In vitro inhibitory analysis of consensus siRNAs against NS3 gene of hepatitis C virus 1a genotype

Objective To explore inhibitory effects of genome-specific, chemically synthesized siRNAs (small interference RNA) against NS3 gene of hepatitis C virus (HCV) 1a genotype in stable Huh-7 (human hepatoma) cells as well as against viral replication in serum-inoculated Huh-7 cells. Methods Stable Huh-7 cells persistently expressing NS3 gene were produced under antibiotic gentamycin (G418) selection. The cell clones resistant to 1 000 μg antibiotic concentration (G418) were picked as stable cell clones. The NS3 gene expression in stable cell clone was confirmed by RT-PCR and Western blotting. siRNA cell cytotoxicity was determined by MTT cell proliferation assay. Stable cell lines were transfected with sequence specific siRNAs and their inhibitory effects were determined by RT-PCR, real-time PCR and Western blotting. The viral replication inhibition by siRNAs in serum inoculated Huh-7 cells was determined by real-time PCR. Results RT-PCR and Western blot analysis confirmed NS3 gene and protein expression in stable cell lines on day 10, 20 and 30 post transfection. MTT cell proliferation assay revealed that at most concentrated dose tested (50 nmol/L), siRNA had no cytotoxic effects on Huh-7 cells and cell proliferation remained unaffected. As demonstrated by the siRNA time-dependent inhibitory analysis, siRNA NS3-is44 showed maximum inhibition of NS3 gene in stable Huh-7 cell clones at 24 (80%, P = 0.013) and 48 h (75%, P = 0.002) post transfection. The impact of siRNAs on virus replication in serum inoculated Huh-7 cells also demonstrated significant decrease in viral copy number, where siRNA NS3-is44 exhibited 70% (P < 0.05) viral RNA reduction as compared to NS3-is33, which showed a 64% (P < 0.05) decrease in viral copy number. siRNA synergism (NS3-is33 + NS3-is44) decreased viral load by 84% (P < 0.05) as compared to individual inhibition by each siRNA (i.e., 64%–70% (P < 0.05)) in serum-inoculated cells. Synthetic siRNAs mixture (NS5B-is88 + NS3-is33) targeting different region of HCV genome (NS5B and NS3) also decreased HCV viral load by 85% (P < 0.05) as compared to siRNA inhibitory effects alone (70% and 64% respectively, P < 0.05). Conclusions siRNAs directed against NS3 gene significantly decreased mRNA and protein expression in stable cell clones. Viral replication was also vividly decreased in serum infected Huh-7 cells. Stable Huh-7 cells expressing NS3 gene is helpful to develop anti-hepatitis C drug screening assays. siRNA therapeutic potential along with other anti-HCV agents can be considered against hepatitis C.

In vitro inhibitory analysis of consensus siRNAs against NS3 gene of hepatitis C virus 1a genotype

OBJECTIVE@#To explore inhibitory effects of genome-specific, chemically synthesized siRNAs (small interference RNA) against NS3 gene of hepatitis C virus (HCV) 1a genotype in stable Huh-7 (human hepatoma) cells as well as against viral replication in serum-inoculated Huh-7 cells.@*METHODS@#Stable Huh-7 cells persistently expressing NS3 gene were produced under antibiotic gentamycin (G418) selection. The cell clones resistant to 1000 μg antibiotic concentration (G418) were picked as stable cell clones. The NS3 gene expression in stable cell clone was confirmed by RT-PCR and Western blotting. siRNA cell cytotoxicity was determined by MTT cell proliferation assay. Stable cell lines were transfected with sequence specific siRNAs and their inhibitory effects were determined by RT-PCR, real-time PCR and Western blotting. The viral replication inhibition by siRNAs in serum inoculated Huh-7 cells was determined by real-time PCR.@*RESULTS@#RT-PCR and Western blot analysis confirmed NS3 gene and protein expression in stable cell lines on day 10, 20 and 30 post transfection. MTT cell proliferation assay revealed that at most concentrated dose tested (50 nmol/L), siRNA had no cytotoxic effects on Huh-7 cells and cell proliferation remained unaffected. As demonstrated by the siRNA time-dependent inhibitory analysis, siRNA NS3-is44 showed maximum inhibition of NS3 gene in stable Huh-7 cell clones at 24 (80%, P = 0.013) and 48 h (75%, P = 0.002) post transfection. The impact of siRNAs on virus replication in serum inoculated Huh-7 cells also demonstrated significant decrease in viral copy number, where siRNA NS3-is44 exhibited 70% (P < 0.05) viral RNA reduction as compared to NS3-is33, which showed a 64% (P < 0.05) decrease in viral copy number. siRNA synergism (NS3-is33 + NS3-is44) decreased viral load by 84% (P < 0.05) as compared to individual inhibition by each siRNA (i.e., 64%-70% (P < 0.05)) in serum-inoculated cells. Synthetic siRNAs mixture (NS5B-is88 + NS3-is33) targeting different region of HCV genome (NS5B and NS3) also decreased HCV viral load by 85% (P < 0.05) as compared to siRNA inhibitory effects alone (70% and 64% respectively, P < 0.05).@*CONCLUSIONS@#siRNAs directed against NS3 gene significantly decreased mRNA and protein expression in stable cell clones. Viral replication was also vividly decreased in serum infected Huh-7 cells. Stable Huh-7 cells expressing NS3 gene is helpful to develop anti-hepatitis C drug screening assays. siRNA therapeutic potential along with other anti-HCV agents can be considered against hepatitis C.

Incidental parasitic infections in surgically removed appendices: a retrospective analysis

Numerous parasitic infections can cause inflammation of the appendix and can mimic appendicitis clinically. The diagnosis is generally achieved only after surgery. However early diagnosis through stool examination may prevent life-threatening complications. This study investigated the presence of parasitic infections in surgically removed appendices as an etiology of acute appendicitis. A retrospective study included patients who had undergone surgery for acute appendicitis over a period of three years from Jan 2012 to Dec 2014. Demographic data, laboratory investigations, operative data and pathological findings, presence and type of parasites were retrieved. The results showed that out of 1536 patients with appendectomy done, 938 [61.1%] were males and 598 [38.9%] were females. Parasitic infection was demonstrated only in 0.4% [6 patients]. Mean average age of these patients was 12 years. Enterobius vermicularis was present in 4 patients [66% of the parasitic affection] and Schistosoma mansoni in 2 patients [34% of the parasitic affection]. Other etiologies were acute suppurative appendicitis [94.1%], chronic appendicitis [3.1%], tumors [0.3%], tuberculosis [0.2%] and actinomycosis [0.1%]. Appendix was found normal in 2% of patients underwent appendectomy

Methicillin-resistant Staphylococcus aureus in the western region of Saudi Arabia: prevalence and antibiotic susceptibility pattern

Methicillin-resistant Staphylococcus aureus [MRSA] emerged in 1960 and was a problem confined largely to the healthcare setting, or hospital-associated MRSA [HA-MRSA]. In the 1990s, community-associated MRSA [CA-MRSA] infections appeared. In Saudi Arabia, the prevalence of MRSA has increased in the past ten years and severe community-acquired infection has been reported. Our objective was to investigate the prevalence of MRSA and their antibiotic susceptibilities in the western region of Saudi Arabia. A retrospective review of the medical records of 186 S aureus infected patients diagnosed from November 2009 through October 2010. S aureus was Identified based on Gram stain, catalase and coagulase tests. susceptibility testing was performed using antibiotic discs and the VITEK 2 system. MRSA was isolated in 39.5% of the specimens. The isolates were commonly associated with wound, skin, and soft tissue infections [87.3%]. The prevalence of MRSA was highest among patients who were 56 years old or older [52.2%]. CA-MRSA infections represented 31.5% of community S aureus infections, while HA-MRSA accounted 52.6% of hospital S aureus [P=.0029]. All MRSA isolates in our study were susceptible to vancomycin, linozolid and teicoplanin. However, multi-resistance was observed in 29.1% of the isolates and was significantly higher among HA-MRSA [P=.03]. The prevalence of MRSA was 39.5%, and infection was commonly associated with sound, skin, and soft tissue infections. MRSA was more prevalent in hospitals and among older patients. All MRSA susceptible to vancomycin, linozolid and teicoplanin

Frequency and antimicrobial susceptibility of Gram-negative bacteria isolated from 2 hospitals in Makkah, Saudi Arabia

To estimate the prevalence and antibiotic susceptibility of the Gram-negative bacteria isolated from 2 hospitals in Makkah. This study was undertaken in 2 main tertiary care hospitals namely; Al-Noor Specialist Hospital, and Hera Hospital in Makkah, Kingdom of Saudi Arabia from October 2005 to March 2006. A total of 1137 Gram-negative bacteria were identified in non-duplicate clinical specimens obtained from 965 patients of various body sites infections. Demographic data, identity of microorganisms, and antimicrobial susceptibilities were obtained from medical and laboratory records. The most prevalent Gram-negative bacteria were Escherichia coli [31.6%], and Pseudomonas aeruginosa [31.2%], followed by Acinetobacter baumannii [10.8%], Klebsiella pneumoniae [8.3%], Klebsiella sp. [6.2%], Haemophilus influenzae [3.7%], Proteus sp. [3.3%], and Enterobacter sp. [1.9%]. Results demonstrated that Gram-negative bacteria have a high rate of resistance to commonly used antibiotics. Furthermore, multi-drug resistance was also common in this study. Our data showed a high rate of resistance among Gram-negative pathogens in comparison with other countries in the world. The implementation of monitoring programs is an important part of the prevention strategy against the development of antibiotic resistance in hospitals