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1.  Evaluation of the operational challenges in implementing reactive screen-and-treat and implications of reactive case detection strategies for malaria elimination in a region of low transmission in southern Zambia 
Malaria Journal  2016;15:412.
Background
As malaria transmission declines in many regions of sub-Saharan Africa, interventions to identify the asymptomatic reservoir are being deployed with the goals of improving surveillance and interrupting transmission. Reactive case detection strategies, in which individuals with clinical malaria are followed up at their home and household residents and neighbours are screened and treated for malaria, are increasingly used as part of malaria elimination programmes.
Methods
A reactive screen-and-treat programme was implemented by the National Malaria Control Centre in Southern Province, Zambia, in which individuals residing within 140 m of an index case were screened with a malaria rapid diagnostic test (RDT) and treated if positive. The operational challenges during the early stages of implementing this reactive screen-and-treat programme in the catchment area of Macha Hospital in Southern Province, Zambia were assessed using rural health centre records, ground truth evaluation of community health worker performance, and data from serial cross-sectional surveys. The proportion of individuals infected with Plasmodium falciparum who were identified and treated was estimated by simulating reactive screen-and-treat and focal drug administration cascades.
Results
Within the 1st year of implementation, community health workers followed up 32 % of eligible index cases. When index cases were followed up, 66 % of residents were at home in the index households and 58 % in neighbouring households. Forty-one neighbouring households of 26 index households were screened, but only 13 (32 %) were within the 140-m screening radius. The parasite prevalence by RDT was 22 % in index households and 5 % in neighbouring households. In a simulation model with complete follow-up, 22 % of the total infected population would be detected with reactive screen-and-treat but 57 % with reactive focal drug administration.
Conclusions
With limited resources, coverage and diagnostic tools, reactive screen-and-treat will likely not be sufficient to achieve malaria elimination in this setting. However, high coverage with reactive focal drug administration could be efficient at decreasing the reservoir of infection and should be considered as an alternative strategy.
Electronic supplementary material
The online version of this article (doi:10.1186/s12936-016-1460-x) contains supplementary material, which is available to authorized users.
doi:10.1186/s12936-016-1460-x
PMCID: PMC4986207  PMID: 27527347
Malaria elimination; Reactive case detection; Reactive test-and-treat; Zambia; Sub-Saharan Africa
2.  Nutrition Screening Tools and the Prediction of Clinical Outcomes among Chinese Hospitalized Gastrointestinal Disease Patients 
PLoS ONE  2016;11(8):e0159436.
Nutrition risk Screening 2002 (NRS-2002) and Subjective Global Assessment (SGA) are widely used screening tools but have not been compared in a Chinese population. We conducted secondary data analysis of a cross-sectional study which included 332 hospitalized gastrointestinal disease patients, collected by the Gastrointestinal department of Peking Union Medical College Hospital (PUMCH) in 2008. Results of NRS-2002 and SGA screening tools, complications, length of stay (LOS), cost, and death were measured. The agreement between the tools was assessed via Kappa (κ) statistics. The performance of NRS-2002 and SGA in predicting LOS and cost was assessed via linear regression. The complications and death prediction of tools was assessed using receiver operating characteristic (ROC) curves. NRS-2002 and SGA identified nutrition risk at 59.0% and 45.2% respectively. Moderate agreement (κ >0.50) between the two tools was found among all age groups except individuals aged ≤ 20, which only slight agreement was found (κ = 0.087). NRS-2002 (R square 0.130) and SGA (R square 0.140) did not perform differently in LOS prediction. The cost prediction of NRS-2002 (R square 0.198) and SGA (R square 0.190) were not significantly different. There was no difference between NRS-2002 (infectious complications: area under ROC (AUROC) = 0.615, death: AUROC = 0.810) and SGA (infectious complications: AUROC = 0.600, death: AUROC = 0.846) in predicting infectious complication and death, but NRS-2002 (0.738) seemed to perform better than SGA (0.552) in predicting non-infectious complications. The risk of malnutrition among patients was high. NRS-2002 and SGA have similar capacity to predict LOS, cost, infectious complications and death, but NRS-2002 performed better in predicting non-infectious complications.
doi:10.1371/journal.pone.0159436
PMCID: PMC4973995  PMID: 27490480
3.  Soluble human TLR2 ectodomain binds diacylglycerol from microbial lipopeptides and glycolipids 
Innate immunity  2014;21(2):175-193.
Toll-like receptors (TLRs) are key innate immune receptors that recognize conserved features of biological molecules that are found in microbes. In particular, TLR2 has been reported to be activated by different kinds of microbial ligands. To advance our understanding of the interaction of TLR2 with its ligands, the recombinant human TLR2 ectodomain (hTLR2ED) was expressed using a baculovirus/insect cell expression system, and its biochemical as well as ligand binding properties were investigated. The hTLR2ED binds synthetic bacterial and mycoplasmal lipopeptides, lipoteichoic acid (LTA) from Staphylococcus aureus, and synthetic lipoarabinomannan precursors from Mycobacterium at extracellular physiological conditions, in the absence of its co-receptors TLR1 and TLR6. We also determined that lipopeptides and glycolipids cannot bind simultaneously to hTLR2ED and that the phosphatidyl inositol mannoside 2 (Pim2) is the minimal lipoarabinomannan structure for binding to hTLR2ED. Binding of hTLR2ED to Pim4, which contains a diacylglycerol group with one of its acyl chain containing 19 carbon atoms, indicates that hTLR2ED can bind ligands with acyl chains longer than 16 carbon atoms. In summary, our data indicate that diacylglycerol is the ligand moiety of microbial glycolipids and lipoproteins that bind to hTLR2ED and that both types of ligands bind to the same binding site of hTLR2ED. The design of novel inhibitors of TLR2, based on their ability to bind to TLR2 but not activate the TLR2 signaling pathway, may lead to the development of novel treatments for septic shock caused by Gram- positive bacteria.
doi:10.1177/1753425914524077
PMCID: PMC4392886  PMID: 24591200
diacylglycerol ligands; lipoarabinomannan; lipoteichoic acid; FSL-1; TLR2
4.  Targeting indoor residual spraying for malaria using epidemiological data: a case study of the Zambia experience 
Malaria Journal  2016;15:11.
Background
In Zambia and other sub-Saharan African countries affected by ongoing malaria transmission, indoor residual spraying (IRS) for malaria prevention has typically been implemented over large areas, e.g., district-wide, and targeted to peri-urban areas. However, there is a recent shift in some countries, including Zambia, towards the adoption of a more strategic and targeted IRS approach, in coordination with increased emphasis on universal coverage of long-lasting insecticidal nets (LLINs) and effective insecticide resistance management. A true targeted approach would deliver IRS to sub-district areas identified as high-risk, with the goal of maximizing the prevention of malaria cases and deaths.
Results
Together with the Government of the Republic of Zambia, a new methodology was developed applying geographic information systems and satellite imagery to support a targeted IRS campaign during the 2014 spray season using health management information system data.
Discussion/Conclusion
This case study focuses on the developed methodology while also highlighting the significant research gaps which must be filled to guide countries on the most effective strategy for IRS targeting in the context of universal LLIN coverage and evolving insecticide resistance.
doi:10.1186/s12936-015-1073-9
PMCID: PMC4704423  PMID: 26738936
Indoor residual spraying; Targeted IRS; Focal IRS; GIS; Malaria
5.  Bridging Small Molecules to Modified Bacterial Microparticles Using a Disulphide Linkage: MIS416 as a Cargo Delivery System 
PLoS ONE  2015;10(12):e0145403.
MIS416 is an intact minimal cell wall skeleton derived from Proprionibacterium acnes that is phagocytosed by antigen presenting cells, including dendritic cells (DCs). This property allows MIS416 to be exploited as a vehicle for the delivery of peptide antigens or other molecules (for example, nucleic acids) to DCs. We previously showed that covalent (non-cleavable) conjugation of OVA, a model antigen derived from ovalbumin, to MIS416 enhanced immune responses in DCs in vivo, compared to unconjugated MIS416 and OVA. Intracellular trafficking promotes the lysosomal degradation of MIS416, leading to the destruction of MIS416 plus the associated cargos conjugated to MIS416. However, lysosomal degradation of cargo may not be desired for some MIS416 conjugates. Here we have investigated whether a cleavable linkage could facilitate release of the cargo in the cytoplasm of DCs to avoid lysosomal degradation. DCs were treated in vitro with disulfide-containing conjugates, and as hypothesised faster release of SIINFEKL peptide in the cytoplasm of DCs was observed with the inclusion of a disulfide bond between MIS416 and cargo. The inclusion of a cleavable disulfide bond in the conjugates did not significantly alter the amount of SIINFEKL antigens presented on MHC I molecules on DCs as compared with conjugates without a disulfide bond. However, the conjugates containing disulfide-linkages performed either slightly better (p<0.05) than, or the same as conjugates without a disulfide bond with respect to in vitro OT-1 T-cell proliferation induced by the presentation of SIINFEKL antigens on DCs, or DC activation studies, respectively. However, disulfide-containing conjugates were less effective than conjugates without a disulfide bond in in vivo cytotoxicity assays. In conclusion, inclusion of a disulfide bond in MIS416-peptide conjugates was associated with efficient release of peptides in the cytoplasm of DCs, an important consideration for MIS416-mediated delivery of degradation-sensitive cargoes. However, treatment of DCs with disulfide-containing conjugates did not significantly alter the presentation of peptide antigens on MHC class I molecules to T-cells, or greatly enhance antigen-associated T-cell proliferation in vitro.
doi:10.1371/journal.pone.0145403
PMCID: PMC4687933  PMID: 26695183
6.  A mitochondria-targeted derivative of ascorbate: MitoC 
Mitochondrial oxidative damage contributes to a wide range of pathologies. One therapeutic strategy to treat these disorders is targeting antioxidants to mitochondria by conjugation to the lipophilic triphenylphosphonium (TPP) cation. To date only hydrophobic antioxidants have been targeted to mitochondria; however, extending this approach to hydrophilic antioxidants offers new therapeutic and research opportunities. Here we report the development and characterization of MitoC, a mitochondria-targeted version of the hydrophilic antioxidant ascorbate. We show that MitoC can be taken up by mitochondria, despite the polarity and acidity of ascorbate, by using a sufficiently hydrophobic link to the TPP moiety. MitoC reacts with a range of reactive species, and within mitochondria is rapidly recycled back to the active ascorbate moiety by the glutathione and thioredoxin systems. Because of this accumulation and recycling MitoC is an effective antioxidant against mitochondrial lipid peroxidation and also decreases aconitase inactivation by superoxide. These findings show that the incorporation of TPP function can be used to target polar and acidic compounds to mitochondria, opening up the delivery of a wide range of bioactive compounds. Furthermore, MitoC has therapeutic potential as a new mitochondria-targeted antioxidant, and is a useful tool to explore the role(s) of ascorbate within mitochondria.
Graphical abstract
Highlights
•A mitochondria-targeted ascorbate, MitoC, has been developed•MitoC is taken up by energized mitochondria and there recycled•Mitochondrial oxidative damage is decreased by MitoC•MitoC is a useful reagent to explore mitochondrial oxidative stress
doi:10.1016/j.freeradbiomed.2015.07.160
PMCID: PMC4698375  PMID: 26453920
ACR, accumulation ratio; AO, ascorbate oxidase; AFR, ascorbyl free radical; DHA, dehydroascorbic acid; DTPA, diethylenetriaminepenta-acetic acid; FCCP, carbonylcyanide p-(trifluoromethoxy)phenylhydrazone; FCS, foetal calf serum; GSH, glutathione; LAH, linoleic acid hydroperoxide; Δψm, mitochondrial membrane potential; MitoC, mitochondria-targeted ascorbate; MitoDHA, mitochondria-targeted dehydroascorbate; TBARS, thiobarbituric acid reactive species; TFA, trifluoroacetic acid; TPMP, methyltriphenylphosphonium; TPP, triphenylphosphonium; RET, reverse electron transport; RLM, rat liver mitochondria; ROS, reactive oxygen species; RP-HPLC, reverse-phase HPLC; XO, xanthine oxidase; Lipophilic cation; Lipid peroxidation; Mitochondria; Mitochondrial targeting; Ascorbic acid; MitoPerox; MitoC
7.  Malaria surveillance in low-transmission areas of Zambia using reactive case detection 
Malaria Journal  2015;14:465.
Background
Repeat national household surveys suggest highly variable malaria transmission and increasing coverage of high-impact malaria interventions throughout Zambia. Many areas of very low malaria transmission, especially across southern and central regions, are driving efforts towards sub-national elimination.
Case description
Reactive case detection (RCD) is conducted in Southern Province and urban areas of Lusaka in connection with confirmed incident malaria cases presenting to a community health worker (CHW) or clinic and suspected of being the result of local transmission. CHWs travel to the household of the incident malaria case and screen individuals living in adjacent houses in urban Lusaka and within 140 m in Southern Province for malaria infection using a rapid diagnostic test, treating those testing positive with artemether–lumefantrine.
Discussion
Reactive case detection improves access to health care and increases the capacity for the health system to identify malaria infections. The system is useful for targeting malaria interventions, and was instrumental for guiding focal indoor residual spraying in Lusaka during the 2014/2015 spray season. Variations to maximize impact of the current RCD protocol are being considered, including the use of anti-malarials with a longer lasting, post-treatment prophylaxis.
Conclusion
The RCD system in Zambia is one example of a malaria elimination surveillance system which has increased access to health care within rural communities while leveraging community members to build malaria surveillance capacity.
doi:10.1186/s12936-015-0895-9
PMCID: PMC4653936  PMID: 26586264
Malaria surveillance; Reactive case detection; Elimination; Urban; Rural; Community health worker; DHIS2
8.  Community Coverage with Insecticide-Treated Mosquito Nets and Observed Associations with All-Cause Child Mortality and Malaria Parasite Infections 
Randomized trials and mathematical modeling suggest that insecticide-treated mosquito nets (ITNs) provide community-level protection to both those using ITNs and those without individual access. Using nationally representative household survey datasets from 17 African countries, we examined whether community ITN coverage is associated with malaria infections in children < 5 years old and all-cause child mortality (ACCM) among children < 5 years old in households with one or more ITNs versus without any type of mosquito net (treated or untreated). Increasing ITN coverage (> 50%) was protective against malaria infections and ACCM for children in households with an ITN, although this protection was not conferred to children in households without ITNs in these data. Children in households with ITNs were protected against malaria infections and ACCM with ITN coverage > 30%, but this protection was not significant with ITN coverage < 30%. Results suggest that ITNs are more effective with higher ITN coverage.
doi:10.4269/ajtmh.14-0318
PMCID: PMC4228892  PMID: 25200267
9.  Open-source satellite enumeration to map households: planning and targeting indoor residual spraying for malaria 
Malaria Journal  2015;14:345.
Background
Defining the number and location of sprayable structures (houses) is foundational to plan and monitor indoor residual spray (IRS) implementation, a primary intervention used to control the transmission of malaria. Only by mapping the location and type of all sprayable structures can IRS operations be planned, estimates of spray coverage determined, and targeted delivery of IRS to specific locations be achieved. Previously, field-based enumeration has been used to guide IRS campaigns, however, this approach is costly, time-consuming and difficult to scale. As a result, field-based enumeration typically fails to map all structures in a given area, making estimations less reliable and reducing the enumerated coverage.
Methods
Using open source satellite imagery and Geographic Information System software, satellite enumeration was conducted to guide IRS operations in 15 districts (91,302 km2) in northern Zambia during the 2014 spray season. Cost of satellite enumeration was compared to standard enumeration. Enumerated households were sampled to estimate sprayable surface area and wall type from the satellite enumeration using linear and logistic regression, respectively.
Results
In comparison to the traditional field-based enumeration procedure, satellite-based enumeration was 22 times faster, and 10 times less costly. An estimated 98 % of the satellite enumerated buildings correctly classified roof type. Predicted surface area of each household correlated at a value of 0.91 with measured surface area of each household.
Conclusion
For IRS campaigns, high quality and high coverage enumeration data aid in planning, through informed insecticide procurement. Through the identification of geographical areas and populations to target, enumeration data guide operations and assist monitoring and evaluation of IRS through the unbiased estimation of coverage achieved. Satellite enumeration represents a quick, cheap and accurate system to provide these data, and has potential applications beyond IRS for delivery of other targeted or non-targeted interventions (e.g. net distributions, mass drug administration, immunization campaigns, or even sampling frames for field studies).
doi:10.1186/s12936-015-0831-z
PMCID: PMC4574022  PMID: 26376980
Malaria; Indoor residual spraying; Google Earth; Enumerate; Structure; mSpray; Ground-truth
10.  Using Inverse Probability Bootstrap Sampling to Eliminate Sample Induced Bias in Model Based Analysis of Unequal Probability Samples 
PLoS ONE  2015;10(6):e0131765.
In ecology, as in other research fields, efficient sampling for population estimation often drives sample designs toward unequal probability sampling, such as in stratified sampling. Design based statistical analysis tools are appropriate for seamless integration of sample design into the statistical analysis. However, it is also common and necessary, after a sampling design has been implemented, to use datasets to address questions that, in many cases, were not considered during the sampling design phase. Questions may arise requiring the use of model based statistical tools such as multiple regression, quantile regression, or regression tree analysis. However, such model based tools may require, for ensuring unbiased estimation, data from simple random samples, which can be problematic when analyzing data from unequal probability designs. Despite numerous method specific tools available to properly account for sampling design, too often in the analysis of ecological data, sample design is ignored and consequences are not properly considered. We demonstrate here that violation of this assumption can lead to biased parameter estimates in ecological research. In addition, to the set of tools available for researchers to properly account for sampling design in model based analysis, we introduce inverse probability bootstrapping (IPB). Inverse probability bootstrapping is an easily implemented method for obtaining equal probability re-samples from a probability sample, from which unbiased model based estimates can be made. We demonstrate the potential for bias in model-based analyses that ignore sample inclusion probabilities, and the effectiveness of IPB sampling in eliminating this bias, using both simulated and actual ecological data. For illustration, we considered three model based analysis tools—linear regression, quantile regression, and boosted regression tree analysis. In all models, using both simulated and actual ecological data, we found inferences to be biased, sometimes severely, when sample inclusion probabilities were ignored, while IPB sampling effectively produced unbiased parameter estimates.
doi:10.1371/journal.pone.0131765
PMCID: PMC4488419  PMID: 26126211
11.  Enhanced surveillance and data feedback loop associated with improved malaria data in Lusaka, Zambia 
Malaria Journal  2015;14:222.
Background
Accurate and timely malaria data are crucial to monitor the progress towards and attainment of elimination. Lusaka, the capital city of Zambia, has reported very low malaria prevalence in Malaria Indicator Surveys. Issues of low malaria testing rates, high numbers of unconfirmed malaria cases and over consumption of anti-malarials were common at clinics within Lusaka, however. The Government of Zambia (GRZ) and its partners sought to address these issues through an enhanced surveillance and feedback programme at clinic level.
Methods
The enhanced malaria surveillance programme began in 2011 to verify trends in reported malaria, as well as to implement a data feedback loop to improve data uptake, use, and quality. A process of monthly data collection and provision of feedback was implemented within all GRZ health clinics in Lusaka District. During clinic visits, clinic registers were accessed to record the number of reported malaria cases, malaria test positivity rate, malaria testing rate, and proportion of total suspected malaria that was confirmed with a diagnostic test.
Results and discussion
Following the enhanced surveillance programme, the odds of receiving a diagnostic test for a suspected malaria case increased (OR = 1.54, 95 % CI = 0.96–2.49) followed by an upward monthly trend (OR = 1.05, 95 % CI = 1.01–1.09). The odds of a reported malaria case being diagnostically confirmed also increased monthly (1.09, 95 % CI 1.04–1.15). After an initial 140 % increase (95 % CI = 91–183 %), costs fell by 11 % each month (95 % CI = 5.7–10.9 %). Although the mean testing rate increased from 18.9 to 64.4 % over the time period, the proportion of reported malaria unconfirmed by diagnostic remained high at 76 %.
Conclusions
Enhanced surveillance and implementation of a data feedback loop have substantially increased malaria testing rates and decreased the number of unconfirmed malaria cases and courses of ACT consumed in Lusaka District within just two years. Continued support of enhanced surveillance in Lusaka as well as national scale-up of the system is recommended to reinforce good case management and to ensure timely, reliable data are available to guide targeting of limited malaria prevention and control resources in Zambia.
doi:10.1186/s12936-015-0735-y
PMCID: PMC4486393  PMID: 26017275
Surveillance; Malaria; Case management; Elimination; DHIS2; Zambia
12.  Population-Wide Malaria Testing and Treatment with Rapid Diagnostic Tests and Artemether-Lumefantrine in Southern Zambia: A community Randomized Step-Wedge Control Trial Design 
Reducing the human reservoir of malaria parasites is critical for elimination. We conducted a community randomized controlled trial in Southern Province, Zambia to assess the impact of three rounds of a mass test and treatment (MTAT) intervention on malaria prevalence and health facility outpatient case incidence using random effects logistic regression and negative binomial regression, respectively. Following the intervention, children in the intervention group had lower odds of a malaria infection than individuals in the control group (adjusted odds ratio = 0.47, 95% confidence interval [CI] = 0.24–0.90). Malaria outpatient case incidence decreased 17% in the intervention group relative to the control group (incidence rate ratio = 0.83, 95% CI = 0.68–1.01). Although a single year of MTAT reduced malaria prevalence and incidence, the impact of the intervention was insufficient to reduce transmission to a level approaching elimination where a strategy of aggressive case investigations could be used. Mass drug administration, more sensitive diagnostics, and gametocidal drugs may potentially improve interventions targeting the human reservoir of malaria parasites.
doi:10.4269/ajtmh.14-0347
PMCID: PMC4426577  PMID: 25802434
13.  A qualitative study of perceptions of a mass test and treat campaign in Southern Zambia and potential barriers to effectiveness 
Malaria Journal  2015;14:171.
Background
A mass test and treat campaign (MTAT) using rapid diagnostic tests (RDTs) and artemether-lumefantrine (AL) was conducted in Southern Zambia in 2012 and 2013 to reduce the parasite reservoir and progress towards malaria elimination. Through this intervention, community health workers (CHWs) tested all household members with rapid diagnostic tests (RDTs) and provided treatment to those that tested positive.
Methods
A qualitative study was undertaken to understand CHW and community perceptions regarding the MTAT campaign. A total of eight focus groups and 33 in-depth and key informant interviews were conducted with CHWs, community members and health centre staff that participated in the MTAT.
Results
Interviews and focus groups with CHWs and community members revealed that increased knowledge of malaria prevention, the ability to reach people who live far from health centres, and the ability of the MTAT campaign to reduce the malaria burden were the greatest perceived benefits of the campaign. Conversely, the primary potential barriers to effectiveness included refusals to be tested, limited adherence to drug regimens, and inadequate commodity supply. Study respondents generally agreed that MTAT services were scalable outside of the study area but would require greater involvement from district and provincial medical staff.
Conclusions
These findings highlight the importance of increased community sensitization as part of mass treatment campaigns for improving campaign coverage and acceptance. Further, they suggest that communication channels between the Ministry of Health, National Malaria Control Centre and Medical Stores Limited may need to be improved so as to ensure there is consistent supply and management of commodities. Continued capacity building of CHWs and health facility supervisors is critical for a more effective programme and sustained progress towards malaria elimination.
doi:10.1186/s12936-015-0686-3
PMCID: PMC4426174  PMID: 25896068
Mass Test and Treat; Malaria elimination; Qualitative methods
14.  Phylogenetically Driven Sequencing of Extremely Halophilic Archaea Reveals Strategies for Static and Dynamic Osmo-response 
PLoS Genetics  2014;10(11):e1004784.
Organisms across the tree of life use a variety of mechanisms to respond to stress-inducing fluctuations in osmotic conditions. Cellular response mechanisms and phenotypes associated with osmoadaptation also play important roles in bacterial virulence, human health, agricultural production and many other biological systems. To improve understanding of osmoadaptive strategies, we have generated 59 high-quality draft genomes for the haloarchaea (a euryarchaeal clade whose members thrive in hypersaline environments and routinely experience drastic changes in environmental salinity) and analyzed these new genomes in combination with those from 21 previously sequenced haloarchaeal isolates. We propose a generalized model for haloarchaeal management of cytoplasmic osmolarity in response to osmotic shifts, where potassium accumulation and sodium expulsion during osmotic upshock are accomplished via secondary transport using the proton gradient as an energy source, and potassium loss during downshock is via a combination of secondary transport and non-specific ion loss through mechanosensitive channels. We also propose new mechanisms for magnesium and chloride accumulation. We describe the expansion and differentiation of haloarchaeal general transcription factor families, including two novel expansions of the TATA-binding protein family, and discuss their potential for enabling rapid adaptation to environmental fluxes. We challenge a recent high-profile proposal regarding the evolutionary origins of the haloarchaea by showing that inclusion of additional genomes significantly reduces support for a proposed large-scale horizontal gene transfer into the ancestral haloarchaeon from the bacterial domain. The combination of broad (17 genera) and deep (≥5 species in four genera) sampling of a phenotypically unified clade has enabled us to uncover both highly conserved and specialized features of osmoadaptation. Finally, we demonstrate the broad utility of such datasets, for metagenomics, improvements to automated gene annotation and investigations of evolutionary processes.
Author Summary
The ability to adjust to changing osmotic conditions (osmoadaptation) is crucial to the survival of organisms across the tree of life. However, significant gaps still exist in our understanding of this important phenomenon. To help fill some of these gaps, we have produced high-quality draft genomes for 59 osmoadaptation “experts” (extreme halophiles of the euryarchaeal family Halobacteriaceae). We describe the dispersal of osmoadaptive protein families across the haloarchaeal evolutionary tree. We use this data to suggest a generalized model for haloarchaeal ion transport in response to changing osmotic conditions, including proposed new mechanisms for magnesium and chloride accumulation. We describe the evolutionary expansion and differentiation of haloarchaeal general transcription factor families and discuss their potential for enabling rapid adaptation to environmental fluxes. Lastly, we challenge a recent high-profile proposal regarding the evolutionary origins of the haloarchaea by showing that inclusion of additional genomes significantly reduces support for a proposed large-scale horizontal gene transfer into the ancestral haloarchaeon from the bacterial domain. This result highlights the power of our dataset for making evolutionary inferences, a feature which will make it useful to the broader evolutionary community. We distribute our genomic dataset through a user-friendly graphical interface.
doi:10.1371/journal.pgen.1004784
PMCID: PMC4230888  PMID: 25393412
15.  Mannosylation of Virus-Like Particles Enhances Internalization by Antigen Presenting Cells 
PLoS ONE  2014;9(8):e104523.
Internalization of peptides by antigen presenting cells is crucial for the initiation of the adaptive immune response. Mannosylation has been demonstrated to enhance antigen uptake through mannose receptors, leading to improved immune responses. In this study we test the effect of surface mannosylation of protein-based virus-like particles (VLP) derived from Rabbit hemorrhagic disease virus (RHDV) on uptake by murine and human antigen presenting cells. A monomannoside and a novel dimannoside were synthesized and successfully conjugated to RHDV VLP capsid protein, providing approximately 270 mannose groups on the surface of each virus particle. VLP conjugated to the mannoside or dimannoside exhibited significantly enhanced binding and internalization by murine dendritic cells, macrophages and B cells as well as human dendritic cells and macrophages. This uptake was inhibited by the inclusion of mannan as a specific inhibitor of mannose specific uptake, demonstrating that mannosylation of VLP targets mannose receptor-based uptake. Consistent with mannose receptor-based uptake, partial retargeting of the intracellular processing of RHDV VLP was observed, confirming that mannosylation of VLP provides both enhanced uptake and modified processing of associated antigens.
doi:10.1371/journal.pone.0104523
PMCID: PMC4133192  PMID: 25122183
16.  Application of Next-Generation Sequencing to Identify Genes and Mutations Causing Autosomal Dominant Retinitis Pigmentosa (adRP) 
The goal of our research is to identify genes and mutations causing auto-somal dominant retinitis pigmentosa (adRP). For this purpose we established a cohort of more than 250 independently ascertained families with adRP in the Houston Laboratory for Molecular Diagnosis of Inherited Eye Diseases. Affected members of each family were screened for disease-causing mutations in genes and gene regions that are commonly associated with adRP. By this approach, we detected mutations in 65 % of the families, leaving 85 families that are likely to harbor mutations outside of the “common” regions or in novel genes. Of these, 32 families were tested by several types of next-generation sequencing (NGS), including (a) targeted polymerase chain reaction (PCR) NGS, (b) whole exome NGS, and (c) targeted retinal-capture NGS. We detected mutations in 11 of these families (31 %) bringing the total detected in the adRP cohort to 70 %. Several large families have also been tested for linkage using Afymetrix single nucleotide polymorphism (SNP) arrays.
doi:10.1007/978-1-4614-3209-8_16
PMCID: PMC4121110  PMID: 24664689
Retinitis pigmentosa; Next-generation sequencing; Linkage mapping; Mutation prevalence; Retinal gene capture; Whole-exome sequencing
17.  Decreased basal chloride secretion and altered cystic fibrosis transmembrane conductance regulatory protein, Villin, GLUT5 protein expression in jejunum from leptin-deficient mice 
Patients with diabetes and obesity are at increased risk of developing disturbances in intestinal function. In this study, we characterized jejunal function in the clinically relevant leptin-deficient ob/ob mouse, a model of diabetes and obesity. We measured transepithelial short circuit current (Isc), across freshly isolated segments of jejunum from 12-week-old ob/ob and lean C57BL/6J (female and male) mice. The basal Isc was significantly decreased (~30%) in the ob/ob mice (66.5±5.7 μA/cm2 [n=20]) (P< 0.05) compared with their lean counterparts (95.1±9.1 μA/cm2 [n=19]). Inhibition with clotrimazole (100 μM, applied bilaterally) was significantly reduced in the ob/ob mice (−7.92%±3.67% [n=15]) (P<0.05) compared with the lean mice (10.44%±7.92% [n=15]), indicating a decreased contribution of Ca2+-activated K+ (KCa) channels in the ob/ob mice. Inhibition with ouabain (100 μM, applied serosally) was significantly reduced in the ob/ob mice (1.40%±3.61%, n=13) (P< 0.05) versus the lean mice (18.93%±3.76% [n=18]), suggesting a potential defect in the Na+/K+-adenosine triphosphate (ATP)ase pump with leptin-deficiency. Expression of cystic fibrosis transmembrane conductance regulatory protein (CFTR) (normalized to glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) was significantly decreased ~twofold (P<0.05) in the ob/ob mice compared with the leans, whilst crypt depth was unchanged. Villi length was significantly increased by ~25% (P<0.05) in the ob/ob mice compared with the leans and was associated with an increase in Villin and GLUT5 expression. GLUT2 and SGLT-1 expression were both unchanged. Our data suggests that reduced basal jejunal Isc in ob/ob mice is likely a consequence of reduced CFTR expression and decreased activity of the basolateral KCa channel and Na+/K+-ATPase. Understanding intestinal dysfunctions in ob/ob jejunum may allow for the development of novel drug targets to treat obesity and diabetes.
doi:10.2147/DMSO.S63714
PMCID: PMC4112754  PMID: 25092993
intestinal secretion; transport; ob/ob; obese; diabetes; small intestine
18.  Synapse Maturation by Activity-Dependent Ectodomain Shedding of SIRPα 
Nature neuroscience  2013;16(10):1417-1425.
Formation of appropriate synaptic connections is critical for proper functioning of the brain. After initial synaptic differentiation, active synapses are stabilized by neural activity-dependent signals to establish functional synaptic connections. However, the molecular mechanisms underlying activity-dependent synapse maturation remain to be elucidated. Here we show that activity-dependent ectodomain shedding of SIRPα mediates presynaptic maturation. Two target-derived molecules, FGF22 and SIRPα, sequentially organize the glutamatergic presynaptic terminals during the initial synaptic differentiation and synapse maturation stages, respectively, in the mouse hippocampus. SIRPα drives presynaptic maturation in an activity-dependent fashion. Remarkably, neural activity cleaves the extracellular domain of SIRPα, and the shed ectodomain, in turn, promotes the maturation of the presynaptic terminal. This process involves CaM kinase, matrix metalloproteinases, and the presynaptic receptor CD47. Finally, SIRPα-dependent synapse maturation has significant impacts on synaptic function and plasticity. Thus, ectodomain shedding of SIRPα is an activity-dependent trans-synaptic mechanism for the maturation of functional synapses.
doi:10.1038/nn.3516
PMCID: PMC3820962  PMID: 24036914
19.  A mitochondria-targeted mass spectrometry probe to detect glyoxals: implications for diabetes☆ 
Free Radical Biology & Medicine  2014;67(100):437-450.
The glycation of protein and nucleic acids that occurs as a consequence of hyperglycemia disrupts cell function and contributes to many pathologies, including those associated with diabetes and aging. Intracellular glycation occurs after the generation of the reactive 1,2-dicarbonyls methylglyoxal and glyoxal, and disruption of mitochondrial function is associated with hyperglycemia. However, the contribution of these reactive dicarbonyls to mitochondrial damage in pathology is unclear owing to uncertainties about their levels within mitochondria in cells and in vivo. To address this we have developed a mitochondria-targeted reagent (MitoG) designed to assess the levels of mitochondrial dicarbonyls within cells. MitoG comprises a lipophilic triphenylphosphonium cationic function, which directs the molecules to mitochondria within cells, and an o-phenylenediamine moiety that reacts with dicarbonyls to give distinctive and stable products. The extent of accumulation of these diagnostic heterocyclic products can be readily and sensitively quantified by liquid chromatography–tandem mass spectrometry, enabling changes to be determined. Using the MitoG-based analysis we assessed the formation of methylglyoxal and glyoxal in response to hyperglycemia in cells in culture and in the Akita mouse model of diabetes in vivo. These findings indicated that the levels of methylglyoxal and glyoxal within mitochondria increase during hyperglycemia both in cells and in vivo, suggesting that they can contribute to the pathological mitochondrial dysfunction that occurs in diabetes and aging.
Highlights
•A mitochondria-targeted mass spectrometric probe, MitoG, has been developed to measure glyoxal and methylglyoxal.•Using MitoG we show that mitochondrial glyoxal and methylglyoxal can be measured in hyperglycemic cells.•MitoG can also be used in vivo to infer mitochondrial glyoxal and methylglyoxal production in a mouse model of type I diabetes.•These findings suggest that the accumulation of glyoxal and methylglyoxal within mitochondria may contribute to mitochondrial dysfunction in diabetes.
doi:10.1016/j.freeradbiomed.2013.11.025
PMCID: PMC3978666  PMID: 24316194
Mitochondria; Exomarker; Methylglyoxal; Glyoxal; Hyperglycemia; MitoG; Free radicals
20.  A Monte Carlo-based framework enhances the discovery and interpretation of regulatory sequence motifs 
BMC Bioinformatics  2012;13:317.
Background
Discovery of functionally significant short, statistically overrepresented subsequence patterns (motifs) in a set of sequences is a challenging problem in bioinformatics. Oftentimes, not all sequences in the set contain a motif. These non-motif-containing sequences complicate the algorithmic discovery of motifs. Filtering the non-motif-containing sequences from the larger set of sequences while simultaneously determining the identity of the motif is, therefore, desirable and a non-trivial problem in motif discovery research.
Results
We describe MotifCatcher, a framework that extends the sensitivity of existing motif-finding tools by employing random sampling to effectively remove non-motif-containing sequences from the motif search. We developed two implementations of our algorithm; each built around a commonly used motif-finding tool, and applied our algorithm to three diverse chromatin immunoprecipitation (ChIP) data sets. In each case, the motif finder with the MotifCatcher extension demonstrated improved sensitivity over the motif finder alone. Our approach organizes candidate functionally significant discovered motifs into a tree, which allowed us to make additional insights. In all cases, we were able to support our findings with experimental work from the literature.
Conclusions
Our framework demonstrates that additional processing at the sequence entry level can significantly improve the performance of existing motif-finding tools. For each biological data set tested, we were able to propose novel biological hypotheses supported by experimental work from the literature. Specifically, in Escherichia coli, we suggested binding site motifs for 6 non-traditional LexA protein binding sites; in Saccharomyces cerevisiae, we hypothesize 2 disparate mechanisms for novel binding sites of the Cse4p protein; and in Halobacterium sp. NRC-1, we discoverd subtle differences in a general transcription factor (GTF) binding site motif across several data sets. We suggest that small differences in our discovered motif could confer specificity for one or more homologous GTF proteins. We offer a free implementation of the MotifCatcher software package at http://www.bme.ucdavis.edu/facciotti/resources_data/software/.
doi:10.1186/1471-2105-13-317
PMCID: PMC3542263  PMID: 23181585
Motif; Monte Carlo; ChIP-seq; ChIP-chip; Comparative genomics; MEME; STAMP; TFB
21.  Synthesis and Toll-Like Receptor 4 (TLR4) Activity of Phosphatidylinositol Dimannoside Analogues 
Journal of Medicinal Chemistry  2011;54(20):7268-7279.
A series of five PIM2 analogues were synthesized and tested for their ability to activate primary macrophages and modulate LPS signaling. Structural changes included replacement of the fatty acid esters of the phosphatidyl moiety of PIM2 with the corresponding ether or amide. An AcPIM2 analogue possessing an ether linkage was also prepared. The synthetic methodology utilized an orthogonally protected chiral myo-inositol starting material that was conveniently prepared from myo-inositol in just two steps. Important steps in the synthetic protocols included the regio- and α-selective glycosylation of inositol O-6 and introduction of the phosphodiester utilizing phosphoramidite chemistry. Replacement of the inositol core with a glycerol moiety gave compounds described as phosphatidylglycerol dimannosides (PGM2). Biological testing of these PIM compounds indicated that the agonist activity was TLR4 dependent. An ether linkage increased agonist activity, removal of the inositol ring enhanced antagonist activity and the presence of an additional lipid chain enhanced LPS-induced cytokine production in primary macrophages. Furthermore, the interruption of the LPS-induced TLR4/MD-2 2:2 signaling complex formation by PIM2 represents a previously unidentified mechanism involved in the bioactivity of PIM molecules.
doi:10.1021/jm2008419
PMCID: PMC3280216  PMID: 21936536
22.  A quasi-experimental evaluation of an interpersonal communication intervention to increase insecticide-treated net use among children in Zambia 
Malaria Journal  2012;11:313.
Background
This paper presents results from an evaluation of the effect of a community health worker (CHW) –based, interpersonal communication campaign (IPC) for increasing insecticide-treated mosquito net (ITN) use among children in Luangwa District, Zambia, an area with near universal coverage of ITNs and moderate to low malaria parasite prevalence.
Methods
A quasi-experimental community randomized control trial was conducted from 2008 to 2010. CHWs were the unit of randomization. Cross-sectional data were collected from houses in both 2008 and 2010 using simple random sampling of a complete household enumeration of the district. A difference-in -differences approach was used to analyse the data.
Results
ITN use among children <5 years old in households with ≥1 ITN increased overall from 54% in 2008 to 81% in 2010 (χ2 = 96.3, p <0.01); however, there was no difference in increase between the treatment and control arms in 2010 (p >0.05). ITN use also increased among children five to 14 years old from 37% in 2008 to 68% in 2010. There was no indication that the CHW-based intervention activities had a significant effect on increasing ITN use in this context, over and above what is already being done to disseminate information on the importance of using an ITN to prevent malaria infection.
Discussion
ITN use increased dramatically in the district between 2008 and 2010. It is likely that IPC activities in general may have contributed to the observed increase in ITN use, as the increased observed in this study was far higher than the increase observed between 2008 and 2010 malaria indicator survey (MIS) estimates. Contamination across control communities, coupled with linear settlement patterns and subsequent behavioural norms related to communication in the area, likely contributed to the observed increase in net use and null effect in this study.
doi:10.1186/1475-2875-11-313
PMCID: PMC3459708  PMID: 22958441
Evaluation; Insecticide-treated net (ITN); Interpersonal communication campaign (IPC); Community health worker (CHW); Malaria; Zambia
23.  Estimates of child deaths prevented from malaria prevention scale-up in Africa 2001-2010 
Malaria Journal  2012;11:93.
Background
Funding from external agencies for malaria control in Africa has increased dramatically over the past decade resulting in substantial increases in population coverage by effective malaria prevention interventions. This unprecedented effort to scale-up malaria interventions is likely improving child survival and will likely contribute to meeting Millennium Development Goal (MDG) 4 to reduce the < 5 mortality rate by two thirds between 1990 and 2015.
Methods
The Lives Saved Tool (LiST) model was used to quantify the likely impact that malaria prevention intervention scale-up has had on malaria mortality over the past decade (2001-2010) across 43 malaria endemic countries in sub-Saharan African. The likely impact of ITNs and malaria prevention interventions in pregnancy (intermittent preventive treatment [IPTp] and ITNs used during pregnancy) over this period was assessed.
Results
The LiST model conservatively estimates that malaria prevention intervention scale-up over the past decade has prevented 842,800 (uncertainty: 562,800-1,364,645) child deaths due to malaria across 43 malaria-endemic countries in Africa, compared to a baseline of the year 2000. Over the entire decade, this represents an 8.2% decrease in the number of malaria-caused child deaths that would have occurred over this period had malaria prevention coverage remained unchanged since 2000. The biggest impact occurred in 2010 with a 24.4% decrease in malaria-caused child deaths compared to what would have happened had malaria prevention interventions not been scaled-up beyond 2000 coverage levels. ITNs accounted for 99% of the lives saved.
Conclusions
The results suggest that funding for malaria prevention in Africa over the past decade has had a substantial impact on decreasing child deaths due to malaria. Rapidly achieving and then maintaining universal coverage of these interventions should be an urgent priority for malaria control programmes in the future. Successful scale-up in many African countries will likely contribute substantially to meeting MDG 4, as well as succeed in meeting MDG 6 (Target 1) to halt and reverse malaria incidence by 2015.
doi:10.1186/1475-2875-11-93
PMCID: PMC3350413  PMID: 22455864
24.  A workflow for genome-wide mapping of archaeal transcription factors with ChIP-seq 
Nucleic Acids Research  2012;40(10):e74.
Deciphering the structure of gene regulatory networks across the tree of life remains one of the major challenges in postgenomic biology. We present a novel ChIP-seq workflow for the archaea using the model organism Halobacterium salinarum sp. NRC-1 and demonstrate its application for mapping the genome-wide binding sites of natively expressed transcription factors. This end-to-end pipeline is the first protocol for ChIP-seq in archaea, with methods and tools for each stage from gene tagging to data analysis and biological discovery. Genome-wide binding sites for transcription factors with many binding sites (TfbD) are identified with sensitivity, while retaining specificity in the identification the smaller regulons (bacteriorhodopsin-activator protein). Chromosomal tagging of target proteins with a compact epitope facilitates a standardized and cost-effective workflow that is compatible with high-throughput immunoprecipitation of natively expressed transcription factors. The Pique package, an open-source bioinformatics method, is presented for identification of binding events. Relative to ChIP-Chip and qPCR, this workflow offers a robust catalog of protein–DNA binding events with improved spatial resolution and significantly decreased cost. While this study focuses on the application of ChIP-seq in H. salinarum sp. NRC-1, our workflow can also be adapted for use in other archaea and bacteria with basic genetic tools.
doi:10.1093/nar/gks063
PMCID: PMC3378898  PMID: 22323522
25.  Comparison of Lives Saved Tool model child mortality estimates against measured data from vector control studies in sub-Saharan Africa 
BMC Public Health  2011;11(Suppl 3):S34.
Background
Insecticide-treated mosquito nets (ITNs) and indoor-residual spraying have been scaled-up across sub-Saharan Africa as part of international efforts to control malaria. These interventions have the potential to significantly impact child survival. The Lives Saved Tool (LiST) was developed to provide national and regional estimates of cause-specific mortality based on the extent of intervention coverage scale-up. We compared the percent reduction in all-cause child mortality estimated by LiST against measured reductions in all-cause child mortality from studies assessing the impact of vector control interventions in Africa.
Methods
We performed a literature search for appropriate studies and compared reductions in all-cause child mortality estimated by LiST to 4 studies that estimated changes in all-cause child mortality following the scale-up of vector control interventions. The following key parameters measured by each study were applied to available country projections: baseline all-cause child mortality rate, proportion of mortality due to malaria, and population coverage of vector control interventions at baseline and follow-up years.
Results
The percent reduction in all-cause child mortality estimated by the LiST model fell within the confidence intervals around the measured mortality reductions for all 4 studies. Two of the LiST estimates overestimated the mortality reductions by 6.1 and 4.2 percentage points (33% and 35% relative to the measured estimates), while two underestimated the mortality reductions by 4.7 and 6.2 percentage points (22% and 25% relative to the measured estimates).
Conclusions
The LiST model did not systematically under- or overestimate the impact of ITNs on all-cause child mortality. These results show the LiST model to perform reasonably well at estimating the effect of vector control scale-up on child mortality when compared against measured data from studies across a range of malaria transmission settings. The LiST model appears to be a useful tool in estimating the potential mortality reduction achieved from scaling-up malaria control interventions.
doi:10.1186/1471-2458-11-S3-S34
PMCID: PMC3231908  PMID: 21501453

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