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1.  Outdoor-sleeping and other night-time activities in northern Ghana: implications for residual transmission and malaria prevention 
Malaria Journal  2015;14:35.
Despite targeted indoor residual spraying (IRS) over a six-year period and free mass distribution of long-lasting insecticide-treated nets (ITNs), malaria rates in northern Ghana remain high. Outdoor sleeping and other night-time social, cultural and economic activities that increase exposure to infective mosquito bites are possible contributors. This study was designed to document these phenomena through direct observation, and to explore the context in which they occur.
During the late dry season months of February and March 2014, study team members carried out continuous household observations from dusk to dawn in one village in Ghana’s Northern Region and one in Upper West Region. In-depth interviews with health workers and community residents helped supplement observational findings.
Study team members completed observations of 182 individuals across 24 households, 12 households per site. Between the two sites, they interviewed 14 health workers, six community health volunteers and 28 community residents. In early evening, nearly all study participants were observed to be outdoors and active. From 18.00-23.00 hours, socializing, night school, household chores, and small-scale economic activities were common. All-night funerals, held outdoors and attended by large numbers of community members, were commonly reported and observed. Outdoor sleeping was frequently documented at both study sites, with 42% of the study population sleeping outdoors at some time during the night. While interviewees mentioned bed net use as important to malaria prevention, observed use was low for both indoor and outdoor sleeping. Net access within households was 65%, but only 17% of those with access used a net at any time during the night. Participants cited heat as the primary barrier and reported higher net use during the rainy season.
Outdoor sleeping and other night-time activities were extensive, and could significantly increase malaria risk. These findings suggest that indoor-oriented control measures such as ITNs and IRS are insufficient to eliminate malaria in this setting, especially given the low net use observed. Development and evaluation of complementary outdoor control strategies should be prioritized. A research agenda is proposed to quantify the relative risk of outdoor night-time activities and test potential vector control interventions that might reduce that risk.
PMCID: PMC4320825  PMID: 25627277
Malaria; Outdoor-sleeping; Qualitative research; Ghana; West Africa; Insecticide-treated mosquito nets; Long-lasting insecticidal nets; Insecticide-treated bed nets; Indoor residual spraying; Bed net access; Night-time observation; Residual transmission; Outdoor transmission
2.  “It is about how the net looks”: a qualitative study of perceptions and practices related to mosquito net care and repair in two districts in eastern Uganda 
Malaria Journal  2014;13(1):504.
Prolonging net durability has important implications for reducing both malaria transmission and the frequency of net replacement. Protective behaviour, such as net care and repair, offers promise for improving net integrity and durability. Given the potential cost-savings and public health benefit associated with extending the useful life of long-lasting insecticidal nets (LLINs), prevention and mitigation of damage will become ever more critical to ensuring adequate net coverage at the population level.
A qualitative assessment was conducted in two districts in central eastern Uganda in September 2013. Data on household net care and repair behaviour, attitudes and practices were collected from 30 respondents through in-depth interviews (IDIs), observations, photos, and video to gather an in-depth understanding of these behaviours.
Net damage was common and the most cited causes were children and rodents. Responses revealed strong social norms about net cleanliness and aesthetics, and strong expectations that others should care for and repair their own nets. Respondents were receptive and able to repair nets, though longer-term repair methods, such as sewing and patching, were not as commonly reported or observed. Self-reported behaviour was not always consistent with observed or demonstrated behaviour, revealing potential misconceptions and the need for clear and consistent net care and repair messaging.
Respondents considered both aesthetics and malaria protection important when deciding whether, when, and how to care for and repair nets. BCC should continue to emphasize the importance of maintaining net integrity for malaria prevention purposes as well as for maintaining aesthetic appeal. Additional research is needed, particularly surrounding washing, drying, daily storage routines, and gender roles in care and repair, in order to understand the complexity of these behaviours, and refine existing or develop new behaviour change communication (BCC) messages for net care and repair.
PMCID: PMC4301822  PMID: 25519882
Malaria; Qualitative research; Direct observation; BCC; ITN; LLIN; Bed nets; Uganda; Net care and repair
3.  Enzymatic Neutralization of the Chemical Warfare Agent VX: Evolution of Phosphotriesterase for Phosphorothiolate Hydrolysis 
Journal of the American Chemical Society  2013;135(28):10426-10432.
The V-type nerve agents (VX and VR) are among the most toxic substances known. The high toxicity and environmental persistence of VX makes the development of novel decontamination methods particularly important. The enzyme phosphotriesterase (PTE) is capable of hydrolyzing VX but with an enzymatic efficiency more than 5-orders of magnitude lower than with its best substrate, paraoxon. PTE has previously proven amenable to directed evolution for the improvement of catalytic activity against selected compounds through the manipulation of active site residues. Here, a series of sequential two-site mutational libraries encompassing twelve active site residues of PTE was created. The libraries were screened for catalytic activity against a new VX analogue (DEVX), which contains the same thiolate leaving group of VX coupled to a di-ethoxy phosphate core rather than the ethoxy, methylphosphonate core of VX. The evolved catalytic activity with DEVX was enhanced 26-fold relative to wildtype PTE. Further improvements were facilitated by targeted error-prone PCR mutagenesis of Loop-7 and additional PTE variants were identified with up to a 78-fold increase in the rate of DEVX hydrolysis. The best mutant hydrolyzed the racemic nerve agent VX with a value of kcat/Km of 7×104 M−1 s−1; a 230-fold improvement relative to the wild-type PTE. The highest turnover number achieved by the mutants created for this investigation was 137 s−1; an enhancement of 152-fold relative to wild-type PTE. The stereoselectivity for the hydrolysis of the two enantiomers of VX was relatively low. These engineered mutants of PTE are the best catalysts ever reported for the hydrolysis of nerve agent VX.
PMCID: PMC3747228  PMID: 23789980
4.  Trials of Improved Practices (TIPs): A Strategy for Making Long-Lasting Nets Last Longer? 
Long-lasting insecticidal net (LLIN) use is a proven malaria prevention method. Mass distribution has greatly expanded LLIN access in sub-Saharan Africa, but a gap remains between LLIN ownership and use. Furthermore, LLINs wear out more quickly than anticipated. This paper suggests a participatory research strategy—trials of improved practices (TIPs)—that could identify locally appropriate approaches to prolonging net life and increasing effective use. We used TIPs to overcome barriers to optimal net use in the Peruvian Amazon. Working with 15 families in three villages, we tested home treatment of cotton nets, use of an alternative netting fabric, and alternative washing and care instructions. TIPs helped confirm feasibility of these interventions. Although our findings are time- and context-specific, TIPs could help improve consistency and effectiveness of current LLIN use and prolong net lifespan in sub-Saharan Africa and elsewhere. This would help maximize the value of shrinking donor resources for malaria.
PMCID: PMC3752810  PMID: 23530074
5.  Decision-making on intra-household allocation of bed nets in Uganda: do households prioritize the most vulnerable members? 
Malaria Journal  2014;13:183.
Access to insecticide-treated bed nets has increased substantially in recent years, but ownership and use remain well below 100% in many malaria endemic areas. Understanding decision-making around net allocation in households with too few nets is essential to ensuring protection of the most vulnerable. This study explores household net allocation preferences and practices across four districts in Uganda.
Data collection consisted of eight focus group discussions, twelve in-depth interviews, and a structured questionnaire to inventory 107 sleeping spaces in 28 households.
In focus group discussions and in-depth interviews, participants almost unanimously stated that pregnant women, infants, and young children should be prioritized when allocating nets. However, sleeping space surveys reveal that heads of household sometimes receive priority over children less than five years of age when households have too few nets to cover all members.
When asked directly, most net owners highlight the importance of allocating nets to the most biologically vulnerable household members. This is consistent with malaria behaviour change and health education messages. In actual allocation, however, factors other than biological vulnerability may influence who does and does not receive a net.
PMCID: PMC4030011  PMID: 24885653
Malaria; Insecticide-treated bed nets (ITNs); Bed net allocation; Bed net access; Mosquito nets; Uganda; Qualitative research; Net use patterns
6.  Quality of Voluntary Medical Male Circumcision Services during Scale-Up: A Comparative Process Evaluation in Kenya, South Africa, Tanzania and Zimbabwe 
PLoS ONE  2014;9(5):e79524.
The rapid expansion of voluntary medical male circumcision (VMMC) has raised concerns whether health systems can deliver and sustain VMMC according to minimum quality criteria.
Methods and Findings
A comparative process evaluation was used to examine data from SYMMACS, the Systematic Monitoring of the Voluntary Medical Male Circumcision Scale-Up, among health facilities providing VMMC across two years of program scale-up. Site-level assessments examined the availability of guidelines, supplies and equipment, infection control, and continuity of care services. Direct observation of VMMC surgeries were used to assess care quality. Two sample tests of proportions and t-tests were used to examine differences in the percent of facilities meeting requisite preparedness standards and the mean number of directly-observed surgical tasks performed correctly. Results showed that safe, high quality VMMC can be implemented and sustained at-scale, although substantial variability was observed over time. In some settings, facility preparedness and VMMC service quality improved as the number of VMMC facilities increased. Yet, lapses in high performance and expansion of considerably deficient services were also observed. Surgical tasks had the highest quality scores, with lower performance levels in infection control, pre-operative examinations, and post-operative patient monitoring and counseling. The range of scale-up models used across countries additionally underscored the complexity of delivering high quality VMMC.
Greater efforts are needed to integrate VMMC scale-up and quality improvement processes in sub-Saharan African settings. Monitoring of service quality, not just adverse events reporting, will be essential in realizing the full health impact of VMMC for HIV prevention.
PMCID: PMC4011679  PMID: 24801073
7.  “People will say that I am proud”: a qualitative study of barriers to bed net use away from home in four Ugandan districts 
Malaria Journal  2014;13:82.
Despite increased access and ownership, barriers to insecticide-treated bed net (ITN) use persist. While barriers within the home have been well documented, the challenges to net use when sleeping away from home remain relatively unexplored. This study examines common situations in which people sleep away from home and the barriers to ITN use in those situations.
To explore these issues, a group of researchers conducted 28 in-depth interviews and four focus groups amongst adults from net-owning households in four Ugandan districts.
In addition to sleeping outside during hot season, participants identified social events, livelihood activities, and times of difficulty as circumstances in which large numbers of people sleep away from home. Associated challenges to ITN use included social barriers such as fear of appearing proud, logistical barriers such as not having a place to hang a net, and resource limitations such as not having an extra net with which to travel. Social disapproval emerged as an important barrier to ITN use in public settings.
Unique barriers to ITN use exist when people spend the night away from home. It is essential to identify and address these barriers in order to reduce malaria exposure in such situations. For events like funerals or religious “crusades” where large numbers of people sleep away from home, alternative approaches, such as spatial repellents may be more appropriate than ITNs. Additional research is required to identify the acceptability and feasibility of alternative prevention strategies in situations where ITNs are unlikely to be effective.
PMCID: PMC3973854  PMID: 24602371
Malaria; Insecticide-Treated Bednet; Sleeping outdoors; Barriers; Net use; Qualitative research; Funerals; Conflict; Uganda
8.  A systematic genome-wide analysis of zebrafish protein-coding gene function 
Nature  2013;496(7446):494-497.
Since the publication of the human reference genome, the identities of specific genes associated with human diseases are being discovered at an enormous rate. A central problem is that the biological activity of these genes is often unclear. Detailed investigations in vertebrate model organisms, typically mice, have been essential for understanding the activities of many orthologues of these disease-associated genes. Although gene-targeting approaches1-3 and phenotype analysis have led to a detailed understanding of nearly 6,000 protein-coding genes3,4, this number falls significantly short of all >22,000 mouse protein-coding genes5. Similarly, in zebrafish genetics, one-by-one gene studies using positional cloning6, insertional mutagenesis7-9, antisense morpholino oligonucleotides10, targeted re-sequencing11-13 and zinc finger and TAL endonucleases14-17 have made significant contributions to our understanding of the biological activity of vertebrate genes, but the number of genes studied again falls well short of the >26,000 zebrafish protein-coding genes18. Importantly, for both mice and zebrafish, none of these strategies is particularly suited to the rapid generation of knockouts in thousands of genes and the assessment of their biological activity. Enabled by a well-annotated zebrafish reference genome sequence18,19, high-throughput sequencing and efficient chemical mutagenesis, we describe an active project that aims to identify and phenotype disruptive mutations in every zebrafish protein-coding gene. Thus far we have identified potentially disruptive mutations in more than 38% of all known protein coding genes. We have developed a multi-allelic phenotyping scheme to efficiently assess the effects of each allele during embryogenesis and have analysed the phenotypic consequences of over 1000 alleles. All mutant alleles and data are available to the community and our phenotyping scheme is adaptable to phenotypic analysis beyond embryogenesis.
PMCID: PMC3743023  PMID: 23594742
9.  Enzymes for the Homeland Defense: Optimizing Phosphotriesterase for the Hydrolysis of Organophosphate Nerve Agents† 
Biochemistry  2012;51(32):6463-6475.
Phosphotriesterase (PTE) from soil bacteria is known for its ability to catalyze the detoxification of organophosphate pesticides and chemical warfare agents. Most of the organophosphate chemical warfare agents are a mixture of two stereoisomers at the phosphorus center and the SP-enantiomers are significantly more toxic than the RP-enantiomers. In previous investigations PTE variants were created through the manipulation of the substrate binding pockets and these mutants were shown to have greater catalytic activities for the detoxification of the more toxic SP-enantiomers of nerve agent analogs for GB, GD, GF, VX, and VR than the less toxic RP-enantiomers. In this investigation alternate strategies were employed to discover additional PTE variants with significant improvements in catalytic activities relative to the wild type enzyme. Screening and selection techniques were utilized to isolate PTE variants from randomized libraries and site specific modifications. The catalytic activities of these newly identified PTE variants towards the SP-enantiomers of chromophoric analogs of GB, GD, GF, VX, and VR have been improved up to 15,000 fold relative to the wild-type enzyme. The X-ray crystal structures of the best PTE variants were determined. Characterization of these mutants with the authentic G-type nerve agents has confirmed the expected improvements in catalytic activity against the most toxic enantiomers of GB, GD, and GF. The values of kcat/Km for the H257Y/L303T (YT) mutant for the hydrolysis of GB, GD, and GF were determined to be 2 × 106 M−1 s−1, 5 × 105 M−1 s−1, and 8 × 105 M−1 s−1, respectively. The YT mutant is the most proficient enzyme reported thus far for the detoxification of G-type nerve agents. These results support a combinatorial strategy of rational design and directed evolution as a powerful tool to discover more efficient enzymes for the detoxification of organophosphate nerve agents.
PMCID: PMC3447986  PMID: 22809162
10.  Identification of the zebrafish maternal and paternal transcriptomes 
Development (Cambridge, England)  2013;140(13):2703-2710.
Transcription is an essential component of basic cellular and developmental processes. However, early embryonic development occurs in the absence of transcription and instead relies upon maternal mRNAs and proteins deposited in the egg during oocyte maturation. Although the early zebrafish embryo is competent to transcribe exogenous DNA, factors present in the embryo maintain genomic DNA in a state that is incompatible with transcription. The cell cycles of the early embryo titrate out these factors, leading to zygotic transcription initiation, presumably in response to a change in genomic DNA chromatin structure to a state that supports transcription. To understand the molecular mechanisms controlling this maternal to zygotic transition, it is important to distinguish between the maternal and zygotic transcriptomes during this period. Here we use exome sequencing and RNA-seq to achieve such discrimination and in doing so have identified the first zygotic genes to be expressed in the embryo. Our work revealed different profiles of maternal mRNA post-transcriptional regulation prior to zygotic transcription initiation. Finally, we demonstrate that maternal mRNAs are required for different modes of zygotic transcription initiation, which is not simply dependent on the titration of factors that maintain genomic DNA in a transcriptionally incompetent state.
PMCID: PMC3678340  PMID: 23720042
MZT; Maternal; Paternal; Transcriptome; Zebrafish
11.  Early responding dendritic cells direct the local natural killer response to control HSV-1 infection within the cornea 
Dendritic cells (DCs) regulate both innate and adaptive immune responses. Here we exploit the unique avascularity of the cornea to examine a role for local or very early infiltrating DCs in regulating the migration of blood-derived innate immune cells towards herpes simplex virus type 1 (HSV-1) lesions. A single systemic diphtheria toxin (DT) treatment 2 days before HSV-1 corneal infection transiently depleted CD11c+DCs from both the cornea and lymphoid organs of CD11c-DTR bone marrow chimeric mice for up to 24 hours after infection. Transient DC depletion significantly delayedHSV-1 clearance from the corneathrough 6 days post infection(dpi). No further compromise of viral clearance was observed when DCs were continuously depleted throughout the first week of infection. DC depletion did not influenceextravasation of NK cells, inflammatory monocytes, orneutrophils into the peripheral cornea,but did significantly reduce migration of NK cells and inflammatory monocytes, but not neutrophils towards the HSV-1 lesion in the central cornea. Depletion of NK cells resulted in similar loss of viral control to transient DC ablation. Our findings demonstrate resident corneal DC and/or those that infiltrate the cornea during the first 24 hours after HSV-1 infection contribute to the migration of NK cells and inflammatory monocytes into the central cornea, and are consistent with a role for NK cells and possibly inflammatory monocytes, but not PMN in the clearing HSV-1 from the infected cornea.
PMCID: PMC3292873  PMID: 22210909
12.  Community Health Workers Use Malaria Rapid Diagnostic Tests (RDTs) Safely and Accurately: Results of a Longitudinal Study in Zambia 
Malaria rapid diagnostic tests (RDTs) could radically improve febrile illness management in remote and low-resource populations. However, reliance upon community health workers (CHWs) remains controversial because of concerns about blood safety and appropriate use of artemisinin combination therapy. This study assessed CHW ability to use RDTs safely and accurately up to 12 months post-training. We trained 65 Zambian CHWs, and then provided RDTs, job-aids, and other necessary supplies for village use. Observers assessed CHW performance at 3, 6, and 12 months post-training. Critical steps performed correctly increased from 87.5% at 3 months to 100% subsequently. However, a few CHWs incorrectly read faint positive or invalid results as negative. Although most indicators improved or remained stable over time, interpretation of faint positives fell to 76.7% correct at 12 months. We conclude that appropriately trained and supervised CHWs can use RDTs safely and accurately in community practice for up to 12 months post-training.
PMCID: PMC3391058  PMID: 22764292
13.  Studies of the Suitability of Fowlpox as a Decontamination and Thermal Stability Simulant for Variola Major 
Variola major, the causative agent of smallpox, has been eradicated from nature. However, stocks still exist; thus, there is a need for relevant decontamination studies, preferably with nonpathogenic simulants. Previous studies have shown a similarity in response of vaccinia virus and variola major to various decontaminants and thermal inactivation. This study compared vaccinia and fowlpox viruses under similar conditions, using disinfectants and temperatures for which variola major data already existed. Most disinfectants showed similar efficacy against vaccinia and fowlpox, suggesting the utility of fowlpox as a decontamination simulant. Inactivation kinetics studies showed that fowlpox behaved similarly to variola major when treated with 0.1% iodine and 5.7% polyethyleneglycol nonylphenyl ether, 0.025% sodium hypochlorite, 0.05% sodium hypochlorite, and 0.1% cetyltrimethylammonium chloride and 0.05% benzalkonium chloride, but differed in its response to 0.05% iodine and 0.3% polyethyleneglycol nonylphenyl ether and 40% ethanol. Thermal inactivation studies demonstrated that fowlpox is a suitable thermal simulant for variola major between 40°C and 55°C.
PMCID: PMC2817860  PMID: 20148078
14.  Visualisation and Quantification of Morphogen Gradient Formation in the Zebrafish 
PLoS Biology  2009;7(5):e1000101.
During embryonic development, signalling molecules known as morphogens act in a concentration-dependent manner to provide positional information to responding tissues. In the early zebrafish embryo, graded signalling by members of the nodal family induces the formation of mesoderm and endoderm, thereby patterning the embryo into three germ layers. Nodal signalling has also been implicated in the establishment of the dorso-ventral axis of the embryo. Although one can infer the existence of nodal gradients by comparing gene expression patterns in wild-type embryos and embryos in which nodal signalling is diminished or augmented, real understanding can only come from directly observing the gradients. One approach is to determine local ligand concentrations in the embryo, but this is technically challenging, and the presence of inhibitors might cause the effective concentration of a ligand to differ from its actual concentration. We have therefore taken two approaches to visualise a direct response to nodal signalling. In the first, we have used transgenic embryos to study the nuclear accumulation of a Smad2-Venus fusion protein, and in the second we have used bimolecular fluorescence complementation to visualise the formation of a complex between Smad2 and Smad4. This has allowed us to visualise, in living embryos, the formation of a graded distribution of nodal signalling activity. We have quantified the formation of the gradient in time and space, and our results not only confirm that nodal signalling patterns the embryo into three germ layers, but also shed light on its role in patterning the dorso-ventral axis and highlight unexpected complexities of mesodermal patterning.
Author Summary
One of the earliest events in vertebrate embryonic development is the patterning of the embryo into three germ layers: the ectoderm, mesoderm, and endoderm. Morphogens are signalling molecules that act in a concentration-dependent manner to induce the formation of different cell types. Members of the nodal family are thought to form a morphogen gradient in the developing zebrafish embryo and to be essential for pattern formation. Mesoderm and endoderm are believed to develop due to high levels of nodal signalling, while cells experiencing the lowest concentrations of nodal signalling become ectoderm. Although this idea is widely accepted, the formation of a nodal morphogen gradient has never been observed directly, and we have therefore used two different approaches to visualise the intensity of nodal signalling within individual cells. Our approaches have allowed us to visualise a gradient of nodal signalling activity in the developing zebrafish embryo. Quantification of the levels of nodal signalling experienced by individual cells confirms that nodal signalling patterns the animal-vegetal axis of the zebrafish embryo and, in contrast to previous studies, also suggests that it plays a role in patterning the dorso-ventral axis of the zebrafish embryo.
Gradients of nodal signalling in developing zebrafish embryos are visualized using a novel biofluorescence complementation reporter and quantified, demonstrating a role for nodal signalling in dorso-ventral patterning in addition to specifying the animal-vegetal axis.
PMCID: PMC2675906  PMID: 19419239
15.  Improving community health worker use of malaria rapid diagnostic tests in Zambia: package instructions, job aid and job aid-plus-training 
Malaria Journal  2008;7:160.
Introduction of artemisinin combination therapy (ACT) has boosted interest in parasite-based malaria diagnosis, leading to increased use of rapid diagnostic tests (RDTs), particularly in rural settings where microscopy is limited. With donor support, national malaria control programmes are now procuring large quantities of RDTs. The scarcity of health facilities and trained personnel in many sub-Saharan African countries means that limiting RDT use to such facilities would exclude a significant proportion of febrile cases. RDT use by volunteer community health workers (CHWs) is one alternative, but most sub-Saharan African countries prohibit CHWs from handling blood, and little is known about CHW ability to use RDTs safely and effectively. This Zambia-based study was designed to determine: (i) whether Zambian CHWs could prepare and interpret RDTs accurately and safely using manufacturer's instructions alone; (ii) whether simple, mostly pictorial instructions (a "job aid") could raise performance to adequate levels; and (iii) whether a brief training programme would produce further improvement.
The job aid and training programme were based on formative research with 32 CHWs in Luangwa District. The study team then recruited three groups of CHWs in Chongwe and Chibombo districts. All had experience treating malaria based on clinical diagnosis, but only six had prior RDT experience. Trained observers used structured observation checklists to score each participant's preparation of three RDTs. Each also read 10 photographs showing different test results. The first group (n = 32) was guided only by manufacturer's instructions. The second (n = 21) used only the job aid. The last (n = 26) used the job aid after receiving a three-hour training.
Mean scores, adjusted for education, age, gender and experience, were 57% of 16 RDT steps correctly completed for group 1, 80% for group 2, and 92% for group 3. Mean percentage of test results interpreted correctly were 54% (group 1), 80% (group 2), and 93% (group 3). All differences were statistically significant (p < 0.05).
Manufacturer's instructions like those provided with the RDTs used in this study are insufficient to ensure safe and accurate use by CHWs. However, well-designed instructions plus training can ensure high performance. More study is underway to determine how well this performance holds up over time.
PMCID: PMC2547110  PMID: 18718028
16.  Tandem repeat regions within the Burkholderia pseudomallei genome and their application for high resolution genotyping 
BMC Microbiology  2007;7:23.
The facultative, intracellular bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a serious infectious disease of humans and animals. We identified and categorized tandem repeat arrays and their distribution throughout the genome of B. pseudomallei strain K96243 in order to develop a genetic typing method for B. pseudomallei. We then screened 104 of the potentially polymorphic loci across a diverse panel of 31 isolates including B. pseudomallei, B. mallei and B. thailandensis in order to identify loci with varying degrees of polymorphism. A subset of these tandem repeat arrays were subsequently developed into a multiple-locus VNTR analysis to examine 66 B. pseudomallei and 21 B. mallei isolates from around the world, as well as 95 lineages from a serial transfer experiment encompassing ~18,000 generations.
B. pseudomallei contains a preponderance of tandem repeat loci throughout its genome, many of which are duplicated elsewhere in the genome. The majority of these loci are composed of repeat motif lengths of 6 to 9 bp with 4 to 10 repeat units and are predominately located in intergenic regions of the genome. Across geographically diverse B. pseudomallei and B.mallei isolates, the 32 VNTR loci displayed between 7 and 28 alleles, with Nei's diversity values ranging from 0.47 and 0.94. Mutation rates for these loci are comparable (>10-5 per locus per generation) to that of the most diverse tandemly repeated regions found in other less diverse bacteria.
The frequency, location and duplicate nature of tandemly repeated regions within the B. pseudomallei genome indicate that these tandem repeat regions may play a role in generating and maintaining adaptive genomic variation. Multiple-locus VNTR analysis revealed extensive diversity within the global isolate set containing B. pseudomallei and B. mallei, and it detected genotypic differences within clonal lineages of both species that were identical using previous typing methods. Given the health threat to humans and livestock and the potential for B. pseudomallei to be released intentionally, MLVA could prove to be an important tool for fine-scale epidemiological or forensic tracking of this increasingly important environmental pathogen.
PMCID: PMC1853098  PMID: 17397553
17.  Purification and Properties of a Highly Active Organophosphorus Acid Anhydrolase from Alteromonas undina 
A highly active organophosphorus acid anhydrolase from Alteromonas undina was purified to homogeneity and found to be composed of a single polypeptide chain with a molecular weight of 53,000. With diisopropylfluorophosphate as a substrate, the purified enzyme has a specific activity of ∼575 μmol/min/mg of protein. The enzyme has optimum activity at pH 8.0 and 55°C and is stimulated by sulfhydryl reducing agents and manganese. It is capable of rapidly hydrolyzing a wide range of nerve agents and several chromogenic phosphinates.
PMCID: PMC182420  PMID: 16349054

Results 1-17 (17)