Design and validation of small interfering RNA on respiratory syncytial virus M2-2 gene: a potential approach in RNA interference on viral replication

Human respiratory syncytial virus (RSV) is the leading cause of severe lower respiratory tract infection in infants and young children globally and is a significant pathogen of the elderly and immunocompromised. The M2-2 protein of respiratory syncytial virus (RSV) is particularly important in regulation of viral RNA transcription and replication that could be a potential anti-viral candidate against RSV infection. In this study, we designed and validated siRNAs that specifically target the RSV M2-2 gene. Four siRNAs targeting different regions of the M2-2 gene were designed using web tool. In-vitro evaluation of silencing effect was performed by using RSV infected Vero cell line. Viral M2-2 linked GFP recombinant plasmid was co-transfected with non-targeted siRNA, Pooled siRNA, siRNA 1, siRNA 2, siRNA 3 and siRNA 4 using synthetic cationic polymer. The silencing effect of M2-2 gene at the protein level was measured both qualitatively and quantitatively by using fluorescence microscopy and flow cytometry. Meanwhile, the silencing effect at the mRNA level was assessed by using RT-qPCR. This study showed that all four designed siRNAs can effectively and efficiently silence M2-2 gene. siRNA 2 showed the highest (98%) silencing effect on protein level and siRNA 4 with 83.1% at the mRNA level. The viral assay showed no significant cytopathic effects observed after 6 days post-infection with siRNAs. In conclusion, this study showed the effectiveness of siRNA in silencing M2-2 gene both at the protein and mRNA level which could potentially be used as a novel therapeutic agent in the treatment of RSV infection. However, further study is warranted to investigate the silencing effect of M2-2 protein and inhibition of RSV infection

Antibacterial activity of marine source extracts against multidrug resistant organisms

Antimicrobial resistance is the major problem of global dimensions with a significant impact on morbidity,mortality and healthcare-associated costs. The problem has recently been worsened by the steady increase in multiresistant strains and by the restriction of antibiotic discovery and development programs. Methicillin-resistant Staphylococcus aureus, pseudomonads and Escherichia coli are a major nosocomial and community-acquired pathogens for which few existing antibiotics are efficacious. The current study was conducted to investigate antibacterial activity of natural seaweed sources. Approach: Gracilaria changii Euchema denticulatum and sea cucumbers extracts against Methicillin-resistan-Staphylococcus aureus, Escherichia coli, Streptococcus pyogenes, Vibrio cholerae, Pseudomonas aeruginosa and Klebsiella pneumoniae. Results: The Minimal Inhibitory Concentration (MIC) values and Minimal Bactericidal Concentration(MBC) values of methanol extract were used against all assayed bacteria. Results indicated that G. changii, E. denticulatum and sea cucumbers extracts must possess major anti bacterial components against infectious microorganisms. Conclusion: The results obtained indicate that Gracilaria changii and Euchema denticulatum could be a source of natural products with antibiotic modifying activity to be used against multidrug resistant bacteria

Novel in-vitro antimicrobial activity of Vigna radiata (L.)R. Wilczek against highly resistant bacterial and fungal pthogens.

The ever rising resistant bacteria and fungi resulted in finding novel antimicrobial sources and agents. Studies confirmed that mung beans have increased phenolic compounds and enhanced defenses during germination. We hypothesized that antimicrobial activities might be found in sprouts of mung beans (MBS), or Vigna radiata (L.) R. Wilczek. The screening method was conducted using disc diffusion assay against 12 gram negative and positive bacteria, including multiple drug resistant (MDR) bacteria and 12 fungi. It was followed by the evaluation of the minimum inhibitory concentration and the minimum bactericidal concentration or the minimum fungicidal concentration. The screening results revealed potential antibacterial and antifungal activities by MBS extract against 11 out of 12 bacteria and 2 out of 10 fungi including remarkable antimicrobial activity against highly infectious MDR bugs such as Methicilline-resistant Staphylococcus aureus, MDR Escherichia coli O157:H7, MDR Pseudomonas aeruginosa, Klebsiella pneumoniae, S. aureus, and Salmonella Typhimurium as well as against human fungal pathogens, Trichophyton rubrum and Trichoderma harzianum. The potential antimicrobial activity of MBS reflects effective quality and quantity of polyphenolic compounds present after bean germination. This unprecedented study showed that MBS extract is a potential source for novel antimicrobials that are inexpensive and readily available at a large scale for pharmaceutical companies

Application of RT-PCR to detect treated and untreated Staphylococcus aureus genes with marine algae

Methacillin Resistant Staphylococcus aureus (MRSA), Extended Spectrum Beta Lactamase (ESBL) organisms and Multiple Drug Resistant Organism (MDRO). Therefore, this study was designed to explore an alternative antibacterial product derived from seaweed extracts, Gracilaria changii and Euchema denticulatum, through the study of DNA and RNA encoding genes of interest in MRSA and non-MRSA. The target of this study is to amplification of several untreated and treated S. aureus and E. coli genes that are potentially involved in the antibacterial activities through RT-PCR assay. G. changii and E. denticulatum extracts showed inhibitory activity against S. aureus, several genes in this pathogen were chosen to study the effect of both seaweed extracts on the genes through PCR and RT-PCR analysis. However, the predicted inhibitory mechanism of both seaweeds extracts on mecA gene was not fully elucidated in the study. The investigation could scientifically proof the natural products to be potentially potent antibacterial agents

Simplex and triplex polymerase chain reaction (PCR) for identification of three medically important Candida species

Candida species are a major cause of invasive infections in both critically ill and immunocompromised patients. Hence, rapid identification of these pathogens may facilitate specific therapy and patient management. The development of rapid and specific diagnostic methods remains a challenge. Herein, we developed the simplex and triplex polymerase chain reaction (PCR) for the identification of three medically important Candida species namely C. albicans, C. parapsilosis and C. tropicalis. The developed methods target the phospholipase B gene (PLB). The primers designed achieved highly specific identification of the selected species using both the simplex PCR and the triplex PCR formats, which were confirmed by DNA sequencing. The primers did not show any non-specific amplification when tested with DNA from other Candida species and other fungal species such as Aspergillus and Cryptococcus. These results showed that the PLB gene provides a novel target that could be used for the detection of medically important Candida species from clinical specimens.Key words: Candida species, primers, phospholipase B gene (PLB), polymerase chain reaction (PCR)

Inhibition of Growth of Highly Resistant Bacterial and Fungal Pathogens by a Natural Product

The continuous escalation of resistant bacteria against a wide range of antibiotics necessitates discovering novel unconventional sources of antibiotics. B. oleracea L (red cabbage) is health-promoting food with proven anticancer and anti-inflammatory activities. However, it has not been researched adequately for its antimicrobial activity on potential resistant pathogens. The methanol crude extract of B. oleracea L. was investigated for a possible anti-microbial activity. The screening method was conducted using disc diffusion assay against 22 pathogenic bacteria and fungi. It was followed by evaluation of the minimum inhibitory concentration (MIC). Moreover, the antibacterial and the antifungal activities were confirmed using the minimum bactericidal concentration (MBC) and the minimum fungicidal concentration (MFC), respectively. Remarkable, antibacterial activity was evident particularly against highly infectious microorganisms such as Methicillin-resistant Staphylococcus aureus, Escherichia coli O157:H7, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, and Salmonella enterica serovar Typhimurium as well as against human fungal pathogens, Trichophyton rubrum and Aspergillus terreus. Red cabbage is a rich source of phenolic compounds, anthocyanins being the most abundant class, which might explain its potent antimicrobial action. This extract is potentially novel for future antimicrobials, inexpensive, and readily available at a large scale for pharmaceutical companies for further investigation and processing

Detection the antibacterial effect of seaweeds on Staphylococcus aureus DNA repair gene (adaB) and cell wall protein synthesis (sav1017) by molecular approaches

Polymerase Chain reaction amplification of DNA was performed to used to study the presence and effect of treated and untreated Stapylococcus aureus genes sav1017 and adaB with marine seaweeds Gracilaria changii and Euchema denticulatum. From the sequencing analysis, the changes were detected in the gene sequence of adaB and sav1017, genes after treated with either G. changii or E. denticulatum extract, which involved the substitution of the nucleotide base pair and insertion or deletion of the purine or pyrimidine base. The novel of this study is the extract of G. changii and E. denticulatum interrupting the important function in MRSA and non-MRSA isolates so that this pathogen cannot survive longer than usual. This significant finding can be applied to a medical treatment whereby both of these extracts can be used as an alternative treatment for the infection of S. aureus especially to overcome drug resistance treatment problems in MRSA strains

Leptospirosis in human: biomarkers in host immune responses

Leptospirosis remains one of the most widespread zoonotic diseases caused by spirochetes of the genus Leptospira, which accounts for high morbidity and mortality globally. Leptospiral infections are often found in tropical and subtropical regions, with people exposed to contaminated environments or animal reservoirs are at high risk of getting the infection. Leptospirosis has a wide range of clinical manifestations with non-specific signs and symptoms and often misdiagnosed with other acute febrile illnesses at early stage of infection. Despite being one of the leading causes of zoonotic morbidity worldwide, there is still a gap between pathogenesis and human immune responses during leptospiral infection. It still remains obscure whether the severity of the infection is caused by the pathogenic properties of the Leptospira itself, or it is a consequence of imbalance host immune factors. Hence, in this review, we seek to summarize the past and present milestone findings on the biomarkers of host immune response aspects during human leptospiral infection, including cytokine and other immune mediators. A profound understanding of the interlink between virulence factors and host immune responses during human leptospirosis is imperative to identify potential biomarkers for diagnostic and prognostic applications as well as designing novel immunotherapeutic strategies in future

Natural product activity against Methicillin-Resistant Staphylococcus aureus genes

Methicilin Resistant Staphylococcus aureus (MRSA) implicated in many post-surgical and cancer treatment as well hospital and community fatalities need to be treated with an effective alternative antimicrobial agent. In the search for anti-MRSA agent, 2 types of natural products were investigated for inhibitory activity against MRSA. In addition to the bioassay, the activity of the anti-MRSA agent was elucidated based on the effect of both natural products on nucleotide changes of chromosome-encoded genes. In this study, the methanol extract of the red marine algae and the natural pure honey were studied for its antibacterial property based on disc diffusion test and Minimum Inhibitory Concentration (MIC). The effects of both natural products on selected gene sequences of S. aureus’s were determined by RT-PCR analysis. The genes of interest, which have been chosen in this study, are genes that are involved in the antibacterial mechanism including inhibition of cell wall synthesis, protein synthesis and nucleic acid synthesis. Five genes of interest chosen in this study include mecA gene, mecR1 gene, mecI gene, adaB gene and sav1017 gene. The results for antibacterial property showed the methanol extract of a red seaweed and the pure honey, inhibited growth of S. aureus strain according to the inhibition zones around discs saturated with the seaweed extract and pure honey, respectively. The MIC test showed decrease in growth of MRSA isolates after growing in broth incorporated with the extract and honey, respectively. The effect of the inhibitory activity of the natural products on selected gene sequences showed that several nucleotide changes occurred in the sequences of certain genes of interest based on the gene sequences of the cDNA after RT-PCR was carried out on the mRNA of S. aureus treated with the natural products. This research underlined that the inhibition effect of the natural products may be chromosome mediated evidenced by the changes of chromosome-encoded genes. The significant findings on activities of the seaweed extract and pure honey may become very useful in the process to find a better treatment for S. aureus infection especially, for the multiple drug resistant isolates. In addition, it is also, a new finding for natural product discovery through gene-expression analysis

Naturally occurring hepatitis B virus surface antigen mutant variants in Malaysian blood donors and vaccinees

Hepatitis B virus surface mutants are of enormous importance because they are capable of escaping detection by serology and can infect both vaccinated and unvaccinated populations, thus putting the whole population at risk. This study aimed to detect and characterise hepatitis B-escaped mutants among blood donors and vaccinees. One thousand serum samples were collected for this study from blood donors and vaccinees. Hepatitis B surface antigen, antibodies and core antibodies were tested using a commercial enzyme-linked immunosorbent assay (ELISA) kit. DNA detection was performed via nested polymerase chain reaction (PCR), and the S gene was sequenced and analysed using bioinformatics. Of the 1,000 samples that were screened, 5.5 % (55/1,000) were found to be HBsAg-negative and anti-HBc- and HBV DNA-positive. All 55 isolates were found to belong to genotype B. Several mutations were found across all the sequences from synonymous and non-synonymous mutations, with the most nucleotide mutations occurring at position 342, where adenine was replaced by guanine, and cytosine at position 46 was replaced by adenine in 96.4 % and 98 % of the isolates, respectively. Mutation at position 16 of the amino acid sequence was found to be common to all the Malaysian isolates, with 85.7 % of the mutations occurring outside the major hydrophilic region. This study revealed a prevalence of 5.5 % for hepatitis B-escaped mutations among blood donors and vaccinated undergraduates, with the most common mutation being found at position 16, where glutamine was substituted with lysine