Strategies for improving hydrolytic efficiency of crude multienzyme extracts in mushroom processing.

The current study investigated and optimized key process parameters affecting mushroom hydrolysis with crude enzymatic extract. The crude enzyme was prepared by solid-state fermentation of pineapple peels using Aspergillus niger. The reaction parameters viz. time, temperature, pH and enzyme concentration were optimized using the central composite design of the response surface methodology. The model predicted glucose yield of 1.49 mg/mL at optimal pH of 6.5, temperature of 50 °C, enzyme loading of 5 % (v/v), and reaction time of 12 h. Mushroom hydrolysis at the same optimal model conditions, increased glucose yield by 10%. More so supplementing SSF media with 0.2% (w/v) Tween-80 and 0.08% (w/v) yeast extract at moisture level of 70-75% significantly (p value < 0.05) improved hydrolytic efficiency of the crude enzyme extract by 2.2-fold. This study provides baseline data that will be useful in developing a low-cost enzyme-based process for hydrolyzing mushrooms to recover high-value products.

The 'LipoYeasts' project: using the oleaginous yeast Yarrowia lipolytica in combination with specific bacterial genes for the bioconversion of lipids, fats and oils into high-value products.

The oleochemical industry is currently still dominated by conventional chemistry, with biotechnology only starting to play a more prominent role, primarily with respect to the biosurfactants or lipases, e.g. as detergents, or for biofuel production. A major bottleneck for all further biotechnological applications is the problem of the initial mobilization of cheap and vastly available lipid and oil substrates, which are then to be transformed into high-value biotechnological, nutritional or pharmacological products. Under the EU-sponsored LipoYeasts project we are developing the oleaginous yeast Yarrowia lipolytica into a versatile and high-throughput microbial factory that, by use of specific enzymatic pathways from hydrocarbonoclastic bacteria, efficiently mobilizes lipids by directing its versatile lipid metabolism towards the production of industrially valuable lipid-derived compounds like wax esters (WE), isoprenoid-derived compounds (carotenoids, polyenic carotenoid ester), polyhydroxyalkanoates (PHAs) and free hydroxylated fatty acids (HFAs). Different lipid stocks (petroleum, alkane, vegetable oil, fatty acid) and combinations thereof are being assessed as substrates in combination with different mutant and recombinant strains of Y. lipolytica, in order to modulate the composition and yields of the produced added-value products.

Population genetics of the potentially invasive African fruit fly species, Ceratitis rosa and Ceratitis fasciventris (Diptera: Tephritidae).

A set of 10 microsatellite markers was used to survey the levels of genetic variability and to analyse the genetic aspects of the population dynamics of two potentially invasive pest fruit fly species, Ceratitis rosa and C. fasciventris, in Africa. The loci were derived from the closely related species, C. capitata. The degree of microsatellite polymorphism in C. rosa and C. fasciventris was extensive and comparable to that of C. capitata. In C. rosa, the evolution of microsatellite polymorphism in its distribution area reflects the colonization history of this species. The mainland populations are more polymorphic than the island populations. Low levels of differentiation were found within the Africa mainland area, while greater levels of differentiation affect the islands. Ceratitis fasciventris is a central-east African species. The microsatellite data over the Uganda/Kenya spatial scale suggest a recent expansion and possibly continuing gene flow within this area. The microsatellite variability data from C. rosa and C. fasciventris, together with those of C. capitata, support the hypothesis of an east African origin of the Ceratitis spp.

Comparative analysis of microsatellite loci in four fruit fly species of the genus Ceratitis (Diptera: Tephritidae).

The possibility to cross-species amplify microsatellites in fruit flies of the genus Ceratitis was tested with the polymerase chain reaction (PCR) by analysing 23 Ceratitis capitata (Wiedemann) microsatellite markers on the genomic DNA of three other economically important, congeneric species: C. rosa (Karsch), C. fasciventris (Bezzi) and C. cosyra (Walker). Twenty-two primer pairs produced amplification products in at least one of the three species tested. The majority of the products were similar, if not identical in size to those expected in C. capitata. The structures of the repeat motifs and their flanking sequences were examined for a total of 79 alleles from the three species. Sequence analysis revealed the same repeat type as the homologous C. capitata microsatellites in the majority of the loci, suggesting their utility for population analysis across the species range. A total of seven loci were differentially present/absent in C. capitata, C. rosa, C. fasciventris and C. cosyra, suggesting that it may be possible to differentiate these four species using a simple sequence repeat-based PCR assay. It is proposed that medfly-based microsatellite markers could be utilized in the identification and tracing of the geographical origins of colonist pest populations of the four tested species and in the assessment of their risk and invasive potentials; thereby assisting regulatory authorities in implementing quarantine restrictions and other pest control measures.

Repetitive sequences upstream of the pfg27/25 gene determine polymorphism in laboratory and natural lines of Plasmodium falciparum.

The structure of the genomic region located upstream of the gametocyte-specific gene pfg27/25 of Plasmodium falciparum was analysed in laboratory lines and field isolates of the parasite. The gene is located in a subtelomeric region of chromosome 13 in parasite clones 3D7 and HB3. Analysis of laboratory lines and field isolates of P. falciparum indicated that polymorphism upstream of pfg27/25 is mainly due to the structure of a repetitive DNA region located at about half a kilobase from the pfg27/25 coding sequence. Different types of repetitive sequences are present in this region, whose copy number is variable in different parasite lines. In addition a GC-rich sequence element contained in this region, which is proposed to be the startpoint of pfg27/25 mRNA, presents either a direct or a reverse orientation in different parasite lines. Genomic deletions upstream of the pfg27/25 gene are also described in two laboratory lines of the parasite, which eliminate two newly identified malaria genes. orf P and orf Gap, from the genome of these parasites. One of them, orf Gap, deleted from the reference parasite clone 3D7, is abundantly expressed as mature mRNA in asexual parasites. PCR analysis on 64 field isolates of P. falciparum indicated that orf P and orf Gap sequences are present in all tested samples of naturally propagating parasites.

Plasmodium falciparum: purification of the various gametocyte developmental stages from in vitro-cultivated parasites.

Cultivated Plasmodium falciparum gametocytes reach maturity in vitro in approximately 14-16 days, during which they pass through five morphologically distinct developmental stages. Purification of the earlier developmental stages has not been previously reported. We have modified the standard discontinuous Percoll gradient method for the separation of stage IV and V gametocytes to obtain enriched preparations of those and the earlier P. falciparum gametocyte stages. In contrast to the stages II, III, and IV, the mature stage V gametocytes from our gradient readily transformed into gametes. Such preparations may be useful in research studies on the mechanisms that underlie gametocytogenesis.

Antifolate drug resistance and point mutations in Plasmodium falciparum in Kenya.

Due to increased chloroquine resistance, the antifolate/sulpha drug combinations are becoming increasingly important in the chemotherapy of falciparum malaria. However, point mutations in the dihydrofolate reductase gene lead to resistance to the antifolate drugs. We therefore investigated the prevalence of the 6 reported point mutations in this gene among field isolates of Plasmodium falciparum from Kenya, to determine if the mutations correlated with resistance to pyrimethamine and the biguanides cycloguanil and chlorcycloguanil. We used a mutation-specific polymerase chain reaction technique to test for these reported mutations in 21 Kenyan isolates and 4 reference lines. We also amplified and directly sequenced the dihydrofolate reductase coding sequence from these parasites to confirm the results and test for other possible mutations. Of the reported mutations, we found S108N, which is the central mutation of pyrimethamine resistance, and mutations N51I and C59R, which modulate the levels of resistance and may confer decreases in response to cycloguanil that are folate and p-aminobenzoic acid dependent. No isolate possessed the paired point mutations S108T and A16V, or I164L and S108N, which have been associated with cycloguanil resistance in previous studies. These results provided supportive evidence for the combined use of a cycloguanil-class drug (e.g., chlorproguanil) and a sulpha drug (e.g., dapsone) against P.falciparum malaria in Kenya.

Serological investigation of HIV-1 variant subtype strains in transmission in Nairobi.

In a bid to determine the HIV-1 subtype variants in transmission in Nairobi and its possible association with clinical status, we screened 207 confirmed HIV-1 positive patients visiting HIV/AIDS laboratory at the Virus Research Centre in Nairobi between January and March 1994. We used a selfmade ELISA obtained from an established panel of HIV-1 V3 loop peptides (ANRS, France) and derived from seven isolates: MN, HXB2, SC, Z6, Z2, ELI and CDC4. Test samples were obtained from 95 blood donors and medical examination attendees, 57 patients with chronic diarrhoea, 31 confirmed pulmonary tuberculosis, 16 with pneumonia and 12 herpes zoster. Out of the total, 21.5% had antibodies against the MN strain, 19.1% had against the Z2 strain while reaction against the HXB2 strain was observed in 17.2%. SC, CDC4, Z6 and ELI had prevalences of 11.5%, 6.2%, 5.3% and 3.8% respectively. Fifteen per cent of the tested sera showed no reaction to any of the used peptides. Strong and significant associations were observed between the total number of strains a sample react to and the clinical state. We infer that both the North American consensus strains (MN and HXB2) and the African isolates (Z2 and Z6) are predominant in Nairobi. The correlation between antibody reactivity and clinical state is an interesting observation that necessitates an expanded study and, the use of strain specific peptides maybe a sensitive and easier method for use for molecular epidemiological purposes.

PIP: During January-March 1994, in Nairobi, Kenya, the sera of pre-university students, suspected AIDS/advanced HIV-infection cases, and blood donors were screened for HIV-1 antibodies at the Virus Research Centre. All confirmed HIV-1 positive samples were categorized according to the patient's clinical status. A self-made ELISA was obtained from an established panel of HIV-1 V3 loop peptides and derived from seven isolates (MN and HXB2 [North American strains], SC, CDC4, Z2 and Z6 [African strains], and ELI). The sera of the 22 confirmed HIV-1 negative students were used as negative controls. There were 207 confirmed HIV-1 cases (95 blood donors and 112 suspected AIDS/advanced HIV-infection cases). 64 (31%) and 112 (54%) samples reacted to at least 3 strains and no more than 2 strains, respectively. The remaining 31 (15%) samples did not react to any of the 7 peptide strains. Samples with CD4 cell counts greater than 500 x 1 million reacted significantly to more peptide strains than those with CD4 counts below 200 x 1 million (88% vs. 7%). Reactivity to specific strains were 21.5% for MN, 19.1% for Z2, 17.2% for HXB2, 11.5% for SC, 6.2% for CDC 4, 5.5% for Z6, and 3.8% for ELI. Anti-HXB2 antibodies were more common in blood donors than suspected AIDS/advanced HIV-infection cases (22% vs. 13%). AIDS/advanced HIV-infection cases were more likely to have no antibodies than blood donors (21% vs. 7%). A significant association existed between the number of peptide strains a patient could react to and the clinical state (p 0.01). Specifically, 77% of samples with no V3 antibodies to the seven strains had AIDS or advanced HIV infection while 55% of those which had cross reactivity with three or more strains were asymptomatic. Further research is needed to better understand this correlation. These findings suggest that use of strain specific peptides may be a sensitive and easier method for use for molecular epidemiological purposes.