Department of Biochemistry, Cell and Molecular Biology
Permanent URI for this collectionhttp://197.255.125.131:4000/handle/123456789/4848
Browse
30 results
Search Results
Item Selection Dynamics Of Circumsporozoite Protein (Csp) Vaccine Target In Ghana: The Contribution Of Human Leukocyte Antigen (Hla) Variation(University Of Ghana, 2021-07) Kpaka, J.N.Implementation of RTS,S/AS01 vaccine for malaria is underway in three (3) African countries, Ghana, Kenya, and Malawi. This vaccine, which targets the Plasmodium falciparum circumsporozoite protein (CSP) provides partial protection for infants and children against clinical and severe malaria infections. Reasons for this reduced efficacy or immunogenicity are poorly understood, but CSP variation has been implicated. Human leukocyte antigen (HLA) has also been observed to influence RTS,S-mediated protection. This study aims to define the variants of CSP and determine its distribution between Begoro and Cape Coast in Ghana over three years. Further, the influence of HLA genotype in terms of parasite frequency and RTS,S/AS01 response was assessed. About 50μl of peripheral blood was collected from participants in Begoro and Cape Coast in 2014, 2015, and 2016, dried blood spot (DBS) prepared and DNA was extracted. The C-terminal of Plasmodium falciparum CSP and the human leukocyte antigen (HLA) class II gene in humans were deep sequenced. The translated amino acid haplotypes of the CSP were aligned and compared to the reference 3D7 vaccine strain. The HLA class II haplotypes were grouped into superfamily and their association with the CSP variants was ascertained. The CSP haplotypes are evenly distributed between Begoro and Cape Coast. There were 31 Th2R haplotypes in Begoro and 30 Th2R haplotypes in Cape Coast; 15 Th3R haplotypes in Begoro and 13 in Cape Coast. About 83.9% of Th2R and 96.5% of Th3R haplotypes in Begoro are shared with Cape Coast. The amino acid changes with reference to the 3D7 vaccine strain at the Th2R epitope range from 1 to 6 and 1 to 4 at theTh3R epitope. There is a 53% and 60% reduction in the 3D7 Th2R and Th3R haplotypes, respectively, from 2014 to 2016, but 3D7 is still common in Ghana, Kenya, and Malawi. The 3D7 haplotype does not correlate with HLA-DRB1, but there is with HLA-DQA1 and HLA-DPB1. Begoro and Cape Coast are two different ecological zones in Ghana but the parasite population is homogenous. The Th2R epitope of CSP is polymorphic than the Th3R epitope and this higher polymorphism is driving a higher non-synonymous amino acid substitution at the Th2R epitope than the Th3R epitope which may have vaccine implication. A decline in frequency of 3D7 parasite population may also affect the performance in the vaccine in Begoro and Cape Coast. Initial correlations indicate that HLA-DPB1 (01:01/17:01) correlates with the 3D7 vaccine strain, but HLA-DPB1 (01:01/17:01) and other variants of HLA-DQA1 also correlates with other Th2R haplotypes and may compete with the vaccine haplotype for antigen presentation to CD+4 T cells. This may have implications for the efficacy of the RTS,S/AS01 vaccine in Ghana.Item Transmitted Drug Resistance and Co-Receptor Tropism of Circulating HIV-1 Subtypes in Art-Naïve HIV Patients in Ghana(University of Ghana, 2021-06) Quansah, D.N.K.Combination Anti-retroviral therapy (ART) has significantly reduced the burden of human immunodeficiency virus (HIV-1) infection, transforming it from a fatal disease into a manageable chronic one. The clinical benefits of ART are sustained viral suppression increase in CD4 T lymphocyte counts and improved general well-being. However, the emergence of drug resistance poses a strong challenge to the success of ART in managing HIV infection. Drug resistance (DR) to anti-retrovirals (ARVs) is caused by the high replication capacity of HIV, its error-prone reverse transcriptase and poor compliance by HIV-infected people on ART. In Ghana, reverse transcriptase (RT) and protease inhibitors (PIs) are the main drug combinations. While the transmission of multi-drug resistant variants, determined by genotypic tests, among other HIV-endemic populations informs the choice of drugs in their regimen, clinical management of HIV-1 in Ghana does not include genotypic tests. In this research, we hypothesized that transmitted drug resistant mutants present in treatment-naïve individuals are related to the co-receptors used for cell entry and may hasten ART failure. Sixty-nine treatment-naive HIV-1 infected persons were enrolled from three hospitals in Accra and their clinical histories were obtained. CD4 lymphocytes and HIV-1 viral load were determined for all samples. HIV-1 RNA was extracted from patients’ plasma and HIV Pol region was amplified by conventional PCR and sequenced. The assembled, edited and aligned contigs were used to phylogenetically characterise circulating HIV-1 subtypes. The sequences were submitted to the Stanford University HIV-DR database for analysis of drug resistance mutants. HIV co-receptor preference was measured using TZM-bl indicator cells. The dominant HIV-1 subtype was CRF02_AG (64%), followed by subtype B (26%,) and others, CRF06_cpx, A, C and G, in smaller proportions. This confirms previous reports of CRF)2_AG as predominant subtype and indicates a growing population of the subtype B, which drives the HIV epidemic in Europe and America, in Ghana. Seven mutations on the WHO drug surveillance list were present in nine out of sixty-nine participants enrolled in this study. All seven of them were either non-nucleoside reverse transcriptase (NNRTI) or PI mutations. Quite surprisingly, no nucleoside reverse transcriptase (NRTI) mutations were found. The K103N, which strongly reduces susceptibility to nevirapine and efavirenz was detected in one individual. All PI mutations that were detected were accessory mutations. This was expected since protease inhibitors are not used on a large scale in Ghana. No one individual carried more than one mutation; indicating low levels of transmitted DR among the study population The study participants had mean viral load and CD4 counts of 137,694copies/ml and 409cells/μl respectively and presented no symptoms of disease. Such attributes are indicative of rapid viral replication which is a major driver of drug resistance. Preliminary analysis of the results from the phenotypic assays showed no association between co-receptor phenotypes and identified drug resistance mutations in individuals who carried these mutations. More experiments are needed to confirm these preliminary results. This study has confirmed the predominance of HIV-1 subtype CRF02_AG as the predominant subtype in Ghana while documenting an increase in the circulation of Subtype B. Transmitted HIV drug resistance is still low in Ghana, and this is good for the country’s HIV control programmes. However, continuous surveillance for drug resistance strains among treatment-naïve HIV persons is highly recommended to inform treatment regimens going forward.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 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.Item Immunological Profiling of Untreated and Ivermectin-Treated Onchocerca-Infected Patients in the Oti Region, Ghana(University of Ghana, 2019-07) Labadah, J.Onchocerciasis also referred to as river blindness is a neglected tropical disease that affects the skin and eyes. It is caused by the nematode Onchocerca volvulus. Disease pathology is largely attributed to host immune responses to microfilariae. The efficacy of ivermectin (IVM) is defined by its microfilaricidal effects (clearing > 60% of microfilariae within 24 hours, ~ 100% within 7 days) and its ability to temporarily retard adult parasite embryogenesis at least within 90 days. This study sought to determine the immune profile of untreated microfilariae positive (microfilaremic) and ivermectin treated microfilariae negative (amicrofilaremic) onchocerciasis patients in order to contribute to understanding the pathophysiology (skin lesions, itching, visual impairment, etc.) of the disease. White blood cell differentials were measured for 124 adults; 53 microfilaremic (Mf) and 71 ivermectin treated (1-5 rounds) seropositive amicrofilaremic (Amf) individuals 3 months following last treatment at the Nkwanta North District of Ghana. Subgroups (16 ≤ n ≤20) of both Mf and first-time IVM-treated Amf were assayed for plasma cytokines, total IgE and urine histamine. The geometric mean microfilariae density/milligram skin snip was 22.5 (range 5.8-56.3). Basophil count was lower (p<0.0001) while eosinophil counts were higher (p=0.02) for Mf compared to Amf. Out of 14 cytokines, Interleukin (IL)-13 (p=0.002), IL-8 (p=0.03) and Interferon-gamma (p=0.003) were higher among Mf compared to first time treated Amf. Histamine and IgE were not different between the two groups. IL-13, IL-8 and interferon-gamma and eosinophils were elevated while basophils were significantly lower in microfilaremic onchocerciasis and these parameters may contribute to disease pathophysiology. These indices would be helpful in complementing diagnosis and monitoring treatment efficacy.Item Isolation and Characterization of Antifungal Agents Produced by Wood Decaying Fungi From Ghana(University of Ghana, 2014-07) Adadey, S.M.Human systemic fungal infections have increased in the past decade due to the dramatic increase in immunocompromised patients hence the emergence of resistant fungal strains. It is therefore necessary to discover new antifungal compounds with novel mode of action. This study was aimed at isolating and characterizing antifungal agents from wood decaying fungi (WDF). The growth conditions for maximum production of bioactive agents from WDF were first tested. It was observed that mineral supplements from soil extract supported the growth and metabolite production of WDF. Media richness, culture volume and mineral supplementation were identified as the critical factors that influence the batch to batch consistency of metabolite production in WDF. Collections of 189 WDF were partially screened for their antifungal activity against Candida albicans. They were further screened against Saccharomyces cerevisiae in this project. Based on this screening, 33 of WDF were selected for mycelium isolation. The mycelia of 31 out of the 33 were successfully isolated on agar plates and stored called Plate Mycelium (PM). This served as a continuous fungal inoculum source. The 31 isolated WDF mycelia were then grown in broth and their metabolites were extracted with ethyl acetate. The antifungal activity of each WDF extracts was validated through a phenotypic assay screening using media conditions and mutant S. cerevisaie strains. In the chemical phenotypic array, the sensitivity of the C. albicans and S. cerevisiae cells to the WDF extracts was altered by chemical modifications in the YPD agar plates. This was used as a read out for validating the antifungal activities of the extracts. From the phenotypic screening, the best 6 WDF candidates with highly potent antifungal activity were selected. The mode of action of this top 6 WDF was examined by the antifungal activity pattern displayed against the mutant S. cereviaise cell.
- «
- 1 (current)
- 2
- 3
- »