Fred Wamunyokoli

A Growth-Related Oncogene/CXC Chemokine Receptor 2 Autocrine Loop Contributes to Cellular Proliferation in Esophageal Cancer

Growth-related oncogene (GRO), a member of the CXC chemokine subfamily, plays a major role in inflammation and wound healing. CXC chemokines have been found to be associated with tumorigenesis, angiogenesis, and metastasis. Although elevated expression of GRO has been reported in several human cancers, the expression and role of GRO and its receptor, CXCR2, in esophageal cancer are poorly understood. This study used real-time reverse transcription-PCR (RT-PCR) and immunohistochemical approaches to show that GROalpha, GRObeta, and CXCR2 are up-regulated in esophageal tumor tissue. Furthermore, GROalpha, GRObeta, and CXCR2 are constitutively expressed in WHCO1, an esophageal cancer cell line that was used as a model system here. GRObeta enhances transcription of EGR-1, via the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway, which can be blocked by a specific antagonist of CXCR2 (SB 225002) or specific antibody to GRObeta. WHCO1 cells treated with SB 225002 exhibited a 40% reduction in cell proliferation. A stable WHCO1 GROalpha RNA interference (RNAi) clone displayed a 43% reduction in GROalpha mRNA levels as determined by real-time RT-PCR, reduced levels of GROalpha by fluorescence microscopy, and a 60% reduction in the levels of phosphorylated ERK1/2. A stable clone expressing GRObeta RNAi displayed >95% reduction in GRObeta mRNA levels, reduced levels of GRObeta by fluorescence microscopy, and an 80% reduction in the levels of phosphorylated ERK1/2. Moreover, these GROalpha RNAi- and GRObeta RNAi-expressing clones displayed a 20% and 50% decrease in cell proliferation, respectively. Our results suggest that GROalpha-CXCR2 and GRObeta-CXCR2 signaling contributes significantly to esophageal cancer cell proliferation and that this autocrine signaling pathway may be involved in esophageal tumorigenesis.

Phytochemical screening, anti-oxidant activity and in vitro anticancer potential of ethanolic and water leaves extracts of Annona muricata (Graviola)

OBJECTIVE To determine the phytochemical composition, antioxidant and anticancer activities of ethanolic and water leaves extracts of Annona muricata (A. muricata) from the Eastern Uganda. METHODS Phytochemical screening was conducted using standard qualitative methods and a Chi-square goodness of fit test was used to assign the relative abundance of the different phytochemicals. The antioxidant activity was determined using the 2, 2-diphenyl-2-picrylhydrazyl and reducing power methods whereas the in vitro anticancer activity was determined using three different cell lines. RESULTS Phytochemical screening of the extracts revealed that they were rich in secondary class metabolite compounds such as alkaloids, saponins, terpenoids, flavonoids, coumarins and lactones, anthraquinones, tannins, cardiac glycosides, phenols and phytosterols. Total phenolics in the water extract were (683.69±0.09) μg/mL gallic acid equivalents (GAE) while it was (372.92±0.15) μg/mL GAE in the ethanolic extract. The reducing power was 216.41 μg/mL in the water extract and 470.51 μg/mL GAE in the ethanolic extract. In vitro antioxidant activity IC50 was 2.0456 mg/mL and 0.9077 mg/mL for ethanolic and water leaves extracts of A. muricata respectively. The ethanolic leaves extract was found to be selectively cytotoxic in vitro to tumor cell lines (EACC, MDA and SKBR3) with IC50 values of 335.85 μg/mL, 248.77 μg/mL, 202.33 μg/mL respectively, while it had no cytotoxic effect on normal spleen cells. The data also showed that water leaves extract of A. muricata had no anticancer effect at all tested concentrations. CONCLUSIONS The results showed that A. muricata was a promising new antioxidant and anticancer agent.

Genetic Diversity of Human Enterovirus 68 Strains Isolated in Kenya Using the Hypervariable 3′- End of VP1 Gene

Reports of increasing worldwide circulation of human enterovirus-68 (EV68) are well documented. Despite health concerns posed by resurgence of these viruses, little is known about EV68 strains circulating in Kenya. In this study, we characterized 13 EV68 strains isolated in Kenya between 2008 and 2011 based on the Hypervariable 3′- end of the VP1 gene. Viral RNA was extracted from the isolates and partial VP1 gene amplified by RT-PCR, followed by nucleotide sequencing. Alignment of deduced amino acid sequences revealed substitutions in Kenyan EV68 isolates absent in the prototype reference strain (Fermon). The majority of these changes were present in the BC and DE-loop regions, which are associated with viral antigenicity and virulence. The Kenyan strains exhibited high sequence homology with respect to those from other countries. Natural selection analysis based on the VP1 region showed that the Kenyan EV68 isolates were under purifying selection. Phylogenetic analysis revealed that majority (84.6%) of the Kenyan strains belonged to clade A, while a minority belonged to clades B and C. Overall, our results illustrate that although EV68 strains isolated in Kenya were genetically and antigenically divergent from the prototype strain (Fermon), they were closely related to those circulating in other countries, suggesting worldwide transmissibility. Further, the presence of shared mutations by Kenyan EV68 strains and those isolated in other countries, indicates evolution in the VP1 region may be contributing to increased worldwide detection of the viruses. This is the first study to document circulation of EV68 in Kenya.

Antagonistic effect of alkaloids and saponins on bioactivity in the quinine tree (Rauvolfia caffra sond.): further evidence to support biotechnology in traditional medicinal plants

BackgroundThe Quinine tree (Rauvolfia caffra) is used as a medicinal plant among traditional communities in many countries to manage tumors and other diseases associated with oxidative stress. To validate indigenous knowledge and possibly position this herb for technology uptake and utilization, we established the level of antioxidant activity in R. caffra, and probed for the presence of associated phytochemicals.MethodsAntioxidant activity was determined on 1,1-diphenyl-2-picrylhydrazyl (DPPH) while major phytochemicals were identified by multiple tests on methanol fractions.ResultsR. caffra showed promise as a cure, with antioxidant activity comparable to the commercially used drug quercetin (R. caffra = 79.7% ±1.9; quercetin = 82.6% ± 2.0). However, we found two phytochemicals with possible antagonistic effect: co-occurrence of alkaloids and saponins significantly reduced antioxidant activity (alkaloids only = 63%; alkaloids plus saponins = 15%; steroids, terpenoids and cardiac glycosides = 82%), thus alkaloids and saponins should be exclusive to each other in drug formulations.ConclusionsAntagonistic relationship among phytochemicals would affect the efficacy of crude extracts as used in traditional medicine. Unlike in herbal medicine, use of modern biotechnology in extraction, purification and design of optimal combinations will ensure efficient drug formulations with optimum bioactivity and minimum toxicity. Metabolic pathway engineering under a controlled environment may optimize availability of desired compounds.

Phylogenetic Variants of Rickettsia africae, and Incidental Identification of "Candidatus Rickettsia Moyalensis" in Kenya.

Background Rickettsia africae, the etiological agent of African tick bite fever, is widely distributed in sub-Saharan Africa. Contrary to reports of its homogeneity, a localized study in Asembo, Kenya recently reported high genetic diversity. The present study aims to elucidate the extent of this heterogeneity by examining archived Rickettsia africae DNA samples collected from different eco-regions of Kenya. Methods To evaluate their phylogenetic relationships, archived genomic DNA obtained from 57 ticks a priori identified to contain R. africae by comparison to ompA, ompB and gltA genes was used to amplify five rickettsial genes i.e. gltA, ompA, ompB, 17kDa and sca4. The resulting amplicons were sequenced. Translated amino acid alignments were used to guide the nucleotide alignments. Single gene and concatenated alignments were used to infer phylogenetic relationships. Results Out of the 57 DNA samples, three were determined to be R. aeschlimanii and not R. africae. One sample turned out to be a novel rickettsiae and an interim name of “Candidatus Rickettsia moyalensis” is proposed. The bonafide R. africae formed two distinct clades. Clade I contained 9% of the samples and branched with the validated R. africae str ESF-5, while clade II (two samples) formed a distinct sub-lineage. Conclusions This data supports the use of multiple genes for phylogenetic inferences. It is determined that, despite its recent emergence, the R. africae lineage is diverse. This data also provides evidence of a novel Rickettsia species, Candidatus Rickettsia moyalensis.

Expression Levels of Odorant Receptor Genes in the Savanna Tsetse Fly, Glossina morsitans morsitans

Abstract Tsetse flies (Glossina) are vectors of African trypanosomiasis. Olfaction plays a critical role in Glossina behavior, including larviposition, feeding, and reproduction. Odorant receptors (ORs) are important in insect chemoreception as they bind volatile odorants and transport them to olfactory receptor neurons to elicit behavioral response.To better understand Glossina chemoreception, we used quantitative polymerase chain reaction to examine the expression levels of ORs in female and male Glossina morsitans morsitans Wiedemann, 1850 (Diptera: Glossinidae) antennae and legs. Results showed that G. m. morsitans ORs code for a transmembrane domain and are involved in odorant binding. The ORs had homologs in Drosophila, mosquitoes, other Glossina species, and the reduced number of tsetse ORs could be linked to its restricted blood-feeding diet. The OR genes were more highly expressed in antennae than the legs with GmmOR33 and GmmOR45 transcript levels being high in the female and male antennae, respectively, whereas GmmOR26 and GmmOR34 levels were high in female and male G. m. morsitans legs, respectively. These findings identified sex- and tissue-specific G. m. morsitans ORs. The expression levels of OR genes in female and male G. m. morsitans could be conserved in function with the antenna being the main olfactory organ.Thus, this study provides a blueprint to explore the functional roles of tsetse ORs with the potential to identify molecular targets that can be used to control the vector based on disruption of its chemosensory system.

In Silico Analysis of Occurrence of Tricorn Protease and Its Homologs

Tricorn protease is an archaeal protease acting downstream of the proteasome and together with its interacting aminopeptidases, degrades oligopeptides to free amino acids thus playing an important role in protein turnover. This study reports a wide distribution of tricorn protease and its homologs in archaea and bacteria. The homologs were identified through a combination of PSI-BLAST, orthology clustering and domain predictions. Functionally important sites were identified through multiple sequence alignment conducted by MAFFT v. 7. The aligned sequences were used to predict the phylogenetic relationship of tricorn protease and its homologs using MEGA v. 7. The functional associations of tricorn protease were predicted through STRING network v.10.0. This study identified several tricorn protease homologs in archaea and in all the bacterial phyla complete with β-propeller, PDZ and catalytic domains. However, in eukaryotes, tricorn protease-like homologs seemed limited to viridiplantae, stramenopile and in a basal metazoa and were classified as non-peptidase homologs with unknown functions. Conserved domain architecture retrieval revealed detectable homology of tricorn protease C-terminal half with the carboxyl-terminal proteases with similar PDZ domains. Therefore, this study predicts functional conservation of tricorn core catalytic domain in prokaryotes and given its role in cellular functions, targeting this protein or its functional homologs in prokaryotic pathogens could lead to development of alternative therapeutic agents.