Antiviral Therapy for Hepatitis C Virus (HCV): Black Mustard Seeds
Hepatitis C Virus (HCV) is an RNA virus, which is considered the main cause of progressive chronic liver disease, cirrhosis, and hepatocellular carcinoma (HCC) worldwide. The number of the patients who are infected with this sleeping virus is increasing rapidly every year, as the unsuitability of the current therapy – interferon α and ribavirin – for most of the genotypes is the main cause of these high rates. Hence, the recent researches are focusing on finding out a new immunotherapy to affect this virus. In this research work, Black Mustard (Brassica nigra) has been used as powdered spice samples to prepare aqueous extracts; One of the included phytochemicals in the black mustard; glucosinolates and their hydrolysis products, was proposed to be used for the HCV patients to prevent the virus progression. Also, the Isothiocyanates are shown with chemotherapeutic and anti-tumor properties. Moreover, some of the structure-related isothiocyanates have the ability to induce the enzyme paraoxonase-1 (PON-1) that is considered hepato-protective agent against liver impairment, inflammation, fibrosis and liver disease mediated by monocyte chemoattractant protein-1 (MCP-1), and is thought to affect the entry of the virus into the hepatocytes. The effect of the black mustard and the produced myrosinase enzyme on the HCV RNA replication is still unknown. In conclusion, the black mustard is thought to affect the progression and the fluidity of the HCV envelope resulting in impairment of viral binding and fusion.
An investigation of the inhibitory potential of Dronedarone on CYP2J2 mediated astemizole metabolism
Dronedarone is an anti-arrhythmic drug approved in 2009 for paroxysmal and persistent atrial fibrillation. It is less toxic than its predecessor Amiodarone as it does not cause systemic toxicity but has the same pharmacological activity. However the administration of dronedarone to permanent AF and heart failure patients leads to increased risk of stroke and cardiac death. The exact mechanism of the toxicity is currently unknown. Extrahepatic Cytochrome P450 enzymes play a dominant role in organ-specific drug metabolism and toxicity. Cytochrome P450 2J2 (CYP2J2) enzyme, a predominant enzyme found in human cardiac myocytes, metabolizes endogenous arachidonic acid (AA) into epoxyeicosatrienoic acids (EETs) which play an important role in maintaining normal cardiac physiology. Inhibition of CYP2J2 and perturbation of AA metabolic pathway could result in exacerbation of cardiac failure. This research aims to find out whether dronedarone inhibits CYP2J2 in a suitable cell model (H9C2) using astemizole as a probe substrate. Our in-house studies using recombinant CYP2J2 enzyme have shown that dronedarone potently inhibits CYP2J2. Rat myoblast cells (H9C2) will be seeded in 12-well plate and differentiated for 4 days. The cells will be then treated with different concentrations of astemizole and incubated for 24 h. The cells will then be harvested, lysed, and the cell lysate will be analyzed using liquid chromatography-mass spectrometry (LCMS). Using multi-reaction monitoring (MRM) on the LCMS, astemizole concentration as well as its CYP2J2-specific metabolite O-desmethylastemizole concentrations will be measured. The presence of O-desmethylastemizole confirms the metabolism of astemizole by CYP2J2 in H9C2 cells. By plotting a Michaelis-Menten kinetics curve, we will be able to determine the Michaelis constant (KM) and maximum rate of reaction (Vmax). H9C2 cells will be then treated with fixed concentration of astemizole while varying the dronedarone concentration. A decrease in metabolite O-desmethylastemizole conce ntration, indicates inhibition of CYP2J2 metabolism by dronedarone. Using this data, Lineweaver-Burke graph will be plotted, to determine the mode and potency of the inhibition. Our preliminary studies showed that the KM value was 2.7μM. This study will be useful in understanding if dronedarone inhibits CYP2J2 which may lead to clinically significant drug-drug interactions, one of the dangers of polypharmacy. Finally this study will shed a new light on the mechanisms for dronedarone mediated cardiac failure exacerbation.
Understanding the Modern Diagnoses of Protein C Deficiency "Pcd" with Unknown Gene Plays a Critical Role in the Inherited Thrombophilia
Protein C deficiency (PCD) is found in 1 out of 200 to 500 persons in the general global population which is also one of the common conditions of Inherited thrombophilia, it’s characterized by an increased tendency of blood to clot in human blood vessels. It is caused by several factors including mutations in the genes involved in thrombin binding, protein c activation and numerous clotting factors. This includes F5 (Factor 5 Leiden) gene on chromosome 1q24.2, F7 (Prothrombin) gene on chromosome 13q34, SERPINC1 (serpin peptidase inhibitor C) on chromosome 1q25.2, SERPIND1 (serpin peptidase inhibitor D) on chromosome 22q11.21, HRG (Histidine Rich Glycoprotein) on chromosome 3q27.3, PLAT (Plasminogen Activator) on chromosome 8q11.21 and THBD (Thrombomodulin) gene on chromosome 20p11.21. In the current study, a three Saudi families with inherited thrombophilia has been recruited to identify the underlying cause of this special condition. Whole exome sequencing, targeting all coding exons of the human genome, was performed using Illumina Nextera library preparation kits followed by paired-end sequencing on Illumina NextSeq500 instrument. Reads quality control was performed and reads were aligned to the reference genome using BWA software. Variants calling and annotation was performed using GATK. All known genes involved in causing inherited thrombophilia All known genes involved in causing PCD were excluded by whole exome sequencing. The genes that were previously reported to be involved in inherited thrombophilia were checked for any causative variant. No mutation has been identified in known genes. identifying a novel gene underlying PCD. The Result of this study will hopefully pave the way to better understanding the disease pathophysiology and help in developing DNA based diagnosis, carrier screening and somatic gene therapy.
Novel Approach to Screening Mutations Causing Retinoblastoma, a Childhood Cancer of Retina
Retinoblastoma (RB) is a childhood retinal cancer caused by mutations in the RB1 gene. Molecular diagnosis is crucial for early detection and treatment. Current DNA diagnostic screening requires substantial amounts of tumour and blood samples. However current screening methods face the challenges of limited DNA templates from minute retinal tumours and too much blood samples drawn from young patients. In addition, the starting DNA template amount and quality are important to ensure confident detection of disease-causing mutations. As the majority of RB1 mutations are unique and distributed throughout the RB1 gene with no real hot spots, the entire gene needs to be thoroughly analysed. This investigation proposes to enrich DNA samples using a whole genome amplification (WGA) step prior to RB1 mutation screening by RB1 gene-specific PCR amplification as well as high resolution melt (HRM) analysis and sequencing. It also identifies RB1 mutations in two RB patients and explores whether WGA and saliva products can be a source of DNA templates for RB1 analysis. In addition, this study was conducted based on the hypotheses that RB1 mutations were the underlying cause of the disease in the two patients, and that the products from WGA could be used specifically for RB1 gene analysis to overcome the constraint of insufficient DNA samples. Two anonymised genomic DNA samples from two unrelated RB patients and five normal healthy DNA samples were used in this project. WGA kits were compared according to three criteria, namely amplification yield, product fragment size and whether DNA is amplifiable. Prior to and after amplification, the optical density of two normal samples was measured to determine the increase in DNA yield. The amplicons were subjected to gel electrophoresis to determine the product fragment size. Exons 6, 14 and 25 of the original and amplified samples undergone PCR, and were examined again using gel electrophoresis to ascertain that the amplicons were amplifiable. Mutation analysis using HRM was carried out with pre-existing primers for all 27 exons and the promoter of RB1. Samples from patients were analysed against 83 saliva DNAs extracted using Oragene•DNA (OG-500) Kit. REPLI-g was observed to produce higher yield and products of reliable fragment size. Single distinct bands were also seen for exons amplified using REPLI-g, indicating that REPLI-g is more accurate and suitable in the amplification of DNA. Abnormal melt profiles were obtained for exon 6 in RB477 and exon 14 in RB572 for HRM. These exons were sequenced to determine the exact mutation. Exon 6 was found to have a splice-site mutation g.607+1G>T, while a point mutation, g.1363C>T (p.Arg455X) was identified in exon 14. Both the uses of saliva as a non-invasive DNA source and the WGA approach for enriching DNA sample for application in RB1 gene analysis have never been reported for RB. Although HRM analysis has been used for other diseases, this is its first instance applied in work on RB1 gene. In short, this report offers novel and promising approaches which would contribute significantly to the molecular analysis of mutations in RB.
Investigating Novel Methods to Reduce Cholesterol Levels
An increase in blood cholesterol contributes to cardiovascular diseases, the number one cause of death worldwide. Statins are currently the most effective in reducing cholesterol levels and treating patients with high cholesterol. However, these pharmaceutical agents have been shown to cause several side effects, prompting the need for a more natural solution to increasing cholesterol levels. Hence, a study was conducted to investigate the ability of lactic acid bacteria in the removal of cholesterol, explore the mechanism for the removal of cholesterol by lactic acid bacteria, and examine the effectiveness of kidney beans and sunflower seeds in inhibiting HMG-CoA reductase in the cholesterol biosynthesis pathway. Results showed that Lactobacillus plantarum was the most effective in reducing cholesterol levels and that the mechanism for cholesterol removal included both the binding to cell wall and active uptake into cells. Sunflower seeds and kidney beans were also shown to be effective in inhibiting HMG-CoA reductase, with sunflower seeds having 100% inhibition of the enzyme, similar to pravastatin, a commercial cholesterol reducing drug, and kidney beans having comparable percentage inhibition of the enzyme compared to pravastatin.
Remedies Recovered from Roof Top Resources
Moss from a roof top was used to treat ear infections in my grandfather’s village. This remedy sparked my curiosity and so I began researching. I was bewildered to discover that the resistance to antibiotics has been labelled as a “Catastrophic Threat” and has been ranked in the same category as terrorism and climate change. Governments globally are urging scientists to identify and produce new antibiotics and reassess novel approaches1. This project aims to evaluate two objectives through the use of several integrated technologies and modified methods: (a) To determine if the extracts, solutions and raw materials derived from Heart wood portion of Picea glauca, Populus tremuloides, Salix spp, Betula papyrifera, Pinus contorta, Quercus alba, Thuja occidentalis, Climacium dendroides, Dicranum fuscescens and Kieselgur, will inhibit the growth of Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, and Escherichia coli. (b) To scientifically reassess my grandfather’s traditional method of treating ear infections using roof moss. The Heartwood portion of each tree was removed using a hammer and mallet. The Heartwood was then burnt to derive the ash and a miter saw was used to make sawdust. The moss was collected, dried and labelled. A Methanol Extraction was performed on all saw dust samples and moss using a Soxhlet Extractor for 24 hours. The ash solutions were diluted, filtered, and neutralized to pH 7. The solvents were evaporated in a Rotary evaporator and the residual material was stored in round bottom flasks. The Kirby Bauer method was modified and a Well Infusion method was devised for the biological assay. The Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, and Escherichia coli were plated using a 0.5 McFarland Standard. Paper filter discs containing 20uL of each extract solution and raw material were precisely placed onto the inoculated plates and incubated for 24 hours. The preliminary results were initially unfavourable, as data could only be collected and analysed for one species; Thuja occidentalis (White Cedar). However, these results were extremely encouraging when the zones of inhibition were measured and analyzed. Confidence Intervals were calculated at 95% and the T-Tests were calculated at a 0.05 alpha level, which indicated significance when compared to the control. The Chi Square values were greater than the critical value of 7.8 and therefore the thorough statistical analysis indicates that the results were not due to chance alone. Literature has indicated that certain components of trees do indeed have antibacterial properties, however there is very limited research specific to the Heartwood portion. Furthermore, I discovered that the Heartwood portion of the White Cedar tree does have certain antibacterial properties that definitely justify further testing. In addition, a combination of examining my grandfather’s possessions and analyzing present data, I can confidently support my grandfather’s traditional method. In conclusion, the use of the Heartwood portion of the White Cedar to combat bacterial infection warrants further exploration. Remedies Recovered from Roof Top Resources may be the solution to this catastrophic threat.
BA-ADA based ROS-responsive nanoparticles for selective drug delivery in cancer cells
Current medical intervention in cancer therapeutic methods has shown risks and side effects with normal tissues. This includes incomplete cancer eradication. In reference to numerous studies and literature reviews, a stimuli-responsive drug delivery system is selected as an innovative, safe and more assured treatment due to its site-specific release ability. This allows specific intervention upon the given stimulus which response to the presenting disease symptoms. Hence, we designed a ROS(Reactive Oxygen Species)-responsive BA-ADA(4-Hydroxyphenylboronic acid pinacol ester and 1-Adamantanecarboxylic acid bonded molecule) nanoparticle delivery system. In our study, ROS-responsive nanoparticle was designed and prepared based on a synthetic molecule from BA and ADA. A therapeutic payload, Doxorubicin, can be loaded into the nanoparticles and it can be selectively released within cancerous tissues whereby ROS level is over-expressed. This will enhance both therapeutic efficiency and reduce side effects. The stability and ROS-responsiveness of the particle were proven in a series of evidence-based experiments. The results showed a significant difference in cell viability during the experiments with healthy and cancerous cell samples. Further research will be required to extend the experiment in vivo.