The multivalent PDZ domain-containing protein PDZK1 regulates transport activity of renal urate-anion exchanger URAT1 via its C terminus

The urate-anion exchanger URAT1 is a member of the organic anion transporter (OAT) family that regulates blood urate level in humans and is targeted by uricosuric and antiuricosuric agents. URAT1 is expressed only in the kidney, where it is thought to participate in tubular urate reabsorption. We found that the multivalent PDZ (PSD-95, Drosophila discs-large protein, Zonula occludens protein 1) domain-containing protein, PDZK1 interacts with URAT1 in a yeast two-hybrid screen. Such an interaction requires the PDZ motif of URAT1 in its extreme intracellular C-terminal region and the first, second, and fourth PDZ domains of PDZK1 as identified by yeast two-hybrid assay, in vitro binding assay and surface plasmon resonance analysis (K(D) = 1.97-514 nM). Coimmunoprecipitation studies revealed that the wild-type URAT1, but not its mutant lacking the PDZ-motif, directly interacts with PDZK1. Colocalization of URAT1 and PDZK1 was observed at the apical membrane of renal proximal tubular cells. The association of URAT1 with PDZK1 enhanced urate transport activities in HEK293 cells (1.4-fold), and the deletion of the URAT1 C-terminal PDZ motif abolished this effect. The augmentation of the transport activity was accompanied by a significant increase in the V(max) of urate transport via URAT1 and was associated with the increased surface expression level of URAT1 protein from HEK293 cells stably expressing URAT1 transfected with PDZK1. Taken together, the present study indicates the novel role of PDZK1 in regulating the functional activity of URAT1-mediated urate transport in the apical membrane of renal proximal tubules.     

Drug resistance-conferring mutations in Mycobacterium tuberculosis from Madang, Papua New Guinea

ABSTRACT: BACKGROUND: Monitoring drug resistance in Mycobacterium tuberculosis is essential to curb the spread of tuberculosis (TB). Unfortunately, drug susceptibility testing is currently not available in Papua New Guinea (PNG) and that impairs TB control in this country. We report for the first time M. tuberculosis mutations associated with resistance to first and second-line anti-TB drugs in Madang, PNG. A molecular cluster analysis was performed to identify M. tuberculosis transmission in that region. RESULTS: Phenotypic drug susceptibility tests showed 15.7% resistance to at least one drug and 5.2% multidrug resistant (MDR) TB. Rifampicin resistant strains had the rpoB mutations D516F, D516Y or S531L; isoniazid resistant strains had the mutations katG S315T or inhA promoter C15T; streptomycin resistant strains had the mutations rpsL K43R, K88Q, K88R), rrs A514C or gidB V77G. The molecular cluster analysis indicated evidence for transmission of resistant strain. CONCLUSIONS: We observed a substantial rate of MDR-TB in the Madang area of PNG associated with mutations in specific genes. A close monitoring of drug resistance is therefore urgently required, particularly in the presence of drug-resistant M. tuberculosis transmission. In the absence of phenotypic drug susceptibility testing in PNG, molecular assays for drug resistance monitoring would be of advantage.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Zingiber petiolatum</Emphasis> (Holttum)

Summary We describe an in vitro propagation protocol for Zingiber petiolatum (Holttum), I. Theilade, a rare species from the southern part of Thailand. Fruits were surface-sterilized and seeds germinated on Murashige and Skoog medium (MS) medium supplemented with 3% sucrose. Three-month-old seedlings were used as initial plant material for in vitro propagation. Terminal buds of the plants were inoculated on MS medium containing 6-benzylaminopurine (BA; 2.2–35.5 μM) alone or in combination with 1-naphthaleneacetic acid (0.5 μM). Eight weeks after inoculation, the cultures were transferred to MS medium without plant growth regulators for 4wk. The cultures transferred from MS medium with 17.8 μM BA revealed the highest shoot induction rate of 6.1±0.7 shoots per explant. Rooting was spontaneously achieved in MS medium without plant growth regulators. Rooted plants were successfully transplanted to soil.     

Factors Affecting Attendance at and Timing of Formal Antenatal Care: Results from a Qualitative Study in Madang,Papua New Guinea

Background

Appropriate antenatal care (ANC) is key for the health of mother and child. However, in Papua New Guinea (PNG), only a third of women receive any ANC during pregnancy. Drawing on qualitative research, this paper explores the influences on ANC attendance and timing of first visit in the Madang region of Papua New Guinea.

Methods

Data were collected in three sites utilizing several qualitative methods: free-listing and sorting of terms and definitions, focus group discussions, in-depth interviews, observation in health care facilities and case studies of pregnant women. Respondents included pregnant women, their relatives, biomedical and traditional health providers, opinion leaders and community members.

Results

Although generally reported to be important, respondents’ understanding of the procedures involved in ANC was limited. Factors influencing attendance fell into three main categories: accessibility, attitudes to ANC, and interpersonal issues. Although women saw accessibility (distance and cost) as a barrier, those who lived close to health facilities and could easily afford ANC also demonstrated poor attendance. Attitudes were shaped by previous experiences of ANC, such as waiting times, quality of care, and perceptions of preventative care and medical interventions during pregnancy. Interpersonal factors included relationships with healthcare providers, pregnancy disclosure, and family conflict. A desire to avoid repeat clinic visits, ideas about the strength of the fetus and parity were particularly relevant to the timing of first ANC visit.

Conclusions

This long-term in-depth study (the first of its kind in Madang, PNG) shows how socio-cultural and economic factors influence ANC attendance. These factors must be addressed to encourage timely ANC visits: interventions could focus on ANC delivery in health facilities, for example, by addressing healthcare staff’s attitudes towards pregnant women.     

Knowledge,Attitudes, and Practices Concerning Malaria in Pregnancy: Results from a Qualitative Study in Madang,Papua New Guinea

Background

Malaria is the leading cause of illness and death in Papua New Guinea (PNG). Infection during pregnancy with falciparum or vivax malaria, as occurs in PNG, has health implications for mother and child, causing complications such as maternal anemia, low birth weight and miscarriage. This article explores knowledge, attitudes and practices concerning malaria during pregnancy and it’s prevention in Madang, PNG, a high prevalence area.

Methods

As part of a qualitative study in Madang, exploring MiP, participatory techniques (free-listing and sorting) were conducted along with focus group discussions, in-depth interviews (with pregnant women, health staff and other community members) and observations in the local community and health facilities.

Results

The main themes explored were attitudes towards and knowledge of MiP, its risks, and prevention. Although there was a general awareness of the term “malaria”, it was often conflated with general sickness or with pregnancy-related symptoms. Moreover, many preventive methods for MiP were related to practices of general healthy living. Indeed, varied messages from health staff about the risks of MiP were observed. In addition to ideas about the seriousness and risk of MiP, other factors influenced the uptake of interventions: availability and perceived comfort of sleeping under insecticide-treated mosquito nets were important determinants of usage, and women’s heavy workload influenced Chloroquine adherence.

Conclusion

The non-specific symptoms of MiP and its resultant conflation with symptoms of pregnancy that are perceived as normal have implications for MiP prevention and control. However, in Madang, PNG, this was compounded by the inadequacy of health staff’s message about MiP.     

Improved Quantification,Propagation, Purification and Storage of the Obligate Intracellular Human Pathogen Orientia tsutsugamushi

Background

Scrub typhus is a leading cause of serious febrile illness in rural Southeast Asia. The causative agent, Orientia tsutsugamushi, is an obligate intracellular bacterium that is transmitted to humans by the bite of a Leptotrombidium mite. Research into the basic mechanisms of cell biology and pathogenicity of O. tsutsugamushi has lagged behind that of other important human pathogens. One reason for this is that O. tsutsugamushi is an obligate intracellular bacterium that can only be cultured in mammalian cells and that requires specific methodologies for propagation and analysis. Here, we have performed a body of work designed to improve methods for quantification, propagation, purification and long-term storage of this important but neglected human pathogen. These results will be useful to other researchers working on O. tsutsugamushi and also other obligate intracellular pathogens such as those in the Rickettsiales and Chlamydiales families.

Methodology

A clinical isolate of O. tsutsugamushi was grown in cultured mouse embryonic fibroblast (L929) cells. Bacterial growth was measured using an O. tsutsugamushi-specific qPCR assay. Conditions leading to improvements in viability and growth were monitored in terms of the effect on bacterial cell number after growth in cultured mammalian cells.

Key results

• Development of a standardised growth assay to quantify bacterial replication and viability in vitro.
• Quantitative comparison of different DNA extraction methods.
• Quantification of the effect on growth of FBS concentration, daunorubicin supplementation, media composition, host cell confluence at infection and frequency of media replacement.
• Optimisation of bacterial purification including a comparison of host cell lysis methods, purification temperature, bacterial yield calculations and bacterial pelleting at different centrifugation speeds.
• Quantification of bacterial viability loss after long term storage and freezing under a range of conditions including different freezing buffers and different rates of freezing.

Conclusions

Here we present a standardised method for comparing the viability of O. tsutsugamushi after purification, treatment and propagation under various conditions. Taken together, we present a body of data to support improved techniques for propagation, purification and storage of this organism. This data will be useful both for improving clinical isolation rates as well as performing in vitro cell biology experiments.     

Characterization and PCR Detection Of Binary,Pir-Like Toxins from Vibrio parahaemolyticus Isolates that Cause Acute Hepatopancreatic Necrosis Disease (AHPND) in Shrimp

Unique isolates of Vibrio parahaemolyticus (VPAHPND) have previously been identified as the causative agent of acute hepatopancreatic necrosis disease (AHPND) in shrimp. AHPND is characterized by massive sloughing of tubule epithelial cells of the hepatopancreas (HP), proposed to be induced by soluble toxins released from VP_AHPND that colonize the shrimp stomach. Since these toxins (produced in broth culture) have been reported to cause AHPND pathology in reverse gavage bioassays with shrimp, we used ammonium sulfate precipitation to prepare protein fractions from broth cultures of VP_AHPND isolates for screening by reverse gavage assays. The dialyzed 60% ammonium sulfate fraction caused high mortality within 24–48 hours post-administration, and histological analysis of the moribund shrimp showed typical massive sloughing of hepatopancreatic tubule epithelial cells characteristic of AHPND. Analysis of the active fraction by SDS-PAGE revealed two major bands at marker levels of approximately 16 kDa (ToxA) and 50 kDa (ToxB). Mass spectrometry analysis followed by MASCOT analysis revealed that both proteins had similarity to hypothetical proteins of V. parahaemolyticus M0605 (contig034 GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"JALL01000066.1","term_id":"576300946","term_text":"JALL01000066.1"}}JALL01000066.1) and similarity to known binary insecticidal toxins called ''Photorhabdus insect related'' proteins A and B (Pir-A and Pir-B), respectively, produced by the symbiotic, nematode bacterium Photorhabdus luminescens. In in vivo tests, it was shown that recombinant ToxA and ToxB were both required in a dose dependent manner to cause AHPND pathology, indicating further similarity to Pir-A and -B. A single-step PCR method was designed for detection of the ToxA gene and was validated using 104 bacterial isolates consisting of 51 VPAHPND isolates, 34 non-AHPND VP isolates and 19 other isolates of bacteria commonly found in shrimp ponds (including other species of Vibrio and Photobacterium). The results showed 100% specificity and sensitivity for detection of VP_AHPND isolates in the test set.