RNAi technology and its use in studying the function of nuclear receptors and coregulators

Until just a few years ago, RNA interference (RNAi) technology was restricted to the research fields of plants, C. elegans or Drosophila. The discovery of gene silencing by in vitro synthesized double-stranded RNA (dsRNA) in mammalian cells has made the use of RNAi possible in nearly the entire life science kingdom. DNA vectors delivering small interfering RNA (siRNA) directed by polymerase III or polymerase II promoters to persistently inhibit target genes expression have extended this technology to study in vivo function of these genes. Recently, RNAi has been used as a powerful tool in the functional analysis of nuclear receptors and their coregulators. This short review will cover studies in this area

A nosocomial transmission of crimean-congo hemorrhagic fever to an attending physician in north kordufan, Sudan

<p>Abstract</p> <p>Background</p> <p>Crimean-Congo hemorrhagic fever (CCHF), a tick-borne disease caused by Crimean-Congo hemorrhagic fever virus (CCHFV), is a member of the genus Nairovirus in the family Bunyaviridae. Recently, CCHFV has been reported as an important emerging infectious viral pathogen in Sudan. Sporadic cases and multiple CCHF outbreaks, associated with nosocomial chain of transmission, have been reported in the Kordufan region of Sudan.</p> <p>Aims</p> <p>To confirm CCHF in an index patient and attending physician in North Kordufan region, Sudan, and to provide some information on virus genetic lineages.</p> <p>Methods</p> <p>Antibody captured ELISA, reverse transcription PCR, partial S segment sequences of the virus and subsequent phylogenetic analysis were used to confirm the CCHFV infection and to determine the virus genetic lineages.</p> <p>Results</p> <p>CCHF was confirmed by monitoring specific IgM antibody and by detection of the viral genome using RT-PCR. Treatment with oral ribavirin, replacement with fluid therapy, blood transfusion and administration of platelets concentrate resulted in rapid improvement of the health condition of the female physician. Phylogenetic analysis of the partial S segment sequences of the 2 CCHFV indicates that both strains are identical and belong to Group III virus lineage, which includes viruses from Africa including, Sudan, Mauritania, South Africa and Nigeria.</p> <p>Conclusion</p> <p>Further epidemiologic studies including, CCHFV complete genome analysis and implementation of improved surveillance are urgently needed to better predict and respond to CCHF outbreaks in the Kordufan region, Sudan.</p

siRNAs Induce Efficient RNAi Response in Bombyx mori Embryos

Short interference RNA (siRNA) is widely used in mammalian cells. In insects, however, reports concerning the suitablility of siRNA in vivo is very limited compared with that of long dsRNA, which is thought to be more effective. There is insufficient information on the essential rules of siRNA design in insects, as very few siRNAs have been tested in this context. To establish an effective method of gene silencing using siRNA in vivo in insects, we determined the effects of siRNA on seven target genes. We designed siRNAs according to a new guideline and injected them into eggs of Bombyx mori. At the mRNA level, the expression of most of these genes was successfully silenced, down to less than half the constitutive level, which in some cases led to the development of distinctive phenotypes. In addition, we observed stronger effect of siRNA both on the mRNA level and the phenotype than that of long dsRNA under comparable conditions. These results indicate that direct injection of siRNA is an effective reverse-genetics tool for the analysis of embryogenesis in vivo in insects

The differential effects of eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids on seizure frequency in patients with drug-resistant epilepsy – A Randomized, double-blind, placebo-controlled trial

Abstract The omega-3 (n-3) fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are known to play an important role in maintenance and modulation of neuronal functions. There is evidence that omega-3 fatty acids may have anticonvulsant effects. The effect of DHA and EPA on seizure rate in patients with DRE was investigated. Methods: A double-blind, randomized, placebo-controlled clinical trial included ninety-nine (n=99) DRE patients, aged 5-16 (n=85) and 17-45 (n=14). After randomization, patients were given two, four or six capsules per day of DHA (417.8 mg DHA and 50.8 mg EPA/capsule, n=33), EPA (385.6 mg EPA and 81.2 mg DHA/capsule, n=33) or placebo (high oleic acid sunflower oil, n=33) for one year. The primary endpoint was the effect of treatment on rate of seizure. Random-effects negative binomial regression models were fitted to model the patients’ total count of seizures per month. The treatment effects on seizure incidence rate ratio was tested after controlling for the covariate effects of gender, age, rate of seizure per week at enrollment, type of seizure and number of AEDs combinations used at enrollment. Results: Fifty-nine patients (n=59) completed the study (59.6%).The average number of seizures per month were 9.7 ± 1.2 in the EPA group, 11.7 ± 1.5 in the DHA group, and 16.6 ± 1.5 in the placebo group. Age, gender and seizure type adjusted seizure incidence rate ratios (IRRs) of the EPA and DHA groups compared with the placebo were 0.61 (CI= 0.42-0.88, p=0.008, 42% reduction) and 0.67 (CI = 0.46-1.0, p= 0.04, 39% reduction), respectively. There was no difference in IRR between the EPA and DHA groups (p=0.56). Both treatment groups had a significantly higher number of seizure-free days compared to placebo (p<0.05). Significance: This study demonstrates that EPA and DHA are effective in reducing seizure frequency in patients with DRE

RNA interference as a key to knockdown overexpressed cyclooxygenase-2 gene in tumour cells

Silencing those genes that are overexpressed in cancer and contribute to the survival and progression of tumour cells is the aim of several researches. Cyclooxygenase-2 (COX-2) is one of the most intensively studied genes since it is overexpressed in most tumours, mainly in colon cancer. The use of specific COX-2 inhibitors to treat colon cancer has generated great enthusiasm. Yet, the side effects of some inhibitors emerging during long-term treatment have caused much concern. Genes silencing by RNA interference (RNAi) has led to new directions in the field of experimental oncology. In this study, we detected sequences directed against COX-2 mRNA, that potently downregulate COX-2 gene expression and inhibit phorbol 12-myristate 13-acetate-induced angiogenesis in vitro in a specific, nontoxic manner. Moreover, we found that the insertion of a specific cassette carrying anti-COX-2 short hairpin RNA sequence into a viral vector (pSUPER.retro) greatly increased silencing potency in a colon cancer cell line (HT29) without activating any interferon response. Phenotypically, COX-2 deficient HT29 cells showed a significant impairment of their in vitro malignant behaviour. Thus, the retroviral approach enhancing COX-2 knockdown, mediated by RNAi, proved to be an useful tool to better understand the role of COX-2 in colon cancer. Furthermore, the higher infection efficiency we observed in tumour cells, if compared to normal endothelial cells, may disclose the possibility to specifically treat tumour cells without impairing endothelial COX-2 activity

Modeling recursive RNA interference.

An important application of the RNA interference (RNAi) pathway is its use as a small RNA-based regulatory system commonly exploited to suppress expression of target genes to test their function in vivo. In several published experiments, RNAi has been used to inactivate components of the RNAi pathway itself, a procedure termed recursive RNAi in this report. The theoretical basis of recursive RNAi is unclear since the procedure could potentially be self-defeating, and in practice the effectiveness of recursive RNAi in published experiments is highly variable. A mathematical model for recursive RNAi was developed and used to investigate the range of conditions under which the procedure should be effective. The model predicts that the effectiveness of recursive RNAi is strongly dependent on the efficacy of RNAi at knocking down target gene expression. This efficacy is known to vary highly between different cell types, and comparison of the model predictions to published experimental data suggests that variation in RNAi efficacy may be the main cause of discrepancies between published recursive RNAi experiments in different organisms. The model suggests potential ways to optimize the effectiveness of recursive RNAi both for screening of RNAi components as well as for improved temporal control of gene expression in switch off-switch on experiments

Efficient Construction of an Inverted Minimal H1 Promoter Driven siRNA Expression Cassette: Facilitation of Promoter and siRNA Sequence Exchange

RNA interference (RNAi), mediated by small interfering RNA (siRNA), is an effective method used to silence gene expression at the post-transcriptional level. Upon introduction into target cells, siRNAs incorporate into the RNA-induced silencing complex (RISC). The antisense strand of the siRNA duplex then "guides" the RISC to the homologous mRNA, leading to target degradation and gene silencing. In recent years, various vector-based siRNA expression systems have been developed which utilize opposing polymerase III promoters to independently drive expression of the sense and antisense strands of the siRNA duplex from the same template.We show here the use of a ligase chain reaction (LCR) to develop a new vector system called pInv-H1 in which a DNA sequence encoding a specific siRNA is placed between two inverted minimal human H1 promoters (approximately 100 bp each). Expression of functional siRNAs from this construct has led to efficient silencing of both reporter and endogenous genes. Furthermore, the inverted H1 promoter-siRNA expression cassette was used to generate a retrovirus vector capable of transducing and silencing expression of the targeted protein by>80% in target cells.The unique design of this construct allows for the efficient exchange of siRNA sequences by the directional cloning of short oligonucleotides via asymmetric restriction sites. This provides a convenient way to test the functionality of different siRNA sequences. Delivery of the siRNA cassette by retroviral transduction suggests that a single copy of the siRNA expression cassette efficiently knocks down gene expression at the protein level. We note that this vector system can potentially be used to generate a random siRNA library. The flexibility of the ligase chain reaction suggests that additional control elements can easily be introduced into this siRNA expression cassette

Thermodynamic analysis of 5′ and 3′ single- and 3′ double-nucleotide overhangs neighboring wobble terminal base pairs

Thermodynamic parameters are reported for duplex formation of 40 self-complementary RNA duplexes containing wobble terminal base pairs with all possible 3′ single and double-nucleotide overhangs, mimicking the structures of short interfering RNAs (siRNA) and microRNAs (miRNA). Based on nearest neighbor analysis, the addition of a single 3′ dangling nucleotide increases the stability of duplex formation up to 1 kcal/mol in a sequence-dependent manner. The addition of a second dangling nucleotide increases the stability of duplexes closed with wobble base pairs in an idiosyncratic manner. The results allow for the development of a nearest neighbor model, which improves the predication of free energy and melting temperature for duplexes closed by wobble base pairs with 3′ single or double-nucleotide overhangs. Phylogenetic analysis of naturally occurring miRNAs was performed. Selection of the effector miR strand of the mature miRNA duplex appears to be dependent on the orientation of the GU closing base pair rather than the identity of the 3′ double-nucleotide overhang. Thermodynamic parameters for the 5′ single terminal overhangs adjacent to wobble closing base pairs are also presented