Edward Muge

Identification of Specific Markers Linked to Regional Differentiation of Warburgia ugandensis

Abstract Warburgia ugandensis is an important African medicinal tree. The species population has shown a high genetic differentiation in the Kenya’s Rift Valley. Nine populations were analysed by Bulk Segregant Analysis employing Random Amplified Polymorphic DNA marker technique to identify regional differentiation-linked markers within and across Kenyan Rift Valley. Five primers showed putative East and West genetic differentiation. Diagnostic markers were isolated, cloned, sequenced and compared with Genbank sequences using BLAST algorithms. Three, (WarburgiaIC15E, WarburgiaIC55E and WarburgiaIC28W) sequences showed homology to plant and bacterial-like chromosomal sequences with low E-values. Sequence alignment indicated conserved protein domains of plants and bacteria-like sequences. Phylogenetic analysis revealed high rates of genetic distances (H” 0.8) and a low rate of disparity indices of (0), suggesting some evolutionary forces behind demographic differentiation. These imply that genetic differentiation observed might be due to genetic mutants in certain domains of chromosome that may have some implication on genome functionality.

Predicted HIV-1 coreceptor usage among Kenya patients shows a high tendency for subtype d to be cxcr4 tropic

BackgroundCCR5 antagonists have clinically been approved for prevention or treatment of HIV/AIDS. Countries in Sub-Saharan Africa with the highest burden of HIV/AIDS are due to adopt these regimens. However, HIV-1 can also use CXCR4 as a co-receptor. There is hence an urgent need to map out cellular tropism of a country’s circulating HIV strains to guide the impending use of CCR5 antagonists.ObjectivesTo determine HIV-1 coreceptor usage among patients attending a comprehensive care centre in Nairobi, Kenya.MethodsBlood samples were obtained from HIV infected patients attending the comprehensive care centre, Kenyatta National Hospital in years 2008 and 2009. The samples were separated into plasma and peripheral blood mononuclear cells (PBMCs). Proviral DNA was extracted from PBMCs and Polymerase Chain reaction (PCR) done to amplify the HIV env fragment spanning the C2-V3 region. The resultant fragment was directly sequenced on an automated sequencer (ABI, 3100). Co-receptor prediction of the env sequences was done using Geno2pheno[co-receptor], and phylogenetic relationships determined using CLUSTALW and Neighbor Joining method.ResultsA total of 67 samples (46 treatment experienced and 21 treatment naive) were successfully amplified and sequenced. Forty nine (73%) sequences showed a prediction for R5 tropism while 18(27%) were X4 tropic. Phylogenetic analysis showed that 46(69%) were subtype A, 11(16%) subtype C, and 10(15%) subtype D. No statistical significant associations were observed between cell tropism and CD4+ status, patient gender, age, or treatment option. There was a tendency for more X4 tropic strains being in the treatment experienced group than the naive group: Of 46 treatment experiencing participants, 14(30%) harboured X4, compared with 4(19%) of 21 of the treatment-naïve participants, the association is however not statistically significant (p = 0.31). However, a strong association was observed between subtype D and CXCR4 co- receptor usage (p = 0.015) with 6(60%) of the 10 subtype D being X4 tropic and 4(40%) R5 tropic.ConclusionHIV-1 R5 tropic strains were the most prevalent in the study population and HIV infected patients in Kenya may benefit from CCR5 antagonists. However, there is need for caution where subtype D infection is suspected or where antiretroviral salvage therapy is indicated.

Genetic Variation of Kenyan Populations of Warburgia ugandensis, an Important East African Highlands Medicinal Tree Species

Abstract Warburgia ugandensis is an important medicinal tree species in Eastern Africa used to treat several ailments. Wild populations are under great threat due to unsustainable harvesting for medicines and indiscriminate felling of trees for timber and firewood. There is an urgent need for developing and implementing conservation strategies of this species and information on genetic structure is a crucial input. Analysis of molecular variance (AMOVA), which employed 141 AFLP markers revealed most genetic variation to be among individuals within populations (59%, P < 0.0001), but variation among populations (41%, P < 0.0001) was highly significant as well. Constrained ordination analysis illustrating the relationship among populations showed a clear distinction between W. ugandensis from Uganda and western Kenya and other W. ugandensis populations. No correspondence was shown in some cases on pair-wise genetic distances and geographic distances among populations. These findings suggested that conservation strategies for the species in Kenya should place relatively more emphasis on the revealed genetic structuring within the country.

Screening and Characterization of Hydrocarbonoclastic Bacteria Isolated from Oil-contaminated Soils from Auto Garages

Release of petroleum oil and its products into the environment is a worldwide concern. The present study focused on isolation, molecular identification, morphological and biochemical characterization of bacteria possessing hydrocarbon-degrading properties. The study also aimed at optimizing appropriate culture conditions for the isolates as well as screening for alkane hydroxylase enzyme. Out of twenty one microbes isolated, nine were selected based on their ability to utilize different hydrocarbons. The isolates were observed to mineralize heating oil, hexane, octane, toluene and diesel oil. PCR amplification of 16S rDNA gene revealed that the isolates belong to six different genera; Pseudomonas, Acinetobacter, Klebsiella, Enterobacter, Salmonella and Ochrobactrum. Based on their ability to degrade diesel oil, three isolates were selected and their growth conditions optimized. Optimum degradation was recorded at less than 1% substrate concentration, pH 7 and temperature range of between 30°C and 37°C and using yeast extract as nitrogen source. GC-MS analyses confirmed their diesel oil degrading properties. Alkane hydroxylase gene from one of the isolates (isolate 1C) was successfully amplified indicating its catabolic capabilities in degrading alkanes. Overall, the characterized isolates may constitute potential candidates for biotechnological application in environmental cleanup of petroleum contaminants.

Comparative Analysis of Phytochemical Composition and Antioxidant Activities of Methanolic Extracts of Leaves, Roots and Bark of Jackfruit (Artocapus heterophyllus) from Selected Regions in Kenya and Uganda

Aim: To determine and compare the phytochemical composition and antioxidant activities of methanolic extracts of Jackfruit roots, leaves and bark, found in selected regions of Kenya and Uganda. Study Design: A total of 192 samples of leaves, roots and barks, were obtained from 64 different Original Research Article Ojwang et al.; JABB, 16(1): 1-13, 2017; Article no.JABB.37355 2 Jackfruit trees. The samples were then dried and extracted using methanol. Place and Duration of Study: The study was carried out at The University of Nairobi, Chiromo campus, Department of Biochemistry in Nairobi, Kenya. The research was carried out from May 2016 to June 2017. Methodology: The composition of phenolic and tannin contents were determined using FolinCiocalteu method. Flavonoids content was determined through the formation of the flavonoids– aluminium complex assay. Antioxidant activity was determined through the DPPH scavenging activity and the reducing power of the extracts. Results: The phytochemical composition had significant variation at (P=.05) in all the different parts of the plant. The roots had the highest phenolic content (67.3759.00 mg/g), while the bark region had the least (38.14 -28.34 mg/g). The flavonoid content was also highest in the roots (10.74 7.31 mg/g) and lowest in the bark region (3.09 – 1.49 mg/g). The tannin contents were equally higher in the roots (3.88 2.69 mg/g), and lowest in bark (0.93 -0.52 mg/g). The DPPH scavenging activity was also highest in the roots (66 -72%), while that of the bark was (24 40%). The reducing power of the roots, leaves and bark ranged from (114.38 – 93.62 μg/ml), (71.63 -67.04 μg/ml) and (54.16 33.15 μg/ml) respectively. Conclusion: The findings suggest that Jackfruit roots, leaves and bark are natural antioxidants and they have significant variation in composition of phytochemical compounds.

Isolation and Characterization of a Thermostable Cellulase from Bacillus licheniformis Strain Vic Isolated from Geothermal Wells in the Kenyan Rift Valley

Cellulolytic microorganisms such as fungi and bacteria are responsible for much of the cellulose degradation in soils. Despite the vast number of cellulase producers, there is a deficiency of microorganisms that can produce significant amount of the three components of a cellulase system. i.e. CMCases, exoglucanases and β-glucosidases to efficiently degrade cellulose to fermentable products. Soil samples were collected from two sites of the Kenyan Rift valley; Hell’s gate National Park and a geothermal well in Eburru hill at depths of 155 m, 156 m and 157 m. Isolation of cellulolytic bacteria was carried out using minimal media supplemented with 1% CMC. Isolates obtained were characterized using both morphological and molecular methods. A quantitative cellulolytic test was carried out to select the isolate with the highest specific activity. Additionally the cell-free supernatant was used for functional studies to determine the isolate’s cellulase system components, optimum temperature, pH and time stability. Phylogenetic analyses indicated that all isolates associated mainly with members of the Bacillus licheniformis. All isolates were capable of utilizing CMC, avicel and Cellobiose as their sole carbon sources. Isolate 3 from a depth of 156 m in the well was identified as Bacillus licheniformis strain vic and selected for further functional studies. This isolate was found to bear the three enzyme components of a cellulase system. A cell free extract was noted to hydrolysecellobiose, Avicel and CMC with enzyme specific activities of 0.46878 U/mg, 0.18784 U/mg and 0.13571 U/mg respectively. Optimum temperature for activity measured over 60 minutes was found to be 60C with relatively high activity at both 70C and 80C. The optimum pH at the predetermined optimum temperature was found to be pH 5. The crude enzyme extract was stable for eight hours of incubation at 60C and pH 5 retaining 92.2% of its activity. This makes B.licheniformis strain vic a quite attractive isolate for potential application in industries.