Kenneth G. Laing

Mefloquine resistance in Plasmodium falciparum and increased pfmdr1 gene copy number.

BACKGROUNDnThe borders of Thailand harbour the worlds most multidrug resistant Plasmodium falciparum parasites. In 1984 mefloquine was introduced as treatment for uncomplicated falciparum malaria, but substantial resistance developed within 6 years. A combination of artesunate with mefloquine now cures more than 95% of acute infections. For both treatment regimens, the underlying mechanisms of resistance are not known.nnnMETHODSnThe relation between polymorphisms in the P falciparum multidrug resistant gene 1 (pfmdr1) and the in-vitro and in-vivo responses to mefloquine were assessed in 618 samples from patients with falciparum malaria studied prospectively over 12 years. pfmdr1 copy number was assessed by a robust real-time PCR assay. Single nucleotide polymorphisms of pfmdr1, P falciparum chloroquine resistance transporter gene (pfcrt) and P falciparum Ca2+ ATPase gene (pfATP6) were assessed by PCR-restriction fragment length polymorphism.nnnFINDINGSnIncreased copy number of pfmdr1 was the most important determinant of in-vitro and in-vivo resistance to mefloquine, and also to reduced artesunate sensitivity in vitro. In a Cox regression model with control for known confounders, increased pfmdr1 copy number was associated with an attributable hazard ratio (AHR) for treatment failure of 6.3 (95% CI 2.9-13.8, p<0.001) after mefloquine monotherapy and 5.4 (2.0-14.6, p=0.001) after artesunate-mefloquine therapy. Single nucleotide polymorphisms in pfmdr1 were associated with increased mefloquine susceptibility in vitro, but not in vivo.nnnINTERPRETATIONnAmplification in pfmdr1 is the main cause of resistance to mefloquine in falciparum malaria.nnnRELEVANCE TO PRACTICEnMultidrug resistant P falciparum malaria is common in southeast Asia, but difficult to identify and treat. Genes that encode parasite transport proteins maybe involved in export of drugs and so cause resistance. In this study we show that increase in copy number of pfmdr1, a gene encoding a parasite transport protein, is the best overall predictor of treatment failure with mefloquine. Increase in pfmdr1 copy number predicts failure even after chemotherapy with the highly effective combination of mefloquine and 3 days artesunate. Monitoring of pfmdr1 copy number will be useful in epidemiological surveys of drug resistance in P falciparum, and potentially for predicting treatment failure in individual patients.

The mRNA of the translationally controlled tumor protein P23/TCTP is a highly structured RNA, which activates the dsRNA-dependent protein kinase PKR

The dsRNA-activated protein kinase PKR is involved in signal transduction pathways that mediate cellular processes as diverse as cell growth and differentiation, the stress response, and apoptosis. PKR was originally described as an interferon-inducible elF2alpha kinase involved in the antiviral defense mechanism of the cell. The interaction of the kinase with specific viral RNAs has been studied in much detail, but information about cellular mRNAs, which are able to bind and activate PKR, is scarce. In search for such cellular mRNAs, we developed a cloning strategy to identify individual mRNA species from the dsRNA-rich fraction of Daudi cell poly(A)+ RNA. Two out of five cDNA clones we obtained contained sequences derived from the mRNA of the translationally controlled tumor protein P23/TCTP, indicating that this mRNA is present in the dsRNA-rich fraction. Secondary structure predictions and gel electrophoretic mobility investigations on P23/TCTP transcripts confirmed the potential of this mRNA to form extensive secondary structure. A full-length P23 transcript, but not a truncated version thereof, was able to bind to PKR in vitro and in vivo. Transient transfection experiments in human 293 cells showed that coexpression of full-length P23 mRNA leads to partial inhibition of the expression of a beta-galactosidase reporter gene in trans. Additional coexpression of a dominant negative mutant of PKR or of adenovirus VA1 RNA suppressed this inhibition, indicating that it is mediated by PKR. Studies on P23/TCTP expression in cells from PKR-knockout mice suggest that P23/TCTP mRNA translation is regulated by PKR. Hence, our results demonstrate that the mRNA of P23/TCTP may both activate PKR and be subject to translational regulation by this kinase.

Regulation of the interferon-inducible eIF-2α protein kinase by small RNAs

This review describes the structure and function of the double-stranded RNA-dependent protein kinase (PKR) and its interaction with RNA activators and inhibitors. The abilities of small virally-encoded RNAs such as VAI RNA of adenovirus, the Epstein-Barr virus encoded (EBER) RNAs and the Tat-responsive region RNA of HIV-1 to bind to and regulate PKR are reviewed, and the physiological implications of such regulation for the control of viral replication and cell growth are discussed. The potential effects on the activity of PKR of other proteins that bind double-stranded RNA and/or small viral and cellular RNAs are also considered.

Glass slide microarrays for bacterial genomes

Abstract The acquisition of complete bacterial genome sequence has coincided with important advances in the development of high-density nucleic acid arrays and hybridization technology. Although ‘chip’ technology holds great promise for the future, at the present time spotted arrays represent an accessible format with sufficient reporter element density to interrogate bacterial genomes by comparative genomics and gene expression profiling. A rapidly growing literature using microarrays in functional genomics studies on bacteria confirms the reality and robust nature of spotted DNA microarrays for small genome organisms and also supports the use of total RNA samples for transcriptome analysis. However, microarray experimental systems are an emerging technology and optimization of nearly every step is an ongoing process. Thus, current methods are subject to development and have limitations and pitfalls. This should not be an argument against their widespread use, since the power of arrays as they are now is of undisputed biological significance and may be regarded as a proven technology. The future challenges for microarrays lies with mathematical and statistical analysis of high-dimensional data sets that are produced even in simple biological experiments.

Fidelity and reproducibility of antisense RNA amplification for the study of gene expression in human CD34+ haemopoietic stem and progenitor cells

Summary. Microarrays provide a powerful tool for the study of haemopoietic stem and progenitor cells (HSC). Because of the low frequency of HSC, it is rarely feasible to obtain enough mRNA for microarray hybridizations, and amplification will be necessary. Antisense RNA (aRNA) amplification is reported to give high‐fidelity amplification, but most studies have used only qualitative validation. Before applying aRNA amplification to the study of HSC, we wished to determine its fidelity and reproducibility, and whether statistically significant results can be obtained. We found that aRNA amplification introduced biases into relative RNA abundance. However, these biases were extremely consistent, and valid comparisons could be made, if amplified RNA was compared with amplified RNA. By applying this method to the effect of interferon‐γ and tumour necrosis factor‐α on normal primary CD34+ HSC, biologically significant differences could be detected, including potential mechanisms for resistance of CD34+ cells to CD95‐mediated apoptosis and evidence of the differentiating effects of the cytokines. Differences of twofold or less were detected, and most of these differences attained statistical significance after triplicate experiments. These data demonstrate that aRNA amplification can be used with microarray hybridization to study the transcriptional profiles of small numbers of primary CD34+ HSC.

Identification of Streptococcus suis Meningitis through Population-Based Surveillance, Togo, 2010–2014

During 2010–2014, we enrolled 511 patients with suspected bacterial meningitis into surveillance in 2 districts of northern Togo. We identified 15 persons with Streptococcus suis infection; 10 had occupational contact with pigs, and 12 suffered neurologic sequelae. S. suis testing should be considered in rural areas of the African meningitis belt.

Microarrays for Microbes: the BμG@S Approach

The Bacterial Microarray Group at St George’s Hospital Medical School (BμG@S; http://bugs. sghms.ac.uk) has been funded by The Wellcome Trust as part of the Resources for Functional Genomics Initiative. A 5 year programme grant entitled ‘A Multi-Collaborative Microbial Pathogen Microarray Facility’ was awarded to Dr Philip Butcher and Joseph Mangan (St. George’s Hospital Medical School), Professor Brendan Wren (London School of Hygiene and Tropical Medicine), Professor Neil Stoker (Royal Veterinary College, London) and Dr Keith Vass (Beatson Institute/Glasgow University). The aim of this funding is to enable a community of collaborating researchers with a common interest in bacterial pathogenesis to have rapid, flexible and cost-effective access to current microarray technology. Rather than each individual group having to invest in the equipment and the lengthy learning and optimization process involved in microarray set-up, the aim was to centralize the expertise within one group that would then be made freely available to collaborating groups as a functional genomics resource. The project was funded to produce whole-genome DNA microarrays for 12 bacterial pathogens within 2 years and provide training and support in the use of the arrays over a period of 5 years. This article provides an overview of the project organization, the technology and support involved in producing and using the microarrays, future developments and a summary of the progress to date.