afsS is a target of AfsR, a transcriptional factor with ATPase activity that globally controls secondary metabolism in Streptomyces coelicolor A3 (2) cally separable, and the two functions are modulated by phosphorylation on serine and threonine residues.Introduction afsR, encoding a 993-amino-acid protein, was cloned from Streptomyces coelicolor A3(2) as a gene that caused Streptomyces lividans to overproduce the pigmented antibiotics actinorhodin and undecylprodigiosin and A-factor (Horinouchi et al., 1983;Horinouchi and Beppu, 1984). The cloned gene 'awakened' transcription of the actinorhodin biosynthetic pathway in S. lividans, thus causing the host to produce actinorhodin (Horinouchi et al., 1989). The cloned gene had originally been thought to represent afsB, as it phenotypically complemented an afsB mutation (Horinouchi et al., 1983). Later, Stein and Cohen (1989) found by genetic analysis that the cloned DNA did not correspond to the afsB locus and renamed it afsR. It also induced overproduction of the calciumdependent antibiotic (CDA) (Horinouchi et al., 1989;. Disruption of the S. coelicolor A3(2) chromosomal afsR gene resulted in significant, but not complete, loss of pigment production. Floriano and Bibb (1996) also observed reduced production of actinorhodin, undecylprodigiosin and CDA in an afsR disruptant. We have thus called afsR a global regulatory gene for secondary metabolite formation.The N-terminal portion of AfsR shows similarity to ActII-ORF4 (Fernández-Moreno et al., 1991), RedD (Narva and Feitelson, 1990), DnrI (Stutzman-Engwall et al., 1992) and CcaR (Pérez-Llarena et al., 1997), all of which are pathway-specific transcriptional regulators that activate transcription of the genes in the respective gene clusters through DNA binding to specific nucleotide sequences (Wietzorrek and Bibb, 1997). In fact, ActII-ORF4 has been shown to recognize and bind specific regions in the actinorhodin biosynthetic gene cluster, thus activating the transcription of act genes (Arias et al., 1999). DnrI also binds promoter regions in the daunorubicin biosynthetic gene cluster and activates their transcription (Tang et al., 1996). However, afsR cannot substitute for the pathwayspecific regulators actII-ORF4 and redD, but may operate independently of these regulatory proteins to influence antibiotic production (Floriano and Bibb, 1996). AfsR was therefore thought to bind some specific DNA sequences

Molecular detection has overcome limitations of microscopic examination by providing greater sensitivity and specificity in Plasmodium species detection. The objective of the present study was to develop a quantitative real-time polymerase chain reaction coupled with high-resolution melting (qRT-PCR-HRM) assay for rapid, accurate and simultaneous detection of all five human Plasmodium spp. A pair of primers targeted the 18S SSU rRNA gene of the Plasmodium spp. was designed for qRT-PCR-HRM assay development. Analytical sensitivity and specificity of the assay were evaluated. Samples collected from 229 malaria suspected patients recruited from Sabah, Malaysia were screened using the assay and results were compared with data obtained using PlasmoNexTM, a hexaplex PCR system. The qRT-PCR-HRM assay was able to detect and discriminate the five Plasmodium spp. with lowest detection limits of 1–100 copy numbers without nonspecific amplifications. The detection of Plasmodium spp. in clinical samples using this assay also achieved 100% concordance with that obtained using PlasmoNexTM. This indicated that the diagnostic sensitivity and specificity of this assay in Plasmodium spp. detection is comparable with those of PlasmoNexTM. The qRT-PCR-HRM assay is simple, produces results in two hours and enables high-throughput screening. Thus, it is an alternative method for rapid and accurate malaria diagnosis.

Thalassemia can lead to severe transfusion-dependent anemia, and it is the most common genetic disorder in Malaysia. This paper aims to determine the prevalence of thalassemia in the Kadazandusuns, the largest indigenous group in Sabah, East Malaysia. α- and β-thalassemia were confirmed in 33.6% and 12.8%, of the individuals studied respectively. The high prevalence of α- and β-thalassemia in the Kadazandusuns indicates that thalassemia screening, genetic counseling, and prenatal diagnosis should be included as part of their healthcare system. This preliminary paper serves as a baseline for further investigations into the health and genetic defects of the major indigenous population in Sabah, East Malaysia.

Malaria remains one of the major killers of humankind and persists to threaten the lives of more than one-third of the world's population. Given that human malaria can now be caused by five species of Plasmodium, i.e., Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale, and the recently included Plasmodium knowlesi, there is a critical need not only to augment global health efforts in malaria control but also, more importantly, to develop a rapid, accurate, species-sensitive/species-specific, and economically effective diagnostic method for malaria caused by these five species. Therefore, in the present study, a straightforward single-step hexaplex PCR system targeting five human Plasmodium 18S small-subunit rRNAs (ssu rRNAs) was designed, and the system successfully detected all five human malaria parasites. In addition, this system enables the differentiation of single infection as well as mixed infections up to the two-species level. This assay was validated with 50 randomly blinded test and 184 clinical samples suspected to indicate malaria. This hexaplex PCR system is not only an ideal alternative for routine malaria diagnosis in laboratories with conventional PCR machines but also adds value to diagnoses when there is a lack of an experienced microscopist or/and when the parasite morphology is confusing. Indeed, this system will definitely enhance the accuracy and accelerate the speed in the diagnosis of malaria, as well as improve the efficacy of malaria treatment and control, in addition to providing reliable data from epidemiological surveillance studies.

BackgroundMalaria is a vector borne-parasitic disease transmitted through the bite of the infective female Anopheles mosquitoes. Five Plasmodium species have been recognized by World Health Organization (WHO) as the causative agents of human malaria. Generally, microscopic examination is the gold standard for routine malaria diagnosis. However, molecular PCR assays in many cases have shown improvement on the sensitivity and specificity over microscopic or other immunochromatographic assays.MethodsThe present study attempts to screen 207 suspected malaria samples from patients seeking treatment in clinics around Sabah state, Malaysia, using two panels of multiplex PCRs, conventional PCR system (PlasmoNex™) and real-time PCR based on hydrolysis probe technology. Discordance results between two PCR assays were further confirmed by sequencing using 18S ssu rRNA species-specific primers.ResultsOf the 207 malaria samples, Plasmodium knowlesi (73.4% vs 72.0%) was the most prevalent species based on two PCR assays, followed by Plasmodium falciparum (15.9% vs 17.9%), and Plasmodium vivax (9.7% vs 7.7%), respectively. Neither Plasmodium malariae nor Plasmodium ovale was detected in this study. Nine discrepant species identification based on both the PCR assays were further confirmed through DNA sequencing. Species-specific real-time PCR only accurately diagnosed 198 of 207 (95.7%) malaria samples up to species level in contrast to PlasmoNex™ assay which had 100% sensitivity and specificity based on sequencing results.ConclusionsMultiplex PCR accelerate the speed in the diagnosis of malaria. The PlasmoNex™ PCR assay seems to be more accurate than real-time PCR in the speciation of all five human malaria parasites. The present study also showed a significant increase of the potential fatal P. knowlesi infection in Sabah state as revealed by molecular PCR assays.

BackgroundPlasmodium knowlesi is a simian malaria parasite that is widespread in humans in Malaysian Borneo. However, little is known about the incidence and distribution of this parasite in the Sandakan division, Malaysian Borneo. Therefore, the aim of the present epidemiological study was to investigate the incidence and distribution of P. knowlesi as well as other Plasmodium species in this division based on a most recent developed hexaplex PCR system (PlasmoNex™).MethodsA total of 189 whole blood samples were collected from Telupid Health Clinic, Sabah, Malaysia, from 2008 to 2011. All patients who participated in the study were microscopically malaria positive before recruitment. Complete demographic details and haematological profiles were obtained from 85 patients (13 females and 72 males). Identification of Plasmodium species was conducted using PlasmoNex™ targeting the 18S ssu rRNA gene.ResultsA total of 178 samples were positive for Plasmodium species by using PlasmoNex™. Plasmodium falciparum was identified in 68 samples (38.2%) followed by 64 cases (36.0%) of Plasmodium vivax, 42 (23.6%) cases of P. knowlesi, two (1.1%) cases of Plasmodium malariae and two (1.1%) mixed-species infections (i e, P. vivax/P. falciparum). Thirty-five PlasmoNex™ positive P. knowlesi samples were misdiagnosed as P. malariae by microscopy. Plasmodium knowlesi was detected in all four districts of Sandakan division with the highest incidence in the Kinabatangan district. Thrombocytopaenia and anaemia showed to be the most frequent malaria-associated haematological complications in this study.ConclusionsThe discovery of P. knowlesi in Sandakan division showed that prospective studies on the epidemiological risk factors and transmission dynamics of P. knowlesi in these areas are crucial in order to develop strategies for effective malaria control. The availability of advanced diagnostic tool PlasmoNex™ enhanced the accuracy and accelerated the speed in the diagnosis of malaria.

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In Streptomyces coelicolor A3(2), a protein serine/threonine kinase (AfsK) and its target protein (AfsR) control secondary metabolism. AfsK and AfsR homologues (AfsK-g and AfsR-g) from Streptomyces griseus showed high endto-end similarity in amino acid sequence with the respective S. coelicolor A3(2) proteins, as determined by cloning and nucleotide sequencing. AfsK-g and a fusion protein between AfsK-g and thioredoxin (TRX-AfsK-g) produced in high yield as inclusion bodies in Escherichia coli were solubilized with urea, purified by column chromatography and then refolded to an active form by dialysis to gradually remove the urea. AfsR-g was also fused to glutathione S-transferase (GST-AfsR-g) ; the fusion product in the soluble fraction in E. coli was purified. Incubation of AfsK-g or TRX-AfsK-g in the presence of [γ-32 P]ATP yielded autophosphorylated products containing phosphoserine and phosphothreonine residues. In addition, TRX-AfsK-g phosphorylated serine and threonine residues of GST-AfsR-g in the presence of [γ-32 P]ATP. Disruption of chromosomal afsK-g had no effect on A-factor or streptomycin production, irrespective of the culture conditions. The afsK-g disruptants did not form aerial mycelium or spores on media containing glucose at concentrations higher than 1 %, but did form spores on mannitol-and glycerol-containing media ; this suggests that afsK-g is essential for morphogenesis in the presence of glucose. Introduction of afsK-g restored aerial mycelium formation in the disruptants. The phenotype of afsR-g disruptants was similar to that of afsK-g disruptants ; introduction of afsR-g restored the defect in aerial mycelium formation on glucose-containing medium. Thus the AfsK/AfsR system in S. griseus is conditionally needed for morphological differentiation, whereas in S. coelicolor A3(2) it is conditionally involved in secondary metabolism.