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Melanocyte stem cells (McSCs) are the undifferentiated melanocytic cells of the mammalian hair follicle (HF) responsible for recurrent generation of a large number of differentiated melanocytes during each HF cycle. HF McSCs reside in both the CD34+ bulge/lower permanent portion (LPP) and the CD34- secondary hair germ (SHG) regions of the HF during telogen. Using Dct-H2BGFP mice, we separate bulge/LPP and SHG McSCs using FACS with GFP and anti-CD34 to show that these two subsets of McSCs are functionally distinct. Genome-wide expression profiling results support the distinct nature of these populations, with CD34- McSCs exhibiting higher expression of melanocyte differentiation genes and with CD34+ McSCs demonstrating a profile more consistent with a neural crest stem cell. In culture and in vivo, CD34- McSCs regenerate pigmentation more efficiently whereas CD34+ McSCs selectively exhibit the ability to myelinate neurons. CD34+ McSCs, and their counterparts in human skin, may be useful for myelinating neurons in vivo, leading to new therapeutic opportunities for demyelinating diseases and traumatic nerve injury.

•HRD dogs accurately identified grave soil samples in 711 trials at 92.8% accuracy.•Grave soils to 915 days PMI were identified by HRD dogs with at least 75% accuracy.•Soils with bodies removed post 667 days were identified by HRD dogs accurately.•Soil solutions from grave soils were identified with 96.8% accuracy by HRD dogs. Decomposing human remains alter the environment through deposition of various compounds comprised of a variety of chemical constituents. Human remains detection (HRD) dogs are trained to indicate the odor of human remains. Residual odor from previously decomposing human remains may remain in the soil and on surfaces long after the remains are gone. This study examined the ability of eight nationally certified HRD dogs (four dual purpose and four single purpose) to detect human remains odor in soil from under decomposing human remains as well as soils which no longer contained human remains, soils which had been cold water extracted and even the extraction fluid itself. The HRD dogs were able to detect the odor of human remains successfully above the level of chance for each soil ranging between 75% and 100% accurate up to 667 days post body removal from soil surface. No significant performance accuracy was found between the dual and single purpose dogs. This finding indicates that even though there may not be anything visually observable to the human eye, residual odor of human remains in soil can be very recalcitrant and therefore detectible by properly trained and credentialed HRD dogs. Further research is warranted to determine the parameters of the HRD dogs capabilities and in determining exactly what they are smelling.

Extensive genetic diversity plays a role in immune evasion, and antibody responses can be strain-specific or broadly reactive depending on the epitope. Controlled human malaria infection (CHMI) allows investigation of immune responses to variant parasite proteins after a single infection with a known strain. We designed a novel diversity-reflecting peptide microarray containing 638,817 unique peptides representing 22,655 variants of 227 proteins from 23 P genome assemblies and 379 field isolates. Using this array, we probed sera from 38 malaria naïve adults before and 28 days after CHMI with one of two genetically distinct strains, NF54 (n = 21) or 7G8 (n = 17). We examined fold-increase in antibody response (intensity) and cross-reactivity to protein variants (breadth). ABCPred was used to predict linear epitopes for all 227 proteins. We used MEME to identify enriched motifs in regions of high intensity or breadth, which were presumed to be potential epitopes. While the two CHMI groups had similar intensity of responses to all proteins on the array, 20 proteins on the array had differential breadth of responses and participants infected with 7G8 strain had a higher breadth of responses to 17 of them. Of 543 ABCPred-predicted epitopes, 66 overlapped with MEME-identified epitopes, six of which were highly cross-reactive with >95% of peptide variants serorecognized by at least one CHMI group. Overall, we found most antibody responses to be comparable after infection with the NF54 strain or 7G8 strain, but we saw notable differences for ~10% of proteins on the array. While many MEME-identified epitopes from highly cross-reactive proteins were asparagine rich, an epitope from PF3D7_1033200 (ETRAMP10.2) was not. Highly cross-reactive responses to ETRAMP10.2 could be further characterized and ETRAMP10.2 could be considered for inclusion in a next generation vaccine.

Malaria vectors in sub-Saharan Africa have proven themselves very difficult adversaries in the global struggle against malaria. Decades of anti-vector interventions have yielded mixed results--with successful reductions in transmission in some areas and limited impacts in others. These varying successes can be ascribed to a lack of universally effective vector control tools, as well as the development of insecticide resistance in mosquito populations. Understanding the impact of vector control on mosquito populations is crucial for planning new interventions and evaluating existing ones. However, estimates of population size changes in response to control efforts are often inaccurate because of limitations and biases in collection methods. Attempts to evaluate the impact of vector control on mosquito effective population size (N(e)) have produced inconclusive results thus far. Therefore, we obtained data for 13-15 microsatellite markers for more than 1,500 mosquitoes representing multiple time points for seven populations of three important vector species--Anopheles gambiae, An. melas, and An. moucheti--in Equatorial Guinea. These populations were exposed to indoor residual spraying or long-lasting insecticidal nets in recent years. For comparison, we also analyzed data from two populations that have no history of organized vector control. We used Approximate Bayesian Computation to reconstruct their demographic history, allowing us to evaluate the impact of these interventions on the effective population size. In six of the seven study populations, vector control had a dramatic impact on the effective population size, reducing N(e) between 55%-87%, the exception being a single An. melas population. In contrast, the two negative control populations did not experience a reduction in effective population size. This study is the first to conclusively link anti-vector intervention programs in Africa to sharply reduced effective population sizes of malaria vectors.

Host gene and protein expression impact susceptibility to clinical malaria, but the balance of immune cell populations, cytokines and genes that contributes to protection, remains incompletely understood. Little is known about the determinants of host susceptibility to clinical malaria at a time when acquired immunity is developing. We analyzed peripheral blood mononuclear cells (PBMCs) collected from children who differed in susceptibility to clinical malaria, all from a small town in Mali. PBMCs were collected from children aged 4-6 years at the start, peak and end of the malaria season. We characterized the immune cell composition and cytokine secretion for a subset of 20 children per timepoint (10 children with no symptomatic malaria age-matched to 10 children with >2 symptomatic malarial illnesses), and gene expression patterns for six children (three per cohort) per timepoint. We observed differences between the two groups of children in the expression of genes related to cell death and inflammation; in particular, inflammatory genes such as CXCL10 and STAT1 and apoptotic genes such as XAF1 were upregulated in susceptible children before the transmission season began. We also noted higher frequency of HLA-DR+ CD4 T cells in protected children during the peak of the malaria season and comparable levels cytokine secretion after stimulation with malaria schizonts across all three time points. This study highlights the importance of baseline immune signatures in determining disease outcome. Our data suggests that differences in apoptotic and inflammatory gene expression patterns can serve as predictive markers of susceptibility to clinical malaria.

The malaria mosquito Anopheles gambiae has a high preference for human hosts, a characteristic that contributes greatly to its capacity for transmitting human malaria. A sibling species, An. quadriannulatus, has a quite different host preference and feeds mostly on bovids. For this reason it does not contribute to human malaria transmission. Host seeking in mosquitoes is modulated by the olfactory system, which is primarily housed in the antennae and maxillary palps. Therefore, the detection of differing host odors by sibling species may be reflected in the expression level of the olfactory genes involved. Accordingly, we compared the transcriptomes of the antennae and maxillary palps of An. gambiae and An. quadriannulatus. We identified seven relatively abundant olfactory receptors, nine ionotropic receptors and three odorant binding proteins that are substantially up-regulated in An. gambiae antennae. Interestingly, we find that the maxillary palps of An. gambiae contain a species-specific olfactory receptor, Or52, and five An. gambiae-specific gustatory receptors (AgGr48-52) that are relatively abundant. These five gustatory receptors are also expressed in An. gambiae antennae, although at lower level, indicating a likely role in olfaction, rather than gustation. We also document an approximately three-fold higher overall expression of olfaction genes in the maxillary palps of An. quadriannulatus, indicating an important role of this organ in the olfaction system of this species. Finally, the expression of the CO2 receptor genes is five to six-fold higher in the zoophilic An. quadriannulatus, implying a much higher sensitivity for detecting CO2. These results identify potential human host preference genes in the malaria vector An. gambiae. Interestingly, species-specific expression of several gustatory receptors in the olfactory organs indicate a role in olfaction rather than gustation. Additionally, a more expansive role for maxillary palps in olfaction is implicated than previously thought, albeit more so in the zoophilic An. quadriannulatus.

Malaria parasites display antigens on the surface of infected red blood cells in the human host that facilitate attachment to blood vessels, contributing to the severity of infection. These antigens are highly variable, allowing the parasite to evade the immune system. var genes encode Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1) antigens. These highly diverse antigens are displayed on the surface of infected erythrocytes and play a critical role in immune evasion and sequestration of infected erythrocytes. Studies of var expression using non-leukocyte-depleted blood are challenging because of the predominance of host genetic material and lack of conserved var segments. Our goal was to enrich for parasite RNA, allowing de novo assembly of var genes and detection of expressed novel variants. We used two overall approaches: (i) enriching for total mRNA in the sequencing library preparations and (ii) enriching for parasite RNA with a custom capture array based on Roche’s SeqCap EZ enrichment system. The capture array was designed with probes based on the whole 3D7 reference genome and an additional >4,000 full-length var gene sequences from other P. falciparum strains. We tested each method on RNA samples from Malian children with severe or uncomplicated malaria infections. All reads mapping to the human genome were removed, the remaining reads were assembled de novo into transcripts, and from these, var -like transcripts were identified and annotated. The capture array produced the longest maximum length and largest numbers of var gene transcripts in each sample, particularly in samples with low parasitemia. Identifying the most-expressed var gene sequences in whole-blood clinical samples without the need for extensive processing or generating sample-specific reference genome data is critical for understanding the role of PfEMP1s in malaria pathogenesis. IMPORTANCE Malaria parasites display antigens on the surface of infected red blood cells in the human host that facilitate attachment to blood vessels, contributing to the severity of infection. These antigens are highly variable, allowing the parasite to evade the immune system. Identifying these expressed antigens is critical to understanding the development of severe malarial disease. However, clinical samples contain limited amounts of parasite genetic material, a challenge for sequencing efforts further compounded by the extreme diversity of the parasite surface antigens. We present a method that enriches for these antigen sequences in clinical samples using a custom capture array, requiring minimal processing in the field. While our results are focused on the malaria parasite Plasmodium falciparum , this approach has broad applicability to other highly diverse antigens from other parasites and pathogens such as those that cause giardiasis and leishmaniasis.

Populations of Drosophila melanogaster face significant mortality risks from parasitoid wasps that use species-specific strategies to locate and survive in hosts. We tested the hypothesis that parasitoids with different strategies select for alternative host defense characteristics and in doing so contribute to the maintenance of fitness variation and produce trade-offs among traits. We characterized defense traits of Drosophila when exposed to parasitoids with different host searching behaviors (Aphaereta sp. and Leptopilina boulardi). We used host larvae with different natural alleles of the gene Dopa decarboxylase (Ddc), a gene controlling the production of dopamine and known to influence the immune response against parasitoids. Previous population genetic analyses indicate that our focal alleles are maintained by balancing selection. Genotypes exhibited a trade-off between the immune response against Aphaereta sp. and the ability to avoid parasitism by L. boulardi. We also identified a trade-off between the ability to avoid parasitism by L. boulardi and larval competitive ability as indicated by differences in foraging and feeding behavior. Genotypes differed in dopamine levels potentially explaining variation in these traits. Our results highlight the potential role of parasitoid biodiversity on host fitness variation and implicate Ddc as an antagonistic pleiotropic locus influencing larval fitness traits.

On Bioko Island, Equatorial Guinea, indoor residual spraying (IRS) has been part of the Bioko Island Malaria Control Project since early 2004. Despite success in reducing childhood infections, areas of high transmission remain on the island. We therefore examined fluctuations in the effective population size (Ne) of the malaria vector Anopheles gambiae in an area of persistent high transmission over two spray rounds. We analyzed data for 13 microsatellite loci from 791 An. gambiae specimens collected at six time points in 2009 and 2010 and reconstructed the demographic history of the population during this period using approximate Bayesian computation (ABC). Our analysis shows that IRS rounds have a large impact on Ne, reducing it by 65%–92% from prespray round Ne. More importantly, our analysis shows that after 3–5 months, the An. gambiae population rebounded by 2818% compared shortly following the spray round. Our study underscores the importance of adequate spray round frequency to provide continuous suppression of mosquito populations and that increased spray round frequency should substantially improve the efficacy of IRS campaigns. It also demonstrates the ability of ABC to reconstruct a detailed demographic history across only a few tens of generations in a large population.