Estimating and Mapping the Population at Risk of Sleeping Sickness

Background

Human African trypanosomiasis (HAT), also known as sleeping sickness, persists as a public health problem in several sub-Saharan countries. Evidence-based, spatially explicit estimates of population at risk are needed to inform planning and implementation of field interventions, monitor disease trends, raise awareness and support advocacy. Comprehensive, geo-referenced epidemiological records from HAT-affected countries were combined with human population layers to map five categories of risk, ranging from “very high” to “very low,” and to estimate the corresponding at-risk population.

Results

Approximately 70 million people distributed over a surface of 1.55 million km² are estimated to be at different levels of risk of contracting HAT. Trypanosoma brucei gambiense accounts for 82.2% of the population at risk, the remaining 17.8% being at risk of infection from T. b. rhodesiense. Twenty-one million people live in areas classified as moderate to very high risk, where more than 1 HAT case per 10,000 inhabitants per annum is reported.

Discussion

Updated estimates of the population at risk of sleeping sickness were made, based on quantitative information on the reported cases and the geographic distribution of human population. Due to substantial methodological differences, it is not possible to make direct comparisons with previous figures for at-risk population. By contrast, it will be possible to explore trends in the future. The presented maps of different HAT risk levels will help to develop site-specific strategies for control and surveillance, and to monitor progress achieved by ongoing efforts aimed at the elimination of sleeping sickness.     

Hydrogen/deuterium exchange studies of native rabbit MM-CK dynamics

Creatine kinase (CK) isoenzymes catalyse the reversible transfer of a phosphoryl group from ATP onto creatine. This reaction plays a very important role in the regulation of intracellular ATP concentrations in excitable tissues. CK isoenzymes are highly resistant to proteases in native conditions. To appreciate localized backbone dynamics, kinetics of amide hydrogen exchange with deuterium was measured by pulse-labeling the dimeric cytosolic muscle CK isoenzyme. Upon exchange, the protein was digested with pepsin, and the deuterium content of the resulting peptides was determined by liquid chromatography coupled to mass spectrometry (MS). The deuteration kinetics of 47 peptides identified by MS/MS and covering 96% of the CK backbone were analyzed. Four deuteration patterns have been recognized: The less deuterated peptides are located in the saddle-shaped core of CK, whereas most of the highly deuterated peptides are close to the surface and located around the entrance to the active site. Their exchange kinetics are discussed by comparison with the known secondary and tertiary structures of CK with the goal to reveal the conformational dynamics of the protein. Some of the observed dynamic motions may be linked to the conformational changes associated with substrate binding and catalytic mechanism.     

Polythiophenes inhibit prion propagation by stabilizing prion protein (PrP) aggregates

Luminescent conjugated polymers (LCPs) interact with ordered protein aggregates and sensitively detect amyloids of many different proteins, suggesting that they may possess antiprion properties. Here, we show that a variety of anionic, cationic, and zwitterionic LCPs reduced the infectivity of prion-containing brain homogenates and of prion-infected cerebellar organotypic cultured slices and decreased the amount of scrapie isoform of PrP(C) (PrP(Sc)) oligomers that could be captured in an avidity assay. Paradoxically, treatment enhanced the resistance of PrP(Sc) to proteolysis, triggered the compaction, and enhanced the resistance to proteolysis of recombinant mouse PrP(23-231) fibers. These results suggest that LCPs act as antiprion agents by transitioning PrP aggregates into structures with reduced frangibility. Moreover, ELISA on cerebellar organotypic cultured slices and in vitro conversion assays with mouse PrP(23-231) indicated that poly(thiophene-3-acetic acid) may additionally interfere with the generation of PrP(Sc) by stabilizing the conformation of PrP(C) or of a transition intermediate. Therefore, LCPs represent a novel class of antiprion agents whose mode of action appears to rely on hyperstabilization, rather than destabilization, of PrP(Sc) deposits.     

Ultraconserved elements in the human genome: association and transmission analyses of highly constrained single-nucleotide polymorphisms

Ultraconserved elements in the human genome likely harbor important biological functions as they are dosage sensitive and are able to direct tissue-specific expression. Because they are under purifying selection, variants in these elements may have a lower frequency in the population but a higher likelihood of association with complex traits. We tested a set of highly constrained SNPs (hcSNPs) distributed genome-wide among ultraconserved and nearly ultraconserved elements for association with seven traits related to reproductive (age at natural menopause, number of children, age at first child, and age at last child) and overall [longevity, body mass index (BMI), and height] fitness. Using up to 24,047 European-American samples from the National Heart, Lung, and Blood Institute Candidate Gene Association Resource (CARe), we observed an excess of associations with BMI and height. In an independent replication panel the most strongly associated SNPs showed an 8.4-fold enrichment of associations at the nominal level, including three variants in previously identified loci and one in a locus (DENND1A) previously shown to be associated with polycystic ovary syndrome. Finally, using 1430 family trios, we showed that the transmissions from heterozygous parents to offspring of the derived alleles of rare (frequency ≤0.5%) hcSNPs are not biased, particularly after adjusting for the rates of genotype missingness and error in the data. The lack of transmission bias ruled out an immediately and strongly deleterious effect due to the rare derived alleles, consistent with the observation that mice homozygous for the deletion of ultraconserved elements showed no overt phenotype. Our study also illustrated the importance of carefully modeling potential technical confounders when analyzing genotype data of rare variants.     

Acyl chains of phospholipase d transphosphatidylation products in Arabidopsis cells: a study using multiple reaction monitoring mass spectrometry

Background

Phospholipases D (PLD) are major components of signalling pathways in plant responses to some stresses and hormones. The product of PLD activity is phosphatidic acid (PA). PAs with different acyl chains do not have the same protein targets, so to understand the signalling role of PLD it is essential to analyze the composition of its PA products in the presence and absence of an elicitor.

Methodology/Principal findings

Potential PLD substrates and products were studied in Arabidopsis thaliana suspension cells treated with or without the hormone salicylic acid (SA). As PA can be produced by enzymes other than PLD, we analyzed phosphatidylbutanol (PBut), which is specifically produced by PLD in the presence of n-butanol. The acyl chain compositions of PBut and the major glycerophospholipids were determined by multiple reaction monitoring (MRM) mass spectrometry. PBut profiles of untreated cells or cells treated with SA show an over-representation of 160/18∶2- and 16∶0/18∶3-species compared to those of phosphatidylcholine and phosphatidylethanolamine either from bulk lipid extracts or from purified membrane fractions. When microsomal PLDs were used in in vitro assays, the resulting PBut profile matched exactly that of the substrate provided. Therefore there is a mismatch between the acyl chain compositions of putative substrates and the in vivo products of PLDs that is unlikely to reflect any selectivity of PLDs for the acyl chains of substrates.

Conclusions

MRM mass spectrometry is a reliable technique to analyze PLD products. Our results suggest that PLD action in response to SA is not due to the production of a stress-specific molecular species, but that the level of PLD products per se is important. The over-representation of 160/18∶2- and 16∶0/18∶3-species in PLD products when compared to putative substrates might be related to a regulatory role of the heterogeneous distribution of glycerophospholipids in membrane sub-domains.     

Tandem purification of two HDL-associated partner proteins in human plasma, paraoxonase (PON1) and phosphate binding protein (HPBP) using hydroxyapatite chromatography

Human plasma paraoxonase (PON1) is calcium-dependent enzyme that hydrolyses esters, including organophosphates and lactones, and exhibits anti-atherogenic properties. Human phosphate binding protein (HPBP) was discovered as contaminant during crystallization trials of PON1. This observation and uncertainties for the real activities of PON1 led us to re-evaluate the purity of PON1 preparations. We developed a hydroxyapatite chromatography for the separation of both HDL-associated proteins. We confirmed that: (1) HPBP is strongly associated to PON1 in HDL, and generally both proteins are co-purified; (2) standard purification protocols of PON1 lead to impure enzyme; (3) hydroxyapatite chromatography allows the simultaneous purification of PON1 and HPBP.     

Nuclear autoantigenic sperm protein (NASP), a linker histone chaperone that is required for cell proliferation

A multichaperone nucleosome-remodeling complex that contains the H1 linker histone chaperone nuclear autoantigenic sperm protein (NASP) has recently been described. Linker histones (H1) are required for the proper completion of normal development, and NASP transports H1 histones into nuclei and exchanges H1 histones with DNA. Consequently, we investigated whether NASP is required for normal cell cycle progression and development. We now report that without sufficient NASP, HeLa cells and U2OS cells are unable to replicate their DNA and progress through the cell cycle and that the NASP(-/-) null mutation causes embryonic lethality. Although the null mutation NASP(-/-) caused embryonic lethality, null embryos survive until the blastocyst stage, which may be explained by the presence of stored NASP protein in the cytoplasm of oocytes. We conclude from this study that NASP and therefore the linker histones are key players in the assembly of chromatin after DNA replication.     

Divalent metal ion complexes of S100B in the absence and presence of pentamidine

As part of an effort to inhibit S100B, structures of pentamidine (Pnt) bound to Ca²⁺-loaded and Zn²⁺,Ca²⁺-loaded S100B were determined by X-ray crystallography at 2.15 Å (R_free = 0.266) and 1.85 Å (R_free = 0.243) resolution, respectively. These data were compared to X-ray structures solved in the absence of Pnt, including Ca²⁺-loaded S100B and Zn²⁺,Ca²⁺-loaded S100B determined here (1.88 Å; R_free = 0.267). In the presence and absence of Zn²⁺, electron density corresponding to two Pnt molecules per S100B subunit was mapped for both drug-bound structures. One Pnt binding site (site 1) was adjacent to a p53 peptide binding site on S100B (± Zn²⁺), and the second Pnt molecule was mapped to the dimer interface (site 2; ± Zn²⁺) and in a pocket near residues that define the Zn²⁺ binding site on S100B. In addition, a conformational change in S100B was observed upon the addition of Zn²⁺ to Ca²⁺-S100B, which changed the conformation and orientation of Pnt bound to sites 1 and 2 of Pnt-Zn²⁺,Ca²⁺-S100B when compared to Pnt-Ca²⁺-S100B. That Pnt can adapt to this Zn²⁺-dependent conformational change was unexpected and provides a new mode for S100B inhibition by this drug. These data will be useful for developing novel inhibitors of both Ca²⁺- and Ca²⁺,Zn²⁺-bound S100B.