Malaria Burden and Artemisinin Resistance in the Mobile and Migrant Population on the Thai–Myanmar Border, 1999–2011: An Observational Study

Background

The Shoklo Malaria Research Unit has been working on the Thai–Myanmar border for 25 y providing early diagnosis and treatment (EDT) of malaria. Transmission of Plasmodium falciparum has declined, but resistance to artesunate has emerged. We expanded malaria activities through EDT and evaluated the impact over a 12-y period.

Methods and Findings

Between 1 October 1999 and 30 September 2011, the Shoklo Malaria Research Unit increased the number of cross-border (Myanmar side) health facilities from two to 11 and recorded the number of malaria consultations. Changes in malaria incidence were estimated from a cohort of pregnant women, and prevalence from cross-sectional surveys. In vivo and in vitro antimalarial drug efficacy were monitored. Over this period, the number of malaria cases detected increased initially, but then declined rapidly. In children under 5 y, the percentage of consultations due to malaria declined from 78% (95% CI 76–80) (1,048/1,344 consultations) to 7% (95% CI 6.2–7.1) (767/11,542 consultations), p<0.001. The ratio of P. falciparum/P. vivax declined from 1.4 (95% CI 1.3–1.4) to 0.7 (95% CI 0.7–0.8). The case fatality rate was low (39/75,126; 0.05% [95% CI 0.04–0.07]). The incidence of malaria declined from 1.1 to 0.1 episodes per pregnant women-year. The cumulative proportion of P. falciparum decreased significantly from 24.3% (95% CI 21.0–28.0) (143/588 pregnant women) to 3.4% (95% CI 2.8–4.3) (76/2,207 pregnant women), p<0.001. The in vivo efficacy of mefloquine-artesunate declined steadily, with a sharp drop in 2011 (day-42 PCR-adjusted cure rate 42% [95% CI 20–62]). The proportion of patients still slide positive for malaria at day 3 rose from 0% in 2000 to reach 28% (95% CI 13–45) (8/29 patients) in 2011.

Conclusions

Despite the emergence of resistance to artesunate in P. falciparum, the strategy of EDT with artemisinin-based combination treatments has been associated with a reduction in malaria in the migrant population living on the Thai–Myanmar border. Although limited by its observational nature, this study provides useful data on malaria burden in a strategically crucial geographical area. Alternative fixed combination treatments are needed urgently to replace the failing first-line regimen of mefloquine and artesunate. Please see later in the article for the Editors'' Summary     

Genetic Association Analyses of Nitric Oxide Synthase Genes and Neural Tube Defects Vary by Phenotype

Neural tube defects (NTDs) are caused by improper neural tube closure during the early stages of embryonic development. NTDs are hypothesized to have a complex genetic origin and numerous candidate genes have been proposed. The nitric oxide synthase 3 (NOS3) G594T polymorphism has been implicated in risk for spina bifida, and interactions between that single nucleotide polymorphism (SNP) and the methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism have also been observed. To evaluate other genetic variation in the NO pathway in the development of NTDs, we examined all three NOS genes: NOS1, NOS2, and NOS3. Using 3109 Caucasian samples in 745 families, we evaluated association in the overall dataset and within specific phenotypic subsets. Haplotype tagging SNPs in the NOS genes were tested for genetic association with NTD subtypes, both for main effects as well as for the presence of interactions with the MTHFR C677T polymorphism. Nominal main effect associations were found with all subtypes, across all three NOS genes, and interactions were observed between SNPs in all three NOS genes and MTHFR C677T. Unlike the previous report, the most significant associations in our dataset were with cranial subtypes and the AG genotype of rs4795067 in NOS2 (p = 0.0014) and the interaction between the rs9658490 G allele in NOS1 and MTHFR 677TT genotype (p = 0.0014). Our data extend the previous findings by implicating a role for all three NOS genes, independently and through interactions with MTHFR, in risk not only for spina bifida, but all NTD subtypes.     

Cancer cachexia’s metabolic signature in a murine model confirms a distinct entity

Despite recent consensus definitions, lack of specific biomarkers remains a hurdle towards a more accurate and efficient diagnosis of cancer cachexia, distinguishing cachexia as a separate entity from other wasting syndromes. In a previous pilot study, we have shown that cancer-cachectic mice have a unique metabolic fingerprint with distinct glucose and lipid alterations compared to healthy controls. Further metabolomics studies were carried out to investigate differences in metabolic profiles of cancer-cachectic mice to tumor-bearing non-cachectic mice, calorie-restricted mice, and surgically treated cancer-cachectic mice. CD2F1 mice were divided into: (1) Cachexia Group received cachexia-inducing C26 undifferentiated colon carcinoma cells; (2) Tumor-Burden Group received, non-cachectic, P388 lymphoma cells; (3) Caloric-Restriction Group, remaining cancer-free, but subjected to caloric-restriction; (4) Surgery Group, similar to Cachexia Group, but tumors resected mid-experiment; and (5) Control Group aged intact. Baseline, mid-experiment and final serum samples were collected for ¹H NMR spectroscopic analysis. After data reduction, unsupervised principal component analysis and orthogonal projections to latent structures analyses demonstrate that the unique metabolic fingerprint is independent of tumor-burden and distinct from profiles of caloric-restriction and aging. Hyperlipidemia, hyperglycemia, and reduced branched-chain amino acids distinguish cachexia from other groups. Furthermore, the profile of surgically treated mice differs from that of cachectic mice, reverting to a profile more congruent with healthy controls indicating cachexia is amenable to correction where surgical cure is possible. That metabolomic analysis of murine serum is able to differentiate cachexia from tumor-burden and caloric-restriction warrants similar translational investigations in patients to explore cancer cachexia’s unique biomarkers.     

Quantitative Bioluminescent Imaging of Pre-Erythrocytic Malaria Parasite Infection Using Luciferase-Expressing Plasmodium yoelii

The liver stages of Plasmodium parasites are important targets for the development of anti-malarial vaccine candidates and chemoprophylaxis approaches that aim to prevent clinical infection. Analyzing the impact of interventions on liver stages in the murine malaria model system Plasmodium yoelii has been cumbersome and requires terminal procedures. In vivo imaging of bioluminescent parasites has previously been shown to be an effective and non-invasive alternative to monitoring liver stage burden in the Plasmodium berghei model. Here we report the generation and characterization of a transgenic P. yoelii parasite expressing the reporter protein luciferase throughout the parasite life cycle. In vivo bioluminescent imaging of these parasites allows for quantitative analysis of P. yoelii liver stage burden and parasite development, which is comparable to quantitative RT-PCR analysis of liver infection. Using this system, we show that both BALB/cJ and C57BL/6 mice show comparable susceptibility to P. yoelii infection with sporozoites and that bioluminescent imaging can be used to monitor protective efficacy of attenuated parasite immunizations. Thus, this rapid, simple and noninvasive method for monitoring P. yoelii infection in the liver provides an efficient system to screen and evaluate the effects of anti-malarial interventions in vivo and in real-time.     

Adaptation of Mycobacteria to Growth Conditions: A Theoretical Analysis of Changes in Gene Expression Revealed by Microarrays

Background

Microarray analysis is a powerful technique for investigating changes in gene expression. Currently, results (r-values) are interpreted empirically as either unchanged or up- or down-regulated. We now present a mathematical framework, which relates r-values to the macromolecular properties of population-average cells. The theory is illustrated by the analysis of published data for two species; namely, Mycobacterium bovis BCG Pasteur and Mycobacterium smegmatis mc² 155. Each species was grown in a chemostat at two different growth rates. Application of the theory reveals the growth rate dependent changes in the mycobacterial proteomes.

Principal Findings

The r-value r _(i) of any ORF (ORF_(i)) encoding protein p _(i) was shown to be equal to the ratio of the concentrations of p _(i) and so directly proportional to the ratio of the numbers of copies of p _(i) per population-average cells of the two cultures. The proportionality constant can be obtained from the ratios DNA: RNA: protein. Several subgroups of ORFs were identified because they shared a particular r-value. Histograms of the number of ORFs versus the expression ratio were simulated by combining the particular r-values of several subgroups of ORFs. The largest subgroup was ORF_(j) (r _(j)  = 1.00± SD) which was estimated to comprise respectively 59% and 49% of ORFs of M. bovis BCG Pasteur and M. smegmatis mc² 155. The standard deviations reflect the properties of the cDNA preparations investigated.

Significance

The analysis provided a quantitative view of growth rate dependent changes in the proteomes of the mycobacteria studied. The majority of the ORFs were found to be constitutively expressed. In contrast, the protein compositions of the outer permeability barriers and cytoplasmic membranes were found to be dependent on growth rate; thus illustrating the response of bacteria to their environment. The theoretical approach applies to any cultivatable bacterium under a wide range of growth conditions.     

Modulation of Ocular Surface Glycocalyx Barrier Function by a Galectin-3 N-terminal Deletion Mutant and Membrane-Anchored Synthetic Glycopolymers

Background

Interaction of transmembrane mucins with the multivalent carbohydrate-binding protein galectin-3 is critical to maintaining the integrity of the ocular surface epithelial glycocalyx. This study aimed to determine whether disruption of galectin-3 multimerization and insertion of synthetic glycopolymers in the plasma membrane could be used to modulate glycocalyx barrier function in corneal epithelial cells.

Methodology/Principal Findings

Abrogation of galectin-3 biosynthesis in multilayered cultures of human corneal epithelial cells using siRNA, and in galectin-3 null mice, resulted in significant loss of corneal barrier function, as indicated by increased permeability to the rose bengal diagnostic dye. Addition of β-lactose, a competitive carbohydrate inhibitor of galectin-3 binding activity, to the cell culture system, transiently disrupted barrier function. In these experiments, treatment with a dominant negative inhibitor of galectin-3 polymerization lacking the N-terminal domain, but not full-length galectin-3, prevented the recovery of barrier function to basal levels. As determined by fluorescence microscopy, both cellobiose- and lactose-containing glycopolymers incorporated into apical membranes of corneal epithelial cells, independently of the chain length distribution of the densely glycosylated, polymeric backbones. Membrane incorporation of cellobiose glycopolymers impaired barrier function in corneal epithelial cells, contrary to their lactose-containing counterparts, which bound to galectin-3 in pull-down assays.

Conclusions/Significance

These results indicate that galectin-3 multimerization and surface recognition of lactosyl residues is required to maintain glycocalyx barrier function at the ocular surface. Transient modification of galectin-3 binding could be therapeutically used to enhance the efficiency of topical drug delivery.     

Speed of Human Biological Form and Motion Processing

Recent work suggests that biological motion processing can begin within ~110 ms of stimulus onset, as indexed by the P1 component of the event-related potential (ERP). Here, we investigated whether modulation of the P1 component reflects configural processing alone, rather than the processing of both configuration and motion cues. A three-stimulus oddball task was employed to evaluate bottom-up processing of biological motion. Intact point-light walkers (PLWs) or scrambled PLWs served as distractor stimuli, whereas point-light displays of tool motion served as standard and target stimuli. In a second experiment, the same design was used, but the dynamic stimuli were replaced with static point-light displays. The first experiment revealed that dynamic PLWs elicited a larger P1 as compared to scrambled PLWs. A similar P1 increase was also observed for static PLWs in the second experiment, indicating that these stimuli were more salient than static, scrambled PLWs. These findings suggest that the visual system can rapidly extract global form information from static PLWs and that the observed P1 effect for dynamic PLWs is not dependent on the presence of motion cues. Finally, we found that the N1 component was sensitive to dynamic, but not static, PLWs, suggesting that this component reflects the processing of both form and motion information. The sensitivity of P1 to static PLWs has implications for dynamic form models of biological motion processing that posit temporal integration of configural cues present in individual frames of PLW animations.     

Role of the Arabidopsis PIN6 Auxin Transporter in Auxin Homeostasis and Auxin-Mediated Development

Plant-specific PIN-formed (PIN) efflux transporters for the plant hormone auxin are required for tissue-specific directional auxin transport and cellular auxin homeostasis. The Arabidopsis PIN protein family has been shown to play important roles in developmental processes such as embryogenesis, organogenesis, vascular tissue differentiation, root meristem patterning and tropic growth. Here we analyzed roles of the less characterised Arabidopsis PIN6 auxin transporter. PIN6 is auxin-inducible and is expressed during multiple auxin–regulated developmental processes. Loss of pin6 function interfered with primary root growth and lateral root development. Misexpression of PIN6 affected auxin transport and interfered with auxin homeostasis in other growth processes such as shoot apical dominance, lateral root primordia development, adventitious root formation, root hair outgrowth and root waving. These changes in auxin-regulated growth correlated with a reduction in total auxin transport as well as with an altered activity of DR5-GUS auxin response reporter. Overall, the data indicate that PIN6 regulates auxin homeostasis during plant development.     

Comparison of fecal glucocorticoid metabolite concentrations in hand- versus parent-reared whooping cranes (Grus americana)

Endangered whooping cranes (Grus americana) have been produced in captivity for reintroduction programs since the 1980s, using techniques such as artificial insemination, multiple clutching, and captive-rearing to speed recovery efforts. Chicks are often hand-reared (HR) by caretakers in crane costumes, socialized into groups and released together, unlike parent-reared (PR) cranes that are raised individually by a male/female crane pair and released singly. HR cranes historically exhibit greater morbidity rates during development than PR cranes, involving musculoskeletal and respiratory system disease, among others. We hypothesized that HR crane chicks exhibit a higher baseline fecal glucocorticoid metabolite (FGM) concentrations during the development compared with PR chicks. Fecal samples were collected between 15 and 70 days of age from HR (n = 15) and PR (n = 8) chicks to test for differences in FGM concentrations using a radioimmunoassay technique following ethanol extraction for steroids. Linear mixed model analysis suggests increasing age of the chick was associated with an increase in FGM (p < .001). Analysis also supported the interaction between rearing strategy and sex of the crane chick (p < .01). Female PR chicks had greater FGM concentrations than all other groups (PR male, p < .01; HR female, p < .001; and HR male, p < .001). This result suggests that there may be an effect of rearing strategy on stress physiology of whooping crane chicks, especially among females. Further research is needed to investigate whether the FGM concentrations are reflective of true differences in stress physiology of young cranes and whether this may impact health and conservation success.