Optimization of process parameters by response surface methodology to develop a more bioefficacious nanosuspension of Silybum marianum seed extract

Purpose: To develop a nanosuspension drug delivery system to enhance the dissolution rate of Silybum marianum seeds extract. Methods: Central composite design was used to study the effect of the input variables (stabilizer to plant extract ratio, antisolvent to solvent ratio, stirring time) on the dependent variables (mean particle size, polydispersity index (PDI) and zeta potential). The optimized formulation was characterized by Scanning Electron Microscopy (SEM), Atomic Force Microscopy (AFM), Fourier Transformed Infrared Microscopy (FT-IR) and in vitro dissolution testing. Results: The optimized nanosuspension with mean particle size of 137 nm, PDI of 0.327 and zeta potential of -37 mV was obtained. SEM studies revealed irregular shaped particles. AFM studies showed nanosized particles with good surface characteristics. The optimized formulation showed faster dissolution rate than coarse suspension. Conclusion: Results suggested that nanosuspension has remarkable potential for enhancement of the dissolution properties of poorly soluble S. marianum seed extract. Keywords: Silybum marianum; Nanosuspension; Optimization; Dissolution rat

Research article Characterization of broad-spectrum biocontrol efficacy of Bacillus velezensis against Fusarium oxysporum in Triticum aestivum L.

Fungi are the most important phytopathogens that cause yield losses. The mycotoxins released by fungi cause spoilage of stored food consumed by humans and feed supplied to animals. Fungi-antagonistic microbes are gaining attention as potential biocontrol agents (BCAs). This study was designed to isolate bacterial isolates from different crops and evaluate their in vitro antifungal assay against three phytopathogens, plant growth promoting (PGP) characteristics, molecular identification, and in vivo efficiency against the most devastating phytopathogenic fungus Fusarium oxysporum Schltdl. In the in vitro experiment, the 3 isolates BA, GL-1, and 5a out of 360 isolates showed more than 60% inhibitory activity against the selected fungi in this study. On the basis of 16S rRNA sequencing and phylogenetic analysis, BA isolate was identified as Bacillus velezensis. All three isolates produced indole acetic acid (IAA), hydrogen cyanide (HCN), and cellulase enzymes, while the BA and GL-1 isolates also produced siderophores and the BA isolate also produced ammonia. BA was selected on basis of not only Biocontrol efficacy but also maximum PGPR activity compared to GL-1 and 5a. In vivo assay, the isolate BA showed a significant decrease in disease severity caused by Fusarium oxysporum by 64.97% after 100 days of inoculation on wheat (FD-08) seedlings in a greenhouse assay and enhanced the shoot root height, fresh and dry mass. The wide-ranging antagonistic action of Bacillus velezensis isolated from the phyllosphere of wheat crops showed promising fungicidal and plant growth-promoting capabilities, suggesting it can be used as a biofungicide

Effect of combined siRNA of HCV E2 gene and HCV receptors against HCV

<p>Abstract</p> <p>Background/Aim</p> <p>Hepatitis C virus (HCV) is a major threat as almost 3% of the world's population (350 million individual) and 10% of the Pakistani population is chronically infected with this virus. RNA interference (RNAi), a sequence-specific degradation process of RNA, has potential to be used as a powerful alternative molecular therapeutic approach in spite of the current therapy of interferon-α and ribavirin against HCV which has limited efficiency. HCV structural gene E2 is mainly involved in viral cell entry via attachment with the host cell surface receptors i.e., CD81 tetraspanin, low density lipoprotein receptor (LDLR), scavenger receptor class B type 1 (SR-B1), and Claudin1 (CLDN1). Considering the importance of HCV E2 gene and cellular receptors in virus infection and silencing effects of RNAi, the current study was designed to target the cellular and viral factors as new therapeutic options in limiting HCV infection.</p> <p>Results</p> <p>In this study the potential of siRNAs to inhibit HCV-3a replication in serum-infected Huh-7 cells was investigated by combined treatment of siRNAs against the HCV E2 gene and HCV cellular receptors (CD81 and LDLR), which resulted in a significant decrease in HCV viral copy number.</p> <p>Conclusion</p> <p>From the current study it is concluded that the combined RNAi-mediated silencing of HCV E2 and HCV receptors is important for the development of effective siRNA-based therapeutic option against HCV-3a.</p

The development of cost effective 100 base pair prototype DNA ladder using polymerase chain reaction

Background: In genomics, DNA scale is used as a standard unit for the measurement of unknown DNA fragments, plasmids, and PCR products during gel electrophoresis. The 100 base pair DNA ladder is essential and cost-effective in molecular biological research and is available commercially which is too expensive and not easily accessible to a common researcher for laboratory usage.Methods: The main purpose of this study was to report easily and practical method to prepare 100 base pair DNA ladder by simple PCR using pCAMBIA 1301 plasmid as a template which is an effective cost reduction strategy for laboratories. pCAMBIA 1301 was transformed into Escherichia coli (Top 10) bacteria by using heat shock method for high the yield of the plasmid. Bacteria containing our desire plasmid were cultured and plasmid was extracted from bacteria by using kit method. About 10 pairs of primers were designed from the backbone of the plasmid which amplifies 100 to 1000 base pair of PCR product with an interval of 100 base pair fragments. These fragments were optimized by using gradient thermo cycler and PCR products were purified using kit methods. For the stability of 100 base pair DNA ladder, it was placed in seven different buffers.Results: The outcome of this study shown that polymerase chain reaction was able to amplify 10 different types of DNA fragments which ranges from 100 to 1000 base pair with high qualification and size accuracy. PCR products were purified and sequenced. DNA ladder was pooled in seven different buffers and stored at -20°C. These buffers were used to optimize and evaluate the stability of the prototype DNA ladder.Conclusion: Our laboratory made 100base pair DNA ladder is very cost effective, it only cost 11 USD to prepare DNA ladder. This 100 base pair DNA ladder provides an independent quantitative unit that can be used with any biological application or technology, enabling genomes to be measured using a common metric.Keywords: 100 bp DNA ladder, pCAMBIA 1301 plasmid; PCR technique; Gel electrophoresis; Break Even Point Analysis   

A brief review on molecular, genetic and imaging techniques for HCV fibrosis evaluation

<p>Abstract</p> <p>Background</p> <p>Chronic HCV is one of the major causes of morbidity and mortality in the present day world. The assessment of disease progression not only provides useful information for diagnosis and therapeutic supervision judgment but also for monitoring disease. Different invasive and non invasive methods are applied to diagnose the disease from initial to end stage (mild fibrosis to cirrhosis). Although, liver biopsy is still considered as gold standard to identify liver histological stages, an assessment of the disease development based on non-invasive clinical findings is also emerging and this may replace the need of biopsy in near future. This review gives brief insight on non-invasive methods currently available for predicting liver fibrosis in HCV with their current pros and cons to make easier for a clinician to choose better marker to assess liver fibrosis in HCV infected patients.</p> <p>Methods</p> <p>More than 200 studies regarding invasive and noninvasive markers available for HCV liver disease diagnosis were thoroughly reviewed. We examined year wise results of these markers based on their sensitivity, specificity, PPV, NPV and AUROCs.</p> <p>Results</p> <p>We found that in all non-invasive serum markers for HCV, FibroTest, Forn's Index, Fibrometer and HepaScore have high five-year predictive value but with low AUROCs (0.60~0.85) and are not comparable to liver biopsy (AUROC = 0.97). Even though from its beginning, Fibroscan is proved to be best with high AUROCs (> 0.90) in all studies, no single noninvasive marker is able to differentiate all fibrosis stages from end stage cirrhosis. Meanwhile, specific genetic markers may not only discriminate fibrotic and cirrhotic liver but also differentiate individual fibrosis stages.</p> <p>Conclusions</p> <p>There is a need of marker which accurately determines the stage based on simplest routine laboratory test. Genetic marker in combination of imaging technique may be the better non invasive diagnostic method in future.</p

Multiple interval mapping of QTLs and epistasis for iron toxicity tolerance in segregating population of Indica rice

The global average temperature has increased by approximately 0.5 °C, over a last few decades and is projected to continue to increase. Environmental stress factors such as, elevated temperature, salinity, toxic elements (Fe, Al, Cd, Cr, Pb, Zn and As), drought and rising CO2 affect plant growth and make a growing threat to agriculture. Rice is a primary food crop in the world and the establishment of rice crop in acidic soil and in marginal soil is a major goal for the improvement of rice production to fulfill the food security. Among environmental stresses, Fe2+ toxicity is one of the main stresses in limiting the cereal crops production. Tolerant rice genotypes that can tolerate the high concentration of Fe2+ toxicity are the potential source genes for rice tolerance improvement in Fe2+ toxicity. In this research work, the genetic basis of seed germination traits and growth traits was investigated in rice using (multiple interval mapping) MIM. Many rice genotypes serve as source of tolerant against toxic metal ion like Fe2+, could be an important factor in controlling the sever effect of Fe2+ toxicity on germination and seedling growth traits.  The F3 progenies of cross between Fe2+ toxicity tolerant cultivar ‘Pokkali’ and susceptible cultivar ‘Pak basmati’ were test against the optimized level of Fe2+ toxicity at germination, to determine the mode of inheritance to Fe2+ toxicity tolerance. Wide range of continues variation was found in F3 progenies. Among the 49 quantitative germination trait and 23 growth trait loci (QTLs) on chromosomes 1, 2, 4, 6, 8 and 9 linked with tolerance to Fe2+ toxicity was mapped. Additionally, 21 QTLs for germination traits and 9 QTLs for growth traits were classified as major QTLs using MIM. For germination and growth traits, notable epistasis between the chromosome 1, 2, 4, 6 and 11 was detected across germination and growth traits. Our results suggest that the tolerance mechanisms at germination and seedling phases could differ for Fe2+ toxicity. QTLs detected in this study for germination and seedling growth could be a source of new alleles for development of tolerance rice to Fe2+ toxicity varieties and transformation, gene cloning and gene editing in the futur

Zinc supplementation in male infertility

Background: Inferile males have been shown to have lower levels of seminal plasma zinc which have been associated with reduced levels of zinc in their blood. Supplementation improve semen parameters by improving zinc level in blood.  Objective: To fnd out whether zinc supplementation is effective in improving semen parameters in oligo­asthenozoospermic patients. Method: The study was carried out in the Infertility unit of the Dept of Obs & Gynae, BSMMU during the period of March 2011 to February 2012. Seventy five oligo-asthenozoospermic patients having no history of medical treatment were recruited for the study. The patients were divided into two groups by odd and even numbers. Odd numbers received tablet zinc 20 mg twice daily (Group A) and even numbers received placebo (Group B).Serum zinc level and seminal zinc level estimation were done by Graphite Furnace Atomic Absorption Spectropho­tometry and semen analysis was done according to WHO guidelines (1999). Data analysis was done using software SPSS (version 16) by applying ANOVA (PostHock) and Paired Student's 't' test. Results: Serum zinc level was low in oligo­zoospermic patients which showed significant improvement with zinc supplementation (A+ 197.83 mmol/1, P<0.01). Mean (±SD) seminal plasma zinc level showed significant improvement in group A following zinc supplementation (+942.39 mmol/L, P<0.001). The mean increase in sperm count, sperm motility, sperm rapid linear motility, sperm morphology in group A following zinc supplementation for 12 weeks was 14.83 million/ml (P<0.01), 16.30% (P<0.01), 11.96% (P<0.01), 4.26% (P<0.001) respectively, which was statistically significant. Conclusion: The study shows zinc deficiency affects sperm count, sperm motility, rapid linear motility and sperm morphology and with zinc supplementa­tion there can be significant improvement in semen parameters

Anti-apoptotic effect of HCV core gene of genotype 3a in Huh-7 cell line

<p>Abstract</p> <p>Background</p> <p>Hepatitis C virus (HCV) Core protein regulates multiple signaling pathways and alters cellular genes expression responsible for HCV induced pathogenesis leading to hepatocellular carcinoma (HCC). Prevalence of HCV genotype 3a associated HCC is higher in Pakistan as compare to the rest of world; however the molecular mechanism behind this is still unclear. This study has been designed to evaluate the effect of HCV core 3a on apoptosis and cell proliferation which are involved in HCC</p> <p>Methodology</p> <p>We examined the in vitro effect of HCV Core protein of genotype 3a and 1a on cellular genes involved in apoptosis by Real time PCR in liver cell line (Huh-7). We analyzed the effect of HCV core of genotype 1a and 3a on cell proliferation by MTT assay and on phosphrylation of Akt by western blotting in Huh-7 cells.</p> <p>Results</p> <p>The HCV 3a Core down regulates the gene expression of Caspases (3, 8, 9 and 10), Cyto C and p53 which are involved in apoptosis. Moreover, HCV 3a Core gene showed stronger effect in regulating protein level of p-Akt as compared to HCV 1a Core accompanied by enhanced cell proliferation in Huh-7 cell line.</p> <p>Conclusion</p> <p>From the current study it has been concluded that reduced expression of cellular genes involved in apoptosis, increased p-Akt (cell survival gene) and enhanced cell proliferation in response to HCV 3a core confirms anti apoptotic effect of HCV 3a Core gene in Huh-7 that may lead to HCC.</p

A comparison of four fibrosis indexes in chronic HCV: Development of new fibrosis-cirrhosis index (FCI)

<p>Abstract</p> <p>Background</p> <p>Hepatitis C can lead to liver fibrosis and cirrhosis. We compared readily available non-invasive fibrosis indexes for the fibrosis progression discrimination to find a better combination of existing non-invasive markers.</p> <p>Methods</p> <p>We studied 157 HCV infected patients who underwent liver biopsy. In order to differentiate HCV fibrosis progression, readily available AAR, APRI, FI and FIB-4 serum indexes were tested in the patients. We derived a new fibrosis-cirrhosis index (FCI) comprised of ALP, bilirubin, serum albumin and platelet count. FCI = [(ALP × Bilirubin) / (Albumin × Platelet count)].</p> <p>Results</p> <p>Already established serum indexes AAR, APRI, FI and FIB-4 were able to stage liver fibrosis with correlation coefficient indexes 0.130, 0.444, 0.578 and 0.494, respectively. Our new fibrosis cirrhosis index FCI significantly correlated with the histological fibrosis stages F0-F1, F2-F3 and F4 (r = 0.818, p < 0.05) with AUROCs 0.932 and 0.996, respectively. The sensitivity and PPV of FCI at a cutoff value < 0.130 for predicting fibrosis stage F0-F1 was 81% and 82%, respectively with AUROC 0.932. Corresponding value of FCI at a cutoff value ≥1.25 for the prediction of cirrhosis was 86% and 100%.</p> <p>Conclusions</p> <p>The fibrosis-cirrhosis index (FCI) accurately predicted fibrosis stages in HCV infected patients and seems more efficient than frequently used serum indexes.</p