive Randomised Trial (MND-SMART: NCT04302870), an adaptive platform trial. We aim to identify and prioritise candidate drugs which have the best available evidence for efficacy, acceptable safety profiles and are feasible for evaluation within the trial protocol. Methods: We conducted a two-stage systematic review to identify potential neuroprotective interventions. First, we reviewed clinical studies in MND, Alzheimer’s disease, Huntington’s disease, Parkinson’s disease and multiple sclerosis, identifying drugs described in at least one MND publication or publications in two or more other diseases. We scored and ranked drugs using a metric evaluating safety, efficacy, study size and study quality. In stage two, we reviewed efficacy of drugs in MND animal models, multicellular eukaryotic models and human induced pluripotent stem cell (iPSC) studies. An expert panel reviewed candidate drugs over two shortlisting rounds and a final selection round, considering the systematic review findings, late breaking evidence, mechanistic plausibility, safety, tolerability and feasibility of evaluation in MND-SMART. Results: From the clinical review, we identified 595 interventions. 66 drugs met our drug/disease logic. Of these, 22 drugs with supportive clinical and preclinical evidence were shortlisted at round 1. Seven drugs proceeded to round 2. The panel reached a consensus to evaluate memantine and trazodone as the first two arms of MND-SMART. Discussion: For future drug selection, we will incorporate automation tools, text-mining and machine learning techniques to the systematic reviews and consider data generated from other domains, including high-throughput phenotypic screening of human iPSCs

The pipeline project: Pre-publication independent replications of a single laboratory's research pipeline

This crowdsourced project introduces a collaborative approach to improving the reproducibility of scientific research, in which findings are replicated in qualified independent laboratories before (rather than after) they are published. Our goal is to establish a non-adversarial replication process with highly informative final results. To illustrate the Pre-Publication Independent Replication (PPIR) approach, 25 research groups conducted replications of all ten moral judgment effects which the last author and his collaborators had “in the pipeline” as of August 2014. Six findings replicated according to all replication criteria, one finding replicated but with a significantly smaller effect size than the original, one finding replicated consistently in the original culture but not outside of it, and two findings failed to find support. In total, 40% of the original findings failed at least one major replication criterion. Potential ways to implement and incentivize pre-publication independent replication on a large scale are discussed

Data from a pre-publication independent replication initiative examining ten moral judgement effects

We present the data from a crowdsourced project seeking to replicate findings in independent laboratories before (rather than after) they are published. In this Pre-Publication Independent Replication (PPIR) initiative, 25 research groups attempted to replicate 10 moral judgment effects from a single laboratory's research pipeline of unpublished findings. The 10 effects were investigated using online/lab surveys containing psychological manipulations (vignettes) followed by questionnaires. Results revealed a mix of reliable, unreliable, and culturally moderated findings. Unlike any previous replication project, this dataset includes the data from not only the replications but also from the original studies, creating a unique corpus that researchers can use to better understand reproducibility and irreproducibility in science