Identification of novel conserved functional motifs across most Influenza A viral strains

Abstract Background Influenza A virus poses a continuous threat to global public health. Design of novel universal drugs and vaccine requires a careful analysis of different strains of Influenza A viral genome from diverse hosts and subtypes. We performed a systematic in silico analysis of Influenza A viral segments of all available Influenza A viral strains and subtypes and grouped them based on host, subtype, and years isolated, and through multiple sequence alignments we extrapolated conserved regions, motifs, and accessible regions for functional mapping and annotation. Results Across all species and strains 87 highly conserved regions (conservation percentage > = 90%) and 19 functional motifs (conservation percentage = 100%) were found in PB2, PB1, PA, NP, M, and NS segments. The conservation percentage of these segments ranged between 94 - 98% in human strains (the most conserved), 85 - 93% in swine strains (the most variable), and 91 - 94% in avian strains. The most conserved segment was different in each host (PB1 for human strains, NS for avian strains, and M for swine strains). Target accessibility prediction yielded 324 accessible regions, with a single stranded probability > 0.5, of which 78 coincided with conserved regions. Some of the interesting annotations in these regions included sites for protein-protein interactions, the RNA binding groove, and the proton ion channel. Conclusions The influenza virus has evolved to adapt to its host through variations in the GC content and conservation percentage of the conserved regions. Nineteen universal conserved functional motifs were discovered, of which some were accessible regions with interesting biological functions. These regions will serve as a foundation for universal drug targets as well as universal vaccine design.</p

Impact of opioid-free analgesia on pain severity and patient satisfaction after discharge from surgery: multispecialty, prospective cohort study in 25 countries

Background: Balancing opioid stewardship and the need for adequate analgesia following discharge after surgery is challenging. This study aimed to compare the outcomes for patients discharged with opioid versus opioid-free analgesia after common surgical procedures.Methods: This international, multicentre, prospective cohort study collected data from patients undergoing common acute and elective general surgical, urological, gynaecological, and orthopaedic procedures. The primary outcomes were patient-reported time in severe pain measured on a numerical analogue scale from 0 to 100% and patient-reported satisfaction with pain relief during the first week following discharge. Data were collected by in-hospital chart review and patient telephone interview 1 week after discharge.Results: The study recruited 4273 patients from 144 centres in 25 countries; 1311 patients (30.7%) were prescribed opioid analgesia at discharge. Patients reported being in severe pain for 10 (i.q.r. 1-30)% of the first week after discharge and rated satisfaction with analgesia as 90 (i.q.r. 80-100) of 100. After adjustment for confounders, opioid analgesia on discharge was independently associated with increased pain severity (risk ratio 1.52, 95% c.i. 1.31 to 1.76; P &lt; 0.001) and re-presentation to healthcare providers owing to side-effects of medication (OR 2.38, 95% c.i. 1.36 to 4.17; P = 0.004), but not with satisfaction with analgesia (beta coefficient 0.92, 95% c.i. -1.52 to 3.36; P = 0.468) compared with opioid-free analgesia. Although opioid prescribing varied greatly between high-income and low- and middle-income countries, patient-reported outcomes did not.Conclusion: Opioid analgesia prescription on surgical discharge is associated with a higher risk of re-presentation owing to side-effects of medication and increased patient-reported pain, but not with changes in patient-reported satisfaction. Opioid-free discharge analgesia should be adopted routinely

The design of optimal therapeutic small interfering RNA molecules targeting diverse strains of influenza a virus

Motivation: There is an urgent need for new medications to combat influenza pandemics. Methods: Using the genome analysis of the influenza A virus performed previously, we designed and performed a combinatorial exhaustive systematic methodology for optimal design of universal therapeutic small interfering RNA molecules (siRNAs) targeting all diverse influenza A viral strains. The rationale was to integrate the factors for highly efficient design in a pipeline of analysis performed on possible influenza-targeting siRNAs. This analysis selects specific siRNAs that has the ability to target highly conserved, accessible and biologically significant regions. This would require minimal dosage and side effects. Results and Discussion: First, \u3e6000 possible siRNAs were designed. Successive filtration followed where a novel method for siRNA scoring filtration layers was implemented. This method excluded siRNAs below the 90% experimental inhibition mapped scores using the intersection of 12 different scoring algorithms. Further filtration of siRNAs is done by eliminating those with offtargets in the human genome and those with undesirable properties and selecting siRNA targeting highly probable single-stranded regions. Finally, the optimal properties of the siRNA were ensured through selection of those targeting 100% conserved, biologically functional short motifs. Validation of a predicted active (sh114) and a predicted inactive (sh113) (that was filtered out in Stage 8) silencer of the NS1 gene showed significant inhibition of the NS1 gene for sh114, with negligible decrease for sh113 which failed target accessibility. This demonstrated the fertility of this methodology. © The Author 2011. Published by Oxford University Press

In silico design and experimental validation of sirnas targeting conserved regions of multiple hepatitis c virus genotypes

© 2016 ElHefnawi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. RNA interference (RNAi) is a post-transcriptional gene silencing mechanism that mediates the sequence-specific degradation of targeted RNA and thus provides a tremendous opportunity for development of oligonucleotide-based drugs. Here, we report on the design and validation of small interfering RNAs (siRNAs) targeting highly conserved regions of the hepatitis C virus (HCV) genome. To aim for therapeutic applications by optimizing the RNAi efficacy and reducing potential side effects, we considered different factors such as target RNA variations, thermodynamics and accessibility of the siRNA and target RNA, and off-target effects. This aim was achieved using an in silico design and selection protocol complemented by an automated MysiRNA-Designer pipeline. The protocol included the design and filtration of siRNAs targeting highly conserved and accessible regions within the HCV internal ribosome entry site, and adjacent core sequences of the viral genome with high-ranking efficacy scores. Off-target analysis excluded siRNAs with potential binding to human mRNAs. Under this strict selection process, two siRNAs (HCV353 and HCV258) were selected based on their predicted high specificity and potency. These siRNAs were tested for antiviral efficacy in HCV genotype 1 and 2 replicon cell lines. Both in silico-designed siRNAs efficiently inhibited HCV RNA replication, even at low concentrations and for short exposure times (24h); they also exceeded the antiviral potencies of reference siRNAs targeting HCV. Furthermore, HCV353 and HCV258 siRNAs also inhibited replication of patient-derived HCV genotype 4 isolates in infected Huh-7 cells. Prolonged treatment of HCV replicon cells with HCV353 did not result in the appearance of escape mutant viruses. Taken together, these results reveal the accuracy and strength of our integrated siRNA design and selection protocols. These protocols could be used to design highly potent and specific RNAi-based therapeutic oligonucleotide interventions

Rosavin Ameliorates Hepatic Inflammation and Fibrosis in the NASH Rat Model via Targeting Hepatic Cell Death

Background: Non-alcoholic fatty liver disease (NAFLD) represents the most common form of chronic liver disease that urgently needs effective therapy. Rosavin, a major constituent of the Rhodiola Rosea plant of the family Crassulaceae, is believed to exhibit multiple pharmacological effects on diverse diseases. However, its effect on non-alcoholic steatohepatitis (NASH), the progressive form of NAFLD, and the underlying mechanisms are not fully illustrated. Aim: Investigate the pharmacological activity and potential mechanism of rosavin treatment on NASH management via targeting hepatic cell death-related (HSPD1/TNF/MMP14/ITGB1) mRNAs and their upstream noncoding RNA regulators (miRNA-6881-5P and lnc-SPARCL1-1:2) in NASH rats. Results: High sucrose high fat (HSHF) diet-induced NASH rats were treated with different concentrations of rosavin (10, 20, and 30 mg/kg/day) for the last four weeks of dietary manipulation. The data revealed that rosavin had the ability to modulate the expression of the hepatic cell death-related RNA panel through the upregulation of both (HSPD1/TNF/MMP14/ITGB1) mRNAs and their epigenetic regulators (miRNA-6881-5P and lnc-SPARCL1-1:2). Moreover, rosavin ameliorated the deterioration in both liver functions and lipid profile, and thereby improved the hepatic inflammation, fibrosis, and apoptosis, as evidenced by the decreased protein levels of IL6, TNF-α, and caspase-3 in liver sections of treated animals compared to the untreated NASH rats. Conclusion: Rosavin has demonstrated a potential ability to attenuate disease progression and inhibit hepatic cell death in the NASH animal model. The produced effect was correlated with upregulation of the hepatic cell death-related (HSPD1, TNF, MMP14, and ITGB1) mRNAs—(miRNA-6881-5P—(lnc-SPARCL1-1:2) RNA panel

Dose-dependent antiviral effects of HCV IRES-specific siRNAs on the replication of subgenomic HCV replicons expressing a NS5A-GFP fusion protein.

<p>(A) Representative confocal images of GFP replicon cells after transfection with HCV-specific siRNAs. Replicon cells were PFA-fixed, and stained with Hoechst 33432 stain. Images of 0.1 and 50 nM siRNA transfections at 72 h post-transfection (p.t.) are shown (ImageXpress Ultra: 20x magnification). Overlaid images: cell nuclei (blue) and HCV RNA replicating cells (green). GFP replicon cells: (B) Huh-7 Con1, (C) Huh-7 JFH-1, and (D) HuH6 JFH-1 replicon cells were plated and transfected with 0.1–50 nM siRNAs targeting the viral genome and a scrambled control. At 72 h p.t., confocal images of GFP replicon cells were acquired and analyzed using an ImageXpress Ultra microscope and MetaXpress software, respectively. Results were normalized to the level of GFP-positive cells of the scrambled siRNA transfection control, which was set to 100%. The threshold of GFP was defined by treatment of replicon cells with an HCV replication inhibitor, which was set to 100% inhibition (data not shown). Fluorescence intensity above this threshold was considered to indicate active HCV replication. Data are presented as the mean ± SEM values for four wells measured in quadruplicate in two independent experiments (N = 32). Black columns indicate HCV RNA replication (GFP-positive cells); grey columns indicate cell viability (total cell number of Hoechst 33432 stained cell nuclei). The numbers on the bars indicate the residual percentage of GFP-positive cells. Asterisks indicate that the mean values are significantly different between samples (*<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001).</p

Features of siRNAs validated in HCV replicon assays.

<p>siRNA sequences targeting HCV IRES subdomains III and IV.</p

Curing of HCV replicon cells with IRES-specific siRNAs.

<p>(A) Subgenomic HCV JFH-1 replicon cells expressing an NS5A-GFP fusion protein were treated in the absence of G418 twice per week with 50 nM of siRNAs, either individually (scramble, HCV321 or HCV353) or in combination (50 nM HCV321 and 50 nM HCV353) for 8 weeks as depicted. HCV RNA replication was determined twice per week using GFP expression. (B) siRNA transfection was discontinued after 8 weeks, replicon cells were treated with 500 μg/mL G418 for 4 weeks, and resistant cell clones were stained using crystal violet.</p

Flowchart of steps and methodology for <i>in silico</i> siRNA design.

<p>Steps 1–3: HCV IRES sequences of different genotypes were selected and aligned. All possible siRNAs were designed and sorted according to the parameters, as depicted. The candidates were scored according to first- and second-generation algorithms, and the 70% and 90% experimental threshold inhibition scores were determined. Step 4: Multi-stage filtering of siRNAs based on threshold scores, off-targets, palindromes, and repeat-motifs was performed, followed by selection of siRNAs with terminal ends mapped to a crucial IRES loop. Step 5: Additional off-target seed-region matches were filtered, and siRNAs with optimal thermodynamic properties were selected.</p