Engineering the functional fitness of transglycosidases and glycosynthases by directed evolution

The artificial implementation of the Darwinian theory of evolution to create new variants of functional proteins, a process referred to as directed evolution, has acquired many applications in biochemical engineering. Directed evolution is a handy tool in the nascent science of glycobiology, where it is used in the conversion of glycosyl hydrolases into transglycosidases or for improving the transglycosylation behaviour of glycosynthases. This review focuses on recent applications of the directed evolution approach to harness the transglycosidase potential of glycosidases and to enhance the functional fitness of glycosynthases.Key words: Directed evolution, transglycosidases, glycosidases,  glycosynthases, oligosaccharides

Developing Bt maize for resource-poor farmers – Recent advances in the IRMA project

This paper presents an overview of the advances in the IRMA project, which develops insect resistant maize varieties for resource-poor farmers, using both conventional breeding and genetic engineering. The project started in 1999 and is active in product development, impact assessment, andcommunication, all within the Kenya regulatory framework. So far, four application for introduction of tissue or commencement of field research were made to and approved by the National Biosafety Committee (NBC), and Bt maize leaves or seeds genes imported for testing against different stem borer species in bioassays on cut leaves in a biosafety laboratory, in potted plants in a Biosafety Greenhouse, and as whole plants in confined field trials in the Open Quarantine Station (OQS) at KARI Kiboko. All these biosafety facilities were specially built by the project for these evaluations. So far, good control has been realized against four of the five major stem borer species: Chilo partellus, Chilo orichalcociliellus, Eldana saccharina and Sesamia calamistis. Economic  impact assessment demonstrated that stem borers are major constraints and cause substantial losses. Resistant maizevarieties are likely to be adopted and to provide major returns to the investment if resistance against the economically most important species, Busseola fusca, can be found. Otherwise, returns would still be positive but small. Environmental impact research indicate that build-up of resistance against the Bt genes has not developed after that sufficient natural refugia exist in most areas, but suitable strategies acceptable to farmers need to be developed for some. Surveys, stakeholders meetings and other communications indicate that farmers, consumers and other stakeholders are cautiously optimistic about technology. Frequent interaction with the stakeholders and regulatory agencies assures a participative decision-making process and compliance with the strictest scientific and regulatorystandards

Evaluation of the Bacillus cereus Strain 1-p Protease for the Unhairing of Goatskins during Leather Production

The unhairing stage of leather processing is associated with the production of significant amounts of solid and liquid wastes. The use of enzymes to replace the polluting sulphides currently used for unhairing is a viable alternative. Various proteases from different Bacillus cereus strains as well as many other bacterial strains have been used successfully for the unhairing of skins. However, no previous work has assessed the use of the crude alkaline protease extract from Bacillus cereus strain 1-p, a novel Bacillus cereus strain obtained from the shores of Lake Bogoria - a soda lake in Kenya – in the unhairing of goatskins. This study, therefore, evaluates the potential of the protease extract from the Bacillus cereus strain 1-p to unhair goatskins. Optimum variables for unhairing using the protease were investigated. Complete unhairing was achieved within 12 hours at 27°C and pH 12 using the crude enzyme. The period and temperature required for complete unhairing were significantly lower than that of other enzymatic unhairing techniques. Compared to the leather unhaired with sulphide, the leather unhaired with the enzyme did not only show superior organoleptic properties but also recorded comparable or superior physical properties, namely tensile strength (26.94 N/mm2), percentage elongation (76.29%), tear strength (43.59 N/mm), and distension at grain crack and burst (7.9 mm and 8.2 mm respectively). The wastewater from the enzymatic unhairing process recorded a significant reduction in biochemical oxygen demand (78%), chemical oxygen demand (83%), and the wastewater volume (50%) compared to the process that uses sulphide. It was concluded that the use of the crude protease extract from the Bacillus cereus strain 1-p in unhairing goatskins is feasible

Identification of Selected Kinetoplastids 18S rRNA Residues required for Efficient Recruitment of Initiator tRNA Met and AUG Selection in silico

High Resolution 18S rRNA structures of kinetoplastids ribosomes from theoretical methods have provided atomic level details about the process of translation. This process entails detailed information on the mRNA and tRNA binding and decoding centers within the 18S rRNA that was previously not very well understood. We identified residues in selected kinetoplastids 18S rRNA critical in recruiting the first methionyl tRNA to the small ribosome subunit during initiation and comparing them to see the differences. The Kozak sequence presence on eukaryotic mRNAs tethers it to the AUG start codon. Kinetoplastids are a closely related group, and the three chosen exhibited differences in the A-site in terms of position and nucleotides found there. Interactions are found at the A-site (543-UUU-546 for T. cruzi, 560-CCUA-563 for T. brucei, and 540-UUUG-543 for Leishmania major), where the different mRNA get complementary sequences at the 16th helix. The current findings show that each messenger RNA has a sequence that is complementary to the appropriate 18S rRNA sequence, tethering the mRNA to the small ribosomal subunit, which then recruits the bigger subunit. When compared to the Kozak region that flanks the AUG start codon, this method effectively promotes start codon placement

rRNA Platform Technology for Drug Discovery Methods for Identifying Ligands That Target Plasmodium RNA Structural Motifs

Determining the structure of the P. falciparum40s leads to better understanding of the structural basis for its protein-synthesizing roles in the cell. This enables researchers in the field of drug development to run in silico ligand screening experiments using the solved P. falciparum 40S structure as a target against a library of potential anti-malarial compounds. Drug leads identified through this method can lead to further biochemical and In vitro binding studies with the ultimate goal of developing new class of anti-malarial drugs. The use of structure prediction and modeling technologies in this study dramatically reduces the time it takes from target identification to drug lead determination. Furthermore, very many compounds that were previously incapable of being experimentally tested can now be tested in silico against the generated structure. Owing to the increasing utility of bioinformatics and three dimensional structural modeling software, one can accurately build physical models solely from sequence data by unwrapping the information therein on probable motif sites capable of being anchored onto available compounds or aptamers

Evaluation of stem borer resistance management strategies for Bt maize in Kenya based on alternative host refugia

Stem borers are the major insect pests of maize in Kenya. The use of Bacillus thuringiensis (Bt) technology is an effective way of controlling lepidopteran pests. However, the likelihood of development of resistance to the Bt toxins by the target stem borer species is a concern. Forages, sorghum and maize varieties were evaluated for stem borer preference and survivorship in the laboratory and field in four locations in Kenya to identify suitable species and varieties for refugia. The economics of using the different kinds of refugia was also investigated. Vegetation surveys were conducted in 15 districts of Kenya to quantify the area covered by natural refugia. The field and laboratory trials indicated highest egg production, survivorship and more exit holes in all sorghum and maize varieties and some forages. Sorghum, non-Bt Maize, and improved Napier grass varieties Kakamega 1 and Kakamega 2) should be promoted as refugia species in Kenya. Some species and cultivars were identified as cost-effective, flexible, easily adoptable and compatible with farmers’ common production practices. Refugia cultivar with multiple uses is expected to give higher pay-offs than one with single use. However, for successful management of a refugia strategy, strict stewardship is required from appropriate government or community institutions.Key words: Refugia, cost-benefit analysis, Bt-maize, insect pest resistance management

Improvement of the Quality of Cotton Fabric Through Functionalization of Tagetes minuta (TaMi) Dye

Non-functionalized and functionalized Tagetes minuta (TaMi) dye obtained using methanol and water showed varied fastness onto cotton fabric. Furthermore, HPLC profile of the methanol dye showed more peaks and hence was expected to be richer in secondary metabolites that impacted on the quality of the dye. This was confirmed from the IR spectrum which showed more functional groups from this dye as compared to the water dye. The functionalized methanol dye exhibited good fastness onto cotton textile and minimal bleaching effects, as compared to non-functionalized H2O and MeOH dye. Furthermore, the process involved the use of low quantities of the dye material/solution as compared to non-functionalized dye, hence resulting to less impact on environmental degradation. The colors of the textiles produced from the methanol functionalized dye were highly resistant to fading or running. The cotton fabric articles that were tri-mordanted at 80-90 °C and dyed using the functionalized dye exhibited uniform absorption of color of the dye and good fastness. Functionalization of the methanol TaMi dye was obtained by the reaction of the TaMi dye with 1,4-butanediol diglycidyl ether (BDDE). The alum tri-mordanted textile materials produced brighter colors of brown as compared to the singly alum mordanted fabrics. This is due to the tannin factor in tri-mordants that led to the brightening effect. Furthermore, longer duration of time for tri-mordanting might have had a greater impact to the brightness effect. Additionally, characterization of the dye obtained using the two solvents was done using HPLC and IR. The formed functionalized dye has many applications as colorants in textiles, paints, inks, plastics, cosmetic articles and electronic materials. Keywords: Cotton fabric, Tagetes minuta, dye, functionalized, 1,4-butanediol diglycidyl ether, colorants DOI: 10.7176/JNSR/11-16-02 Publication date:August 31st 202

Performance of methylcellulose and Avicel overlays in plaque and focus assays of Chikungunya virus

Background: Chikungunya virus is a re-emerging pathogen that is responsible for Chikungunya fever periodic outbreaks along the Kenyan coast and in other African countries.  Epidemiological data from the World Health Organization show that in 2014-2015, there was a major outbreak of Chikungunya fever in the Americas and Pacific Islands.  Surveillance and correct diagnosis are therefore key in controlling the spread and management of the disease. Plaque and focus assays are key techniques in viral characterization or quantification, and both assays typically require overlay with gelling polymers to limit the spread of viruses in cell culture.  There are anecdotal reports that Avicel may be superior to methylcellulose in assay of Influenza virus. However, it is unclear whether this would apply to other viruses. Objective: The objective of this study was to determine the performance of methylcellulose and Avicel overlays in plaque and focus assays of Chikungunya virus. Methods: Confluent Vero cells were seeded in 6- or 96-well plates for plaque and focus assays respectively. Cells were inoculated with serially diluted Chikungunya virus, and incubated to allow adherence of the virus to the cells. The inoculum was removed; replaced with Avicel or methylcellulose overlay at various concentrations and stained with crystal violet or immunostained.  Statistical significance was computed using the Holm-Sidak test. Results: The size of plaques formed by Chikungunya virus was dependent on the concentration of both Avicel and methylcellulose gels used as overlays, with Avicel overlays giving consistently larger plaques than methylcellulose.  Chikungunya virus formed plaques nearly 2.5 times larger in diameter (2 vs 0.8 mm) with 1.2 % Avicel than with 1.25 % methylcellulose after 60 hr growth.  Plaques formed with Avicel were better defined and easier to count after 48 hr growth period compared to a 60 hr period. However, methycellulose overlays provided smaller, more distinct and better defined foci in focus assays. Conclusion: Both methylcellulose and Avicel are good overlay media for viral assays. Avicel is marginally better for plaque assays while methylcellulose provides more distinct and easier to count foci in focus assays. Key words: Chikungunya virus, plaque assay, focus assay, methylcellulose, Avice

Development of Dromedary Antibody-based Enzyme Linked Immunosorbent Assay for Detecting Chikungunya virus Infections

Background: Chikungunya virus (CHIKV) is an arthropod-borne Togavirus belonging to the genus Alphavirus that is responsible for sporadic worldwide outbreaks of Chikungunya fever, an acute febrile illness often associated with severe polyarthralgia. In Kenya, Chikungunya virus is of great epidemiological concern, with the last major outbreak occurring in 2016 in North Eastern Kenya. Reliable detection of CHIKV infections is key to controlling this re-emerging pathogen, for which no cure currently exists.  Current diagnostic methods for CHIKV employ a combination of tests, particularly immunologic, serologic or virologic techniques.  However, the independent scientific reviews on the validity and sensitivity of currently available commercial assays have been conflicting. Objective: This study aimed to develop and validate a dromedary antibody-based enzyme linked immunosorbent assay for detecting Chikungunya virus infections. Methods: To produce sufficient antigen for camel immunization, Chikungunya virus (strain Lamu 33) was propagated in confluent C6-36 E2 cells using Cytodex microcarrier system. Purified and inactivated CHIKV immunogen was used to inoculate two camels reared at the University of Nairobi farm in Kibwezi, Kenya. Camel serum samples collected over the entire immunization period were assayed for the presence of anti-Chikungunya IgG by indirect ELISA. Purification of camel Heavy Chain IgG antibodies was performed by lectin affinity chromatography on protein A and protein G-Sepharose columns; then conjugated with horse radish peroxidase (HRP). The HRP-conjugated camel Heavy Chain IgG2 and IgG3 were optimized for ELISA, with optical density measured using a microplate reader set at 492nm.   A total of 188 human sera samples were assayed using the developed dromedary-based enzyme linked immunosorbent assay to determine Chikungunya virus infections. Results: The sensitivity of the dromedary HCAb IgG2 assay was 91.3% (95% CI: 0.831 - 0.994); while that for HCAb IgG3 assay was 95.7% (95% CI: 0.898 - 1.01).  The specificity of HCAb IgG2 assay was 92.3% (95% CI: 0.879 - 0.967); while the specificity of HCAb IgG3 method was 90% (95% CI: 0.851 - 0.949). For HCAb IgG2 and IgG3 based assays, the positive predictive values were 79.2% and 75.8 % respectively; while the negative predictive values were 97% and 98.4% for HCAb IgG2 and IgG3 based assays respectively. Conclusion: The camel antibody based assay was found to be reliable assay with very good sensitivity and specificity, and can be deployed for detection of Chikungunya virus infections. Key words: Chikungunya, ELISA, camel antibodies, diagnosi