Department of Biochemistry, Cell and Molecular Biology
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Item Anti-Diabetic And Probiotic Effect Of Kombucha On Alloxan-Induced Diabetic Rats(2019-07) Adade, E.E.Diabetes mellitus, is a metabolic disorder caused by the inability of the beta pancreatic cells to adequately produce insulin or due to insulin resistance of cells. As a result of the increasingly high incidence of diabetes globally, the World Health Organization (WHO) has set timelines and guidelines for the reduction of the risk of mortalities and morbidities associated with non-communicable diseases including diabetes, by the year 2030. However, this agenda is hinged on the availability of affordable, safe and effective alternatives for the management and treatment of these diseases. Hence, there is a need to explore other alternatives to the conventional oral anti-hyperglycemic agents driven by factors such as patient’s preference, demand among others. Kombucha is tea fermented by a symbiotic culture of bacteria and yeasts (SCOBY). Consumers of Kombucha have reported several anecdotal evidences of its medicinal potential. This study seeks to investigate its anti-diabetic and probiotic effect on alloxan-induced diabetic rats. It was hypothesized that Kombucha, being a complex matrix of microorganisms and nutraceuticals, would play an essential role in the management of diabetes. Molecular characterization of the microbiome of Kombucha using shotgun metagenomics (Oxford Nanopore MINION sequencing technology) showed Brettanomyces bruxellensis CBS 2499 as the most abundant species within the microbial community accounting for about 51 % of all reads. Brettanomyces anomalus, Komagataeibacter xylinus NBRC 15237, Bacillus nealsonii AAU1, Zygosaccharomyces bailii CLIB 213, Acetobacter, Gluconobacter and over 300 other genera and species of microorganisms including archaea and viruses were also detected using a combination of REFSEQ and One Codex data bases (OXCDB). In-vivo experiment was used to evaluate the anti-diabetic property, safety and gut microbiome changes of Kombucha. Kombucha was found to perform better than the conventional antidiabetic drugs, metformin and glibenclamide in lowering the fasting blood glucose (FBG) of the diabetic rats. Daily administration of 25 mg/kg and 100 mg/kg of freeze-dried Kombucha tea demonstrated a 5 fold reduction in FBG (p<0.05) and 40% and 50% respective increases in body weight of the alloxan-induced diabetic rats compared to the diabetic control (DC). Histological analysis, shows Kombucha enhances pancreas regeneration and hence the concomitant increase in insulin secretion as demonstrated in the study. Serum lipid profiling showed 100mg/kg Kombucha treatment increases the levels of total cholesterol (16%), high density lipoproteins (HDL) (13%) and low-density lipoproteins (LDL) (10%) but conversely reduces triglyceride level (17%) compared to the DC (p>0.05). Further analyses demonstrated that Kombucha decreases the relative organ (liver and kidney) to body weight ratio in treated animals. In addition, Kombucha was able to reduce significantly the elevated levels of liver enzymes such as Alkaline phosphatase (ALP), Alanine transaminase (ALT) and Aspartate Aminotransferase (AST) as well as renal toxicity indices, creatinine and urea in treated animals. Histology of the kidney and liver also showed that Kombucha has no adverse effect on the morphology and cellular integrity of these organs suggesting its hepatoprotective and renal protective potentials. Urinanalysis also showed reduction of glucose in urine for the 100 mg/kg Kombucha-treated animals. Additionally, Kombucha protects the gut microbiome, most significantly by enhancing the Lactobaccillaceae family of bacteria within the gut and reduces the possibilities of colonization of the gut by other opportunistic bacterial species. The study demonstrated that Kombucha is enriched with diverse microbial population with probiotic value and daily intake of Kombucha may be potentially helpful in the management of diabetes, protection against renal and liver toxicity and offer gut microbiome protection.Item Correlates of Virus Release in HIV-1(University of Ghana, 2019-03) Kissi-Twum, A.A.Integration of HIV-1 within the host genome may result in latent infection, where a replication competent provirus remains in a state of transcriptional silence. The latent reservoir remains the major barrier to cure efforts as to achieve cure, the reservoir should be depleted of all replication competent proviruses. Various assays to measure the latent reservoir rely on virus release and vary widely in their estimations. Quantitative viral outgrowth assay (QVOA) which is the gold standard relies on reactivation of latent proviruses to release virions and thus falls short when proviruses are non-inducible. The correlates of virus release per cell were examined in this study. The TZ-5 zipcoded library which is a Jurkat cell line with one uniquely tagged HIV-1 integrant per cell was used in this study. The library consisted of infected cells expressing HIV-1 gag and pol with an inactivated env gene and vpr-. GFP is expressed as a Nef spliced product to indicate HIV-1 expression. Intracellular Gag staining coupled with the relative amounts of p24 detected in culture supernatant showed GFP expression is an accurate proxy for HIV-1 expression. High-throughput sequencing was carried out on PCR amplified zipcodes. The topmost 74 clones which represents ≥ 97% of the raw sequence reads was analysed in this study. Although cell proliferation is thought to drive HIV-1 persistence, clonal abundance was not found to correlate with high virus release. Rather a higher GFP+ expression as determined by the green proportion correlates with high virus release per cell. Spliced and unspliced mRNA products are needed to productively assemble virions in HIV-1 although Gag which is an unspliced product is the driving force. Clones with higher amounts of unspliced product to spliced had higher green proportions and subsequently overlapped with higher amounts of virus released. Taken together, higher green proportions and unspliced RNA may predict a higher amount of virus released per cell. As green proportion is a measure of overall HIV-1 expression by a clone, incorporating the extent of HIV-1 expression in QVOA measurements will calibrate HIV-1 expression among inducible and noninducible cells in the latent pool and ultimately the decay rate of such clones. The frequency of formation of host-virus read-through and read-in chimeras and packaging into virions was examined as these unusual species can be helpful in integration site determination and ultimately determine genetic correlates of chimeric RNA generation. RNase protection assays showed the presence of chimeric read-through and read-in RNAs both in cells and virus. The genomic context of integration may contribute to our observed findings as more read-in RNA was observed to be produced and packaged into virions than read-through RNA.Item Molecular Subtyping and Gene Expression Analysis of Drug Resistant Invasive Non-Typhoidal Salmonella(University of Ghana, 2019-07) Adjei, R.O.Invasive Non-Typhoidal Salmonella infections have become widespread in sub-Saharan Africa. Predominant hyper-virulent clone S. Typhimurium ST313 is found to be associated with bloodstream invasion and multidrug resistance. Despite its importance, limited information is available on the circulating genotypes causing invasive non-typhoidal salmonellosis in Ghana. A total of fifty-one NTS isolates from pediatric blood samples was used for this study. The resistance profiles of isolates were assessed against 8 classes of antibiotics by disc diffusion method followed by PCR detection of resistance markers. Molecular detection of the presence of a virulence associated gene, st313-td, was used as plausive test to select isolates for Multi-Locus Sequence Typing (MLST) analysis. Additionally, the mechanism of ciprofloxacin resistance was explored by monitoring changes in expression levels of important targeted genes including gyrA, acrA, ydhJ, emrD, and soxR under drug pressure using reverse transcription quantitative PCR. Antibiotic susceptibility testing results showed all isolates were completely susceptible to imipenem, ceftriaxone and ceftazidime. In most cases, multidrug resistance (including reduced susceptibility to fluoroquinolones) were frequently recorded among S. Typhimurium isolates (63%, n=32/51). On the contrary, nine out of ten S. Dublin and the five unknown serovars were susceptible to all antibiotics used for screening. The virulence gene st313-td was detected in only 27% (n=14/51) isolates. Eight (8) other isolates showing varying resistance phenotypes were selected in addition to the 14 positive isolates for typing and analyses by MLST. Out of twenty-two (22) selected isolates, 82% (n=18), 4% (n=1) and 14% (n=3) were S. Typhimurium ST313, S. Typhimurium ST19, and S. Dublin ST 10 respectively. Most importantly, it was observed that about 83% (n=15/18) of S. Typhimurium ST313 clones were multidrug resistant (MDR). Likewise, ten selected isolates for PCR analyses tested positive for the resistance markers; CatA1 (30%), StrA (90%), BlaTem (50%), TetA (30%) & TetB (10%), Sul1 (80%) & Sul2 (100%) and aadA (70%). The normalized transformed mRNA transcript levels showed significant variations in gene expression of gyrA, acrA, ydhJ, emrD and soxR among susceptible, intermediate and laboratory induced resistant isolates. Further, sequence data analysis detected a single nucleotide polymorphism at position 324 resulting in a mutation (Phe → Asp) in the gyr A of clinical intermediate isolate, although there were no significant mutations in acrA, ydhJ, emrD and soxR genes among the three test isolates. From the results, high frequencies of multidrug resistant invasive NTS pathogens carrying plasmid-borne resistance gene markers were found. Also, reduced susceptibility to fluoroquinolones and cephalosporin were observed which calls for increased surveillance and management of antimicrobial resistance to guide treatment. Detection of a SNP in gyrA gene and variations in mRNA levels suggest possible genetic and transcriptional regulation mechanisms employed by Salmonella pathogen to withstand drug stress. Therefore, further investigations are required to understand these mechanisms which will assist in developing alternative therapeutic interventions.Item Detection and Characterization of Epstein Barr Virus in Nasopharyngeal Cancers in Ghana(University of Ghana, 2019-07) Ayee, R.Purpose: Nasopharyngeal cancer (NPC) is a malignant tumor which is commonly associated with Epstein Barr virus (EBV). Two genotypes of EBV, genotype 1 and genotype 2, which are geographically restricted, are implicated in the pathogenesis of NPC. This study aimed at detecting and characterizing EBV genotypes involved in the pathogenesis of NPC in Ghana. Experimental design: Whole blood and biopsy samples were collected from 55 patients diagnosed of NPC by CT scan and endoscopy at the Ear, Nose and Throat Unit (ENT) of the Korle-Bu Teaching Hospital (KBTH). As control subjects, whole blood was collected from 53 individuals confirmed NPC negative or without known oncological diseases by CT scan and endoscopy. Association of NPC with risk factors such as gender, alcohol intake, smoking and consumption of salted fish, was evaluated. EBV was detected by amplification of Epstein Barr nuclear antigen 1 (EBNA-1) and genotyping by amplification of Epstein Barr nuclear antigen 2 (EBNA-2) using primers specific to genotypes 1 or 2. Viral load in blood and biopsy specimen was quantified using real-time polymerase chain reaction (PCR). Results: Risk factors such as gender, consumption of alcohol and smoking were not significantly (p > 0.05) associated with NPC development. There was a significant association (p< 0.05) of consumption of salted fish with NPC development. The frequencies of EBV positivity were 67%, 67% and 92% in NPC blood, NPC biopsies, and control blood samples, respectively. The predominant EBV genotype in blood specimen from cases was EBV genotype 2 (52%) and that of the control group was type 1 (62%). Statistically significant difference (χ2= 72.26, p = 0.001) was observed for the frequency of EBV genotypes in the NPC blood and controls. In biopsy specimen, the predominant EBV genotype was genotype 1 (58%). Median viral load (9×107 copies/ mL) in whole blood of NPC case subjects was significantly higher than median viral load (6×104 copies/ mL) in the whole blood of control subjects (p=0.001). Significantly higher (p < 0.001) median EBV DNA load (9×107 copies/ mL) was observed in NPC whole blood samples compared to that of NPC tumor biopsies (1.58×105 copies/mL). The median viral load of case samples infected with EBV genotype 1 was significantly higher (p = 0.001) than control samples infected with the same EBV genotype (genotype 1 median viral load in cases, 2 ×107 copies/ mL verses 29 ×103 copies/ mL genotype 1 median viral load in controls). Significantly higher (p = 0.001) median viral load was observed in cases infected with EBV genotype 2 when compared to control samples infected with the same EBV genotype (1×108 copies/ mL compared to 18 ×103 copies/ mL in cases and controls, respectively). Conclusions: In summary, gender, consumption of alcohol and smoking were not associated with NPC development in the current study whereas association was established between NPC and salted fish consumption. The frequency of EBV was higher in control subjects than NPC patients, confirming reports suggesting large number of the world’s population being asymptomatic carriers. Gender was not a risk factor of EBV infection in this study. This study identified EBV genotype 2 infection as the virulent genotype in Ghana and a possible risk factor in the development of NPC in Ghanaian patients. EBV genotype 1 was predominant in the control group and consistent with the genotype being relatively less virulent. Detection and quantification of EBV load can be used as a non-invasive biomarker for diagnosis of NPC.Item Characterizing Pathogenic Risk Factors Associated with Breast Cancer in Ghanaian Women(University of Ghana, 2019-07) Buckman, M.A.Globally, the second leading cause of cancer death in women is breast cancer. It is responsible for 16% of all cancer cases and the most common cancer in Ghana. Breast cancer pathogenesis has not been fully elucidated but is however known to have a complex aetiology involving both genetic and environmental factors. Pathogens are one of the environmental factors that have been linked with breast cancer development. Epstein-Barr Virus (EBV), Human Papilloma Virus (HPV) and Mouse Mammary Tumour virus (MMTV) have been implicated in breast cancer. Research has showed that the local microbiome of the host could modulate breast cancer risk, yet it is unknown what microbes (pathogenic or probiotic) inhabit breast tumour tissues in Ghana. The objectives of this study were to detect the presence of HPV, EBV, MMTV and bacteria in breast tumour tissues from Ghanaian women and determine the effect of bacteria on DNA in HeLa and MCF-7 cells. Formalin-Fixed Paraffin-Embedded (FFPE) tissues and fresh breast cancer tissues were obtained from 204 breast cancer patients at the Department of Surgery, Korle-bu Teaching Hospital. Nucleic acid was extracted from the samples and amplified using standard Polymerase Chain Reaction (PCR) to detect the viruses. HPV-positive tumours were sequenced using the Sanger sequencing method. EBV typing of EBV positive samples was done by a nested PCR reaction using EBV-1 and EBV-2 specific primers. Bacteria from fresh breast cancer tissues were obtained and identified using basic biochemical tests and Sanger sequencing. The DNA damage potential of the isolates was tested on HeLa and MCF-7 cells using the histone-2AX phosphorylation assay. HPV was detected in 27 (13.2%) of the 204 cases analyzed. EBV was identified in 66 (32.4%) of the samples. Out of the EBV positive cases, 2 (1%) were positive for EBV-1, 30 (14.7%) were positive for EBV-2, 26 (12.7%) were positive for both EBV-1 and EBV-2 and 8 (3.9%) could not be genotyped using available methods. Co-infection of both HPV and EBV was detected in 11 (5.4%) of the cases. MMTV was not detected in any of the samples analyzed. Microbiome analysis showed relatively high evidence of bacteria belonging to the Staphylococcus and Bacillus species. Staphylococcus sciuri, Staphylococcus epidermidis, Staphylococcus lugdunensis obtained from breast tumours induced DNA double-strand breaks in MCF-7 cells. The diversity and the probable role of the microbiome in breast cancer were also identified. Therefore, the presence of these pathogens showed probable involvement in breast cancer carcinogenesis.Item Hepatitis Delta Virus (HDV) in HIV/HBV Co-Infected Patients in Ghana(University Of Ghana, 2019-07) Attiku, K.O.Within persons infected with HIV worldwide, almost 10% have chronic HBV infection, and about 6% of all HBV infected patients are co-/superinfected with HDV, resulting in accelerated progression of hepatitis towards end stage liver disease. Even though within HIV/HBV infected patients, an increase in HDV infection has been observed, there is inadequate information on HDV prevalence as well as virologic profile in Ghana. This study sought to determine the presence of HDV in HIV/HBV co-infected patients in Ghana. This was a longitudinal purposive study which enrolled 113 patients, who have been co-infected with HIV and HBV, attending clinic at Korle-Bu Teaching Hospital (KBTH) in Ghana. Patient demographic information was obtained using a questionnaire, and 5 mL whole blood collected at two-time points (baseline and 4-6 months afterwards). The sera obtained were tested to confirm the presence of HIV, HBV antibodies/antigens and HBV DNA. Antibodies and viral RNA were also determined for HDV with respective serological and molecular assays. Amplified HBV DNA and HDV RNA were sequenced with the Sanger method and sequence reads analyzed using Sequencher software version 2.3. Phylogenetic analysis was carried out with references from the GenBank to establish the genotypes using the MEGA X software. Out of 113 samples tested, 63 (55.7%) were females and 50 (44.25%) were males. Patients enrolled in this study were within the age range of 24 to 73 years, with a median age of 45 years. A total of 100 (88.5%) samples had detectable HBV surface antigen (HBsAg), and 32/113 had detectable HBV DNA. Nucleotide sequences were obtained for 15 samples, and phylogenetic analysis showed the circulating strains to be HBV genotype E. Of 13 samples that were HBsAg unreactive, 4 (30.8%) had detectable HBV DNA and suggest the presence of an occult infection. HDV was detected in four samples and giving a prevalence of 3.54% in the HIV/HBV cohort used for the study. Two of the HDV positive samples sequenced were HDV genotype 1. In summary, the data suggest the presence and circulation of HDV and occult HBV infection in HIV patients co-infected with HBV in Ghana. The occurrence of occult HBV infection and HDV in the study population is supported by other research studies, and makes it imperative to look out for the presence of HDV and occult HBV in patients co-infected with HIV/HBV.Item Anti-Diabetic and Probiotic Effect of Kombucha on Alloxan-Induced Diabetic Rats(University of Ghana, 2019-07) Adade, E.E.Diabetes mellitus, is a metabolic disorder caused by the inability of the beta pancreatic cells to adequately produce insulin or due to insulin resistance of cells. As a result of the increasingly high incidence of diabetes globally, the World Health Organization (WHO) has set timelines and guidelines for the reduction of the risk of mortalities and morbidities associated with non-communicable diseases including diabetes, by the year 2030. However, this agenda is hinged on the availability of affordable, safe and effective alternatives for the management and treatment of these diseases. Hence, there is a need to explore other alternatives to the conventional oral anti-hyperglycemic agents driven by factors such as patient’s preference, demand among others. Kombucha is tea fermented by a symbiotic culture of bacteria and yeasts (SCOBY). Consumers of Kombucha have reported several anecdotal evidences of its medicinal potential. This study seeks to investigate its anti-diabetic and probiotic effect on alloxan-induced diabetic rats. It was hypothesized that Kombucha, being a complex matrix of microorganisms and nutraceuticals, would play an essential role in the management of diabetes. Molecular characterization of the microbiome of Kombucha using shotgun metagenomics (Oxford Nanopore MINION sequencing technology) showed Brettanomyces bruxellensis CBS 2499 as the most abundant species within the microbial community accounting for about 51 % of all reads. Brettanomyces anomalus, Komagataeibacter xylinus NBRC 15237, Bacillus nealsonii AAU1, Zygosaccharomyces bailii CLIB 213, Acetobacter, Gluconobacter and over 300 other genera and species of microorganisms including archaea and viruses were also detected using a combination of REFSEQ and One Codex data bases (OXCDB). In-vivo experiment was used to evaluate the anti-diabetic property, safety and gut microbiome changes of Kombucha. perform better than the conventional antidiabetic drugs, metformin and glibenclamide in lowering the fasting blood glucose (FBG) of the diabetic rats. Daily administration of 25 mg/kg and 100 mg/kg of freeze-dried Kombucha tea demonstrated a 5 fold reduction in FBG (p<0.05) and 40% and 50% respective increases in body weight of the alloxan-induced diabetic rats compared to the diabetic control (DC). Histological analysis, shows Kombucha enhances pancreas regeneration and hence the concomitant increase in insulin secretion as demonstrated in the study. Serum lipid profiling showed 100mg/kg Kombucha treatment increases the levels of total cholesterol (16%), high density lipoproteins (HDL) (13%) and low-density lipoproteins (LDL) (10%) but conversely reduces triglyceride level (17%) compared to the DC (p>0.05). Further analyses demonstrated that Kombucha decreases the relative organ (liver and kidney) to body weight ratio in treated animals. In addition, Kombucha was able to reduce significantly the elevated levels of liver enzymes such as Alkaline phosphatase (ALP), Alanine transaminase (ALT) and Aspartate Aminotransferase (AST) as well as renal toxicity indices, creatinine and urea in treated animals. Histology of the kidney and liver also showed that Kombucha has no adverse effect on the morphology and cellular integrity of these organs suggesting its hepatoprotective and renal protective potentials. Urinanalysis also showed reduction of glucose in urine for the 100 mg/kg Kombucha-treated animals. Additionally, Kombucha protects the gut microbiome, most significantly by enhancing the Lactobaccillaceae family of bacteria within the gut and reduces the possibilities of colonization of the gut by other opportunistic bacterial species. The study demonstrated that Kombucha is enriched with diverse microbial population with probiotic value and daily intake of Kombucha may be potentially helpful in the management of diabetes, protection against renal and liver toxicity and offer gut microbiome protection.Item Anti-Diabetic and Probiotic Effect of Kombucha on Alloxan-Induced Diabetic(University of Ghana, 2019-07) Adade, E.E.Diabetes mellitus, is a metabolic disorder caused by the inability of the beta pancreatic cells to adequately produce insulin or due to insulin resistance of cells. As a result of the increasingly high incidence of diabetes globally, the World Health Organization (WHO) has set timelines and guidelines for the reduction of the risk of mortalities and morbidities associated with non-communicable diseases including diabetes, by the year 2030. However, this agenda is hinged on the availability of affordable, safe and effective alternatives for the management and treatment of these diseases. Hence, there is a need to explore other alternatives to the conventional oral anti-hyperglycemic agents driven by factors such as patient’s preference, demand among others. Kombucha is tea fermented by a symbiotic culture of bacteria and yeasts (SCOBY). Consumers of Kombucha have reported several anecdotal evidences of its medicinal potential. This study seeks to investigate its anti-diabetic and probiotic effect on alloxan-induced diabetic rats. It was hypothesized that Kombucha, being a complex matrix of microorganisms and nutraceuticals, would play an essential role in the management of diabetes. Molecular characterization of the microbiome of Kombucha using shotgun metagenomics (Oxford Nanopore MINION sequencing technology) showed Brettanomyces bruxellensis CBS 2499 as the most abundant species within the microbial community accounting for about 51 % of all reads. Brettanomyces anomalus, Komagataeibacter xylinus NBRC 15237, Bacillus nealsonii AAU1, Zygosaccharomyces bailii CLIB 213, Acetobacter, Gluconobacter and over 300 other genera and species of microorganisms including archaea and viruses were also detected using a combination of REFSEQ and One Codex data bases (OXCDB). In-vivo experiment was used to evaluate the anti-diabetic property, safety and gut microbiome changes of Kombucha. Kombucha was found to perform better than the conventional antidiabetic drugs, metformin and glibenclamide in lowering the fasting blood glucose (FBG) of the diabetic rats. Daily administration of 25 mg/kg and 100 mg/kg of freeze-dried Kombucha tea demonstrated a 5 fold reduction in FBG (p<0.05) and 40% and 50% respective increases in body weight of the alloxan-induced diabetic rats compared to the diabetic control (DC). Histological analysis, shows Kombucha enhances pancreas regeneration and hence the concomitant increase in insulin secretion as demonstrated in the study. Serum lipid profiling showed 100mg/kg Kombucha treatment increases the levels of total cholesterol (16%), high density lipoproteins (HDL) (13%) and low-density lipoproteins (LDL) (10%) but conversely reduces triglyceride level (17%) compared to the DC (p>0.05). Further analyses demonstrated that Kombucha decreases the relative organ (liver and kidney) to body weight ratio in treated animals. In addition, Kombucha was able to reduce significantly the elevated levels of liver enzymes such as Alkaline phosphatase (ALP), Alanine transaminase (ALT) and Aspartate Aminotransferase (AST) as well as renal toxicity indices, creatinine and urea in treated animals. Histology of the kidney and liver also showed that Kombucha has no adverse effect on the morphology and cellular integrity of these organs suggesting its hepatoprotective and renal protective potentials. Urinanalysis also showed reduction of glucose in urine for the 100 mg/kg Kombucha-treated animals. Additionally, Kombucha protects the gut microbiome, most significantly by enhancing the Lactobaccillaceae family of bacteria within the gut and reduces the possibilities of colonization of the gut by other opportunistic bacterial species. The study demonstrated that Kombucha is enriched with diverse microbial population with probiotic value and daily intake of Kombucha may be potentially helpful in the management of diabetes, protection against renal and liver toxicity and offer gut microbiome protection.Item Identification of Natural Product-Derived Compounds that Inhibit Hiv-1 Entry into Target Cells(University of Ghana, 2019-07) Ugwu, N.P.HIV-1 infection is mediated by the interaction between the host cellular CD4 receptor and co-receptors with HIV Envelope protein (Env). Inhibition of HIV entry into host cell presents a biological target for therapeutic intervention. Broadly neutralizing antibody (bNabs) are potent in neutralizing a broad range of HIV strains by binding to the Env and preventing entry into the host cell. VRC01 is a CD4 binding-site class bNabs that binds to the conserved CD4 binding region of Env. However, using antibodies as therapeutic agents pose challenges of low availability and high cost of production. Natural products that can mimic VRC01 bNabs by interacting with the conserved CD4-binding regions may serve as new generation of HIV-1 entry inhibitors, which are broadly reactive and potently neutralizing. This study aimed to identify natural products that mimic VRC01 broadly neutralizing antibody, by interacting with the CD4-bs of HIV-1 gp120 and inhibit viral entry into a target cell. A library of purchasable compounds derived from natural products was virtually screened against the HIV-1 gp120 of clade A/E recombinant 93TH057 (PDB: 3ngb) using AutoDock Vina, and LIGPLOT was used to elucidate the interactions between the compounds and the HIV-1 gp120 complexes. The compounds with the lowest binding energies were selected and further screened against gp120 of a reference HIV strain (HXB2). Pharmacological profiling of the compounds was undertaken using SwissADME. Ten selected compounds were purchased and used for cell-based antiviral infectivity inhibition assay using HXB2-env pseudotype virus. Three compounds, NP-005114, NP-008297 and NP-007382 inhibited entry of HIV HXB2 pseudotype virus into target cells, while the rest of the compounds showed no inhibitory activity against HIV entry into target cells. NP-005114, extracted from the seed of T. chebula, had IC50 of 10μg/ml. Protein-ligand interaction showed that NP-005114 had inter-molecular hydrogen bond interactions with eight and eleven amino acid residues on the CD4- binding site of recombinant clades A/E and B HIV -1 gp120, respectively. NP-005114 had intermolecular hydrogen and hydrophobic interactions with Asp457, a critical amino acid residue in the Phe43 cavity of the HIV gp120 for recombinant clades A/E and B, respectively. Derived from the leaf of Ginkgo biloba, NP-008297 formed ten and four hydrogen bond interactions with amino acid residues of the CD4-binding site on HIV-1 gp120 of recombinant clades A/E and B, respectively. NP-008297 inhibited viral entry at IC50 of 15.5μg/ml. NP-007382 was derived from bacteria and interacted with five amino acid residues in the CD4-binding site of HIV-1 gp120 of recombinant clades A/E and B, via intermolecular hydrogen bond. The 50% inhibitory concentration of NP-007382 against viral entry into the target cell was 13.1μg/ml. These compounds inhibited the entry of HIV clade B pseudotype (HXB2) with an IC50 which was comparable to that of VRC01 (0.1-50 μg/ml) against HIV clade B pseudotypes and primary isolate. The interaction of the compounds with critical residues of the CD4-binding site of more than one clade of HIV-gp120 demonstrates the possibility of broad entry inhibition of other HIV clades. This is the first report of the characterization of NP-008297, NP-00738, and NP-005114 as HIV-1 entry inhibitors.Item Genetic Diversity of Mycolactone Producing Mycobacteria Causing Buruli Ulcer in Ghana and Côte D’ivoire(University of Ghana, 2019-07) Dogbe, M.Buruli ulcer remains a neglected tropical disease endemic in Africa especially Ghana and Côte d’Ivoire. It is a necrotizing skin and soft tissue infection caused by Mycobacterium ulcerans which produces mycolactone which causes adverse effects associated with disease in humans. Other mycolactone producing bacteria (MPMs) have been identified to cause granulomas in fish and frog. There are limited advanced genetic tools for studying transmission of the diseases and for carrying out molecular epidemiological studies. The mycolactone producing mycobacteria (MPM), M. shinshuense has been reported to cause Buruli ulcer and other studies have revealed several MPM infections other than M. ulcerans in clinical samples. It is therefore imperative to know the exact MPMs that are in circulation by genotypically distinguishing them using molecular tools. Patients with Buruli ulcer-like disease presentation were recruited from endemic communities in Ghana and Côte d’Ivoire. Swabs and FNA samples were screened using primers detecting the insertion sequence 2404 gene and the Enoyl reductase gene. Genotyping was achieved at 16 MU VNTR loci and length polymorphisms arising from differences in tandem repeats for samples were validated using six (6) controls and sequencing. A total one hundred and fifty-nine (159) of the 189 samples were confirmed as Buruli ulcer positive. Genotyping was successful for all controls (100%) and most samples (69%) at all sixteen (16) VNTR loci. Seven (7) MU genotypes designated, A, B, C, D, E, F and G and five MPM genotypes MLF (Mycobacterium liflandii), MHB (Mycobacterium marinum hybrid), MDL (Mycobacterium marinum DL), MCL (Mycobacterium marinum CL) and MSA (Mycobacterium marinum SA) were generated samples were assigned genotypes based on their VNTR profiles. Genotypes C, D and F were present in both Ghana and Côte d’Ivoire. However, genotypes E and MLF were only found in Ghana whiles Genotype A and G were found in only Côte d’Ivoire Genotype D has persisted over the years from 2008 to 2019 comparing this study to published data. These findings support the hypothesis that Mycolactone producing mycobacteria causing Buruli ulcer in Ghana and Côte d’Ivoire are diverse and affirms VNTR typing as a comparably useful tool for differentiating MU strains as well as other MPMs in Buruli ulcer endemic communities.