A quinazoline-based HDAC inhibitor affects gene expression pathways involved in cholesterol biosynthesis and mevalonate in prostate cancer cells.

Chronic inflammation can lead to the development of cancers and resolution of inflammation is an ongoing challenge. Inflammation can result from dysregulation of the epigenome and a number of compounds that modify the epigenome are in clinical use. In this study the anti-inflammatory and anti-cancer effects of a quinazoline epigenetic-modulator compound were determined in prostate cancer cell lines using a non-hypothesis driven transcriptomics strategy utilising the Affymetrix PrimeView® Human Gene Expression microarray. GATHER and IPA software were used to analyse the data and to provide information on significantly modified biological processes, pathways and networks. A number of genes were differentially expressed in both PC3 and DU145 prostate cancer cell lines. The top canonical pathways that frequently arose across both cell lines at a number of time points included cholesterol biosynthesis and metabolism, and the mevalonate pathway. Targeting of sterol and mevalonate pathways may be a powerful anticancer approach.

Characterisation of the pncA gene in Mycobacterium tuberculosis isolates from Gauteng, South Africa.

SETTING: The use of pyrazinamide (PZA) is important for the treatment of Mycobacterium tuberculosis as it is bactericidal to semi-dormant mycobacteria that are not affected by other drugs. The incidence of resistance to PZA and other drugs used in the treatment of M. tuberculosis is increasing in South Africa. OBJECTIVE: To characterise the pncA gene of M. tuberculosis isolates from Gauteng, South Africa, and to develop a rapid diagnostic method. DESIGN: The pncA gene and the putative regulatory gene were characterised by sequence analysis in a total of six PZA susceptible and 15 resistant isolates. The association with classical PZA susceptibility testing and PZase activity was determined. RESULTS: All PZA-resistant isolates were PZase negative as well as resistant to at least one other anti-tuberculosis drugs. Mutations were identified throughout the length of the pncA gene in 10/15 PZA-resistant isolates. Five lacked PZase activity, but the wild type pncA sequence was present. In all six PZase-positive strains, a PZA-susceptible pattern was obtained on BACTEC and the wild type pncA sequence was present. CONCLUSION: Sequencing is an effective means to identify mutations in the pncA gene in M. tuberculosis and therefore resistance to PZA. The fact that some PZA-resistant M. tuberculosis isolates lack mutations in the pncA gene suggests that alternative mechanisms for drug resistance exist. In PZase negative strains with no genetic changes which are resistant to 100 microg/ml and susceptible to 300 microg/ml, 300 microg/ml may be a more reliable breakpoint.

Estimation and management of genetic diversity in small populations of plains zebra (Equus quagga) in KwaZulu-Natal, South Africa.

Plains zebras (Equus quagga antiquorum) occur in few large, but many small, isolated populations in KwaZulu-Natal. Problems identified in small populations include reduced striping patterns on hind quarters, smaller size, elevated mortality rates and high number of still-births. Inbreeding may be implicated. Population viability analysis (PVA) was conducted with a computer model (VORTEX), and DNA and allozyme analyses were conducted to test the findings of the model. Using standard methods, DNA (PCR-RAPD) and allozyme diversity was assessed in blood samples from 72 plains zebra from four KwaZulu-Natal Nature Conservation Services (KZN-NCS) protected areas: Umfolozi Game Reserve (UGR), Albert Falls (AFNR), Vernon Crookes (VCNR) and Harold Johnson (HJNR) Nature Reserves. Populations of the latter three, small-sized (9-110 individuals) populations were seeded from the same source population (UGR: current population of 2000) during the past 25 years. Information from PCR-RAPD and allozyme analyses were compared with each other as well as to that predicted by population genetic modelling (using VORTEX). Allozyme heterozygosities were consistently high in all populations (12.1-12.9%), with no observable losses associated with reduced population size. On the other hand, percentage loss of polymorphism (20-39%) calculated from the PCR-RAPD study appeared to be positively correlated with the loss of heterozygosity predicted by population viability analysis (PVA), and negatively correlated with population size. On the basis of the above results, a policy of translocation was advocated for small, intensely managed populations of zebras, whereby a harem should be translocated every five years for a population size of nine (HJNR), while for a population size of 110 (VCNR) translocations should take place every 15 years if heterozygosity is to be maintained at more than 90% within each population over 100 years.

Genomic mutations in the katG, inhA and aphC genes are useful for the prediction of isoniazid resistance in Mycobacterium tuberculosis isolates from Kwazulu Natal, South Africa.

Genotypic analysis of isoniazid (INH) resistance in 79 isolates of M. tuberculosis (MTB) was undertaken by PCR-single strand conformation polymorphism (SSCP), Msp1 restriction enzyme analysis and sequence analysis of specific regions of three genes (part of the coding sequence of katG, and promoter regions of the inhA operon and ahpC) in order to determine the particular allelic variants within these genes. The epidemiologic relatedness was determined using IS6110 and polymorphic G-C region (PGRS (MTB484(1)) based restriction fragment length polymorphism (RFLP). Mutations in katG, inhA locus and ahpC were identified in 77/79, 19/79 and 10/79 isolates respectively. The ability of PCR-SSCP to detect mutations associated with INH resistance in katG, inhA and ahpC genes was 100% (CI 91.2-99.7%), 98.7% (CI 74.0-99.9%), and 100% (CI 69.2-100%) respectively. Specificity was 100%. All isolates with mutations in the 209 bp fragment of the MTB katG gene containing the Ser315Thr codon were positive by PCR-RFLP using Msp1 enzyme restriction analysis. Sixteen of 19 isolates with alterations on the 3' end of the ribosome binding site upstream of mabA in inhA locus simultaneously harbored Ser315Thr mutations in KatG. In 9/10 isolates, mutations in the ahpC promoter region were located in the 105 bp oxyR-ahpC intergenic region. None of 17 INH drug susceptible isolates harbored mutations in any of the three genetic regions, although the katG1 allele (Arg 463 Leu) was present in one isolate. Characterization by IS6110/PGRS(MTB484(1))RFLP analysis revealed that a number of drug resistant clones are widespread in the community. We conclude that the frequency of the Ser315Thr katG mutation in the local strain population makes the PCR-RFLP MTB katG assay a reliable, rapid and useful method for detecting INH resistance.

Rapid screening of Mycobacterium tuberculosis for susceptibility to rifampicin and streptomycin.

OBJECTIVE: To investigate rapid detection of drug-resistant tuberculosis using the genotypic Inno-LiPA Rif TB assay and a novel, low-cost, bacteriophage-based susceptibility assay. DESIGN: The performance of the microwell phage replication assay (MPRA) on 18 isolates from suspected multidrug-resistant tuberculosis patients was compared to the LiPA assay performed directly on sputum specimens. Mutations in the rpoB gene identified by LiPA that confer resistance to rifampicin (RMP) were confirmed by DNA sequencing, while susceptibilities were confirmed by the proportion method and BACTEC. A further 19 isolates undergoing routine screening for both RMP and streptomycin susceptibility were included for comparison. RESULTS: Susceptibility to RMP was determined for 17/18 (94.4%) sputum specimens tested by LiPA. Correlation between MPRA, molecular and conventional methods was 100% for the detection of RMP susceptibility. However, for susceptibility to streptomycin one discrepant result was found: an isolate susceptible to streptomycin by the proportion method was found resistant by MPRA to 2 microg/ml of streptomycin. Similarly, an isolate initially resistant by MPRA upon re-testing was found susceptible in agreement with the conventional method. CONCLUSION: LiPA enables rapid detection of drug-resistant infection, while MPRA offers simple, low-tech testing of drug susceptibilities that may be appropriate for application in low-income countries.