Environmental Thermal Stress Induces Neuronal Cell Death and Developmental Malformations in Reptiles.

Every stage of organismal life history is being challenged by global warming. Many species are already experiencing temperatures approaching their physiological limits; this is particularly true for ectothermic species, such as lizards. Embryos are markedly sensitive to thermal insult. Here, we demonstrate that temperatures currently experienced in natural nesting areas can modify gene expression levels and induce neural and craniofacial malformations in embryos of the lizard Anolis sagrei. Developmental abnormalities ranged from minor changes in facial structure to significant disruption of anterior face and forebrain. The first several days of postoviposition development are particularly sensitive to this thermal insult. These results raise new concern over the viability of ectothermic species under contemporary climate change. Herein, we propose and test a novel developmental hypothesis that describes the cellular and developmental origins of those malformations: cell death in the developing forebrain and abnormal facial induction due to disrupted Hedgehog signaling. Based on similarities in the embryonic response to thermal stress among distantly related species, we propose that this developmental hypothesis represents a common embryonic response to thermal insult among amniote embryos. Our results emphasize the importance of adopting a broad, multidisciplinary approach that includes both lab and field perspectives when trying to understand the future impacts of anthropogenic change on animal development.

Publisher Correction: Plasmodium falciparum sexual differentiation in malaria patients is associated with host factors and GDV1-dependent genes.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Plasmodium falciparum sexual differentiation in malaria patients is associated with host factors and GDV1-dependent genes.

Plasmodium sexual differentiation is required for malaria transmission, yet much remains unknown about its regulation. Here, we quantify early gametocyte-committed ring (gc-ring) stage, P. falciparum parasites in 260 uncomplicated malaria patient blood samples 10 days before maturation to transmissible stage V gametocytes using a gametocyte conversion assay (GCA). Seventy six percent of the samples have gc-rings, but the ratio of gametocyte to asexual-committed rings (GCR) varies widely (0-78%). GCR correlates positively with parasitemia and is negatively influenced by fever, not hematocrit, age or leukocyte counts. Higher expression levels of GDV1-dependent genes, ap2-g, msrp1 and gexp5, as well as a gdv1 allele encoding H217 are associated with high GCR, while high plasma lysophosphatidylcholine levels are associated with low GCR in the second study year. The results provide a view of sexual differentiation in the field and suggest key regulatory roles for clinical factors and gdv1 in gametocytogenesis in vivo.

The effects of thermal stress on the early development of the lizard Anolis sagrei.

Across the globe terrestrial ectotherms-amphibians and non-avian reptiles-are facing a range of emerging challenges. Increasing global temperatures, in particular, are affecting all aspects of ectotherm biology and life history. Embryonic development is a thermally sensitive period of the organismal lifecycle, yet the impacts of thermal stress on the early development of ectotherms have significantly lagged behind studies of later stages and adult thermal physiology. Morphogenesis, the stage where the major anatomical systems are actively forming, is particularly sensitive to thermal stress, yet is not studied as often as later stages where growth is the primary process happening within the egg. Here, we focus on the effects of thermal stress on the first 12 days of development, the stages of morphogenesis, in the lizard Anolis sagrei. We examine the resiliency of the early developmental stages to heat stress by incubating eggs at temperatures that parallel conditions observed today and predicted over the next 50-100 years of projected climate change. Our results suggest that some anole nests are currently at the thermal limits for which the early embryonic stages can properly develop. Our results emphasize the importance of studying early embryonic stages of development and the importance of studying stage-specific effects of thermal stress on squamate development.

Maduramicin Rapidly Eliminates Malaria Parasites and Potentiates the Gametocytocidal Activity of the Pyrazoleamide PA21A050.

New strategies targeting Plasmodium falciparum gametocytes, the sexual-stage parasites that are responsible for malaria transmission, are needed to eradicate this disease. Most commonly used antimalarials are ineffective against P. falciparum gametocytes, allowing patients to continue to be infectious for over a week after asexual parasite clearance. A recent screen for gametocytocidal compounds demonstrated that the carboxylic polyether ionophore maduramicin is active at low nanomolar concentrations against P. falciparum sexual stages. In this study, we showed that maduramicin has an EC50 (effective concentration that inhibits the signal by 50%) of 14.8 nM against late-stage gametocytes and significantly blocks in vivo transmission in a mouse model of malaria transmission. In contrast to other reported gametocytocidal agents, maduramicin acts rapidly in vitro, eliminating gametocytes and asexual schizonts in less than 12 h without affecting uninfected red blood cells (RBCs). Ring stage parasites are cleared by 24 h. Within an hour of drug treatment, 40% of the normally crescent-shaped gametocytes round up and become spherical. The number of round gametocytes increases to >60% by 2 h, even before a change in membrane potential as monitored by MitoProbe DiIC1 (5) is detectable. Maduramicin is not preferentially taken up by gametocyte-infected RBCs compared to uninfected RBCs, suggesting that gametocytes are more sensitive to alterations in cation concentration than RBCs. Moreover, the addition of 15.6 nM maduramicin enhanced the gametocytocidal activity of the pyrazoleamide PA21A050, which is a promising new antimalarial candidate associated with an increase in intracellular Na(+) concentration that is proposed to be due to inhibition of PfATP4, a putative Na(+) pump. These results underscore the importance of cation homeostasis in sexual as well as asexual intraerythrocytic-stage P. falciparum parasites and the potential of targeting this pathway for drug development.

Plasmodium falciparum gametocyte development 1 (Pfgdv1) and gametocytogenesis early gene identification and commitment to sexual development.

Malaria transmission requires the production of male and female gametocytes in the human host followed by fertilization and sporogonic development in the mosquito midgut. Although essential for the spread of malaria through the population, little is known about the initiation of gametocytogenesis in vitro or in vivo. Using a gametocyte-defective parasite line and genetic complementation, we show that Plasmodium falciparumgametocyte development 1 gene (Pfgdv1), encoding a peri-nuclear protein, is critical for early sexual differentiation. Transcriptional analysis of Pfgdv1 negative and positive parasite lines identified a set of gametocytogenesis early genes (Pfge) that were significantly down-regulated (>10 fold) in the absence of Pfgdv1 and expression was restored after Pfgdv1 complementation. Progressive accumulation of Pfge transcripts during successive rounds of asexual replication in synchronized cultures suggests that gametocytes are induced continuously during asexual growth. Comparison of Pfge gene transcriptional profiles in patient samples divided the genes into two groups differing in their expression in mature circulating gametocytes and providing candidates to evaluate gametocyte induction and maturation separately in vivo. The expression profile of one of the early gametocyte specific genes, Pfge1, correlated significantly with asexual parasitemia, which is consistent with the ongoing induction of gametocytogenesis during asexual growth observed in vitro and reinforces the need for sustained transmission-blocking strategies to eliminate malaria.

Functional analysis of the exported type IV HSP40 protein PfGECO in Plasmodium falciparum gametocytes.

During Plasmodium falciparum infection, host red blood cell (RBC) remodeling is required for the parasite's survival. Such modifications are mediated by the export of parasite proteins into the RBC that alter the architecture of the RBC membrane and enable cytoadherence. It is probable that some exported proteins also play a protective role against the host defense response. This may be of particular importance for the gametocyte stage of the life cycle that is responsible for malaria transmission, since the gametocyte remains in contact with blood as it proceeds through five morphological stages (I to V) during its 12-day maturation. Using microarray analysis, we identified several genes with encoded secretory or export sequences that were differentially expressed during early gametocytogenesis. One of these, PfGECO, encodes a predicted type IV heat shock protein 40 (HSP40) that we show is expressed in gametocyte stages I to IV and is exported to the RBC cytoplasm. HSPs are traditionally induced under stressful conditions to maintain homeostasis, but PfGECO expression was not increased upon heat shock, suggesting an alternate function. Targeted disruption of PfGECO indicated that the gene is not essential for gametocytogenesis in vitro, and quantitative reverse transcriptase PCR (RT-PCR) showed that there was no compensatory expression of the other type IV HSP40 genes. Although P. falciparum HSP40 members are implicated in the trafficking of proteins to the RBC surface, removal of PfGECO did not affect the targeting of other exported gametocyte proteins. This work has expanded the repertoire of known gametocyte-exported proteins to include a type IV HSP40, PfGECO.

The proteasome inhibitor epoxomicin has potent Plasmodium falciparum gametocytocidal activity.

Malaria continues to be a major global health problem, but only a limited arsenal of effective drugs is available. None of the antimalarial compounds commonly used clinically kill mature gametocytes, which is the form of the parasite that is responsible for malaria transmission. The parasite that causes the most virulent human malaria, Plasmodium falciparum, has a 48-h asexual cycle, while complete sexual differentiation takes 10 to 12 days. Once mature, stage V gametocytes circulate in the peripheral blood and can be transmitted for more than a week. Consequently, if chemotherapy does not eliminate gametocytes, an individual continues to be infectious for several weeks after the clearance of asexual parasites. The work reported here demonstrates that nanomolar concentrations of the proteasome inhibitor epoxomicin effectively kill all stages of intraerythrocytic parasites but do not affect the viability of human or mouse cell lines. Twenty-four hours after treatment with 100 nM epoxomicin, the total parasitemia decreased by 78%, asexual parasites decreased by 86%, and gametocytes decreased by 77%. Seventy-two hours after treatment, no viable parasites remained in the 100 or 10 nM treatment group. Epoxomicin also blocked oocyst production in the mosquito midgut. In contrast, the cysteine protease inhibitors epoxysuccinyl-L-leucylamido-3-methyl-butane ethyl ester and N-acetyl-L-leucyl-L-leucyl-L-methioninal blocked hemoglobin digestion in early gametocytes but had no effect on later stages. Moreover, once the cysteine protease inhibitor was removed, sexual differentiation resumed. These findings provide strong support for the further development of proteasome inhibitors as antimalaria agents that are effective against asexual, sexual, and mosquito midgut stages of P. falciparum.

Malaria transmission-blocking antigen, Pfs230, mediates human red blood cell binding to exflagellating male parasites and oocyst production.

Malaria transmission requires that the parasites differentiate into gametocytes prior to ingestion by a mosquito during a blood meal. Once in the mosquito midgut the gametocytes emerge from red blood cells (RBCs), fertilize, develop into ookinetes and finally infectious sporozoites. Gamete surface antigen, Pfs230, is an important malaria transmission-blocking vaccine candidate, but its function has remained unclear. Two clones with distinct Pfs230 gene disruptions (Delta1.356 and Delta2.560) and a clone with a disruption of Pfs48/45 were used to evaluate the role of Pfs230 in the mosquito midgut. Pfs230 disruptants successfully emerge from RBCs and male gametes exflagellate producing microgametes. However, exflagellating Pfs230-minus males, in the presence or absence of Pfs48/45, are unable to interact with RBCs and form exflagellation centres. Oocyst production and mosquito infectivity is also significantly reduced, 96-92% and 76-71% respectively. In contrast, in the Pfs230 disruptants the expression and localization of other known sexual stage-specific antigens, including Pfs48/45, Pfs47, the Pfs230 paralogue (PfsMR5), Pfs16 or Pfs25, were not altered and the Pfs230-minus gametes retained resistance to the alternative pathway of human complement. These results suggest that Pfs230 is the surface molecule on males that mediates RBC binding and plays an important role in oocyst development, a critical step in malaria transmission.

Targeted disruption of Plasmodium falciparum cysteine protease, falcipain 1, reduces oocyst production, not erythrocytic stage growth.

Cysteine proteases are currently targets for drug development in a number of parasitic diseases, including malaria. In Plasmodium falciparum, the parasite responsible for the most virulent form of human malaria, there are four members of the cathepsin L-like family of cysteine proteases. Three of these (falcipains 2A, 2B and 3) are thought to be primarily involved in haemoglobin digestion, whereas falcipain 1 has recently been linked to erythrocyte invasion. Neither their expression nor their role in P. falciparum gametocytogenesis, which is required for malaria transmission, has been evaluated. In this study, RNA transcripts for the falcipain family members were identified as the parasite developed through all five stages of gametocytogenesis. Falcipain 1 transcript was upregulated in gametocytes, while levels of falcipain 2A/2B decreased in late-stage gametocytes and gametes. To evaluate the function of falcipain 1, the gene was disrupted, and clones from independent transformations were isolated. The asexual growth of the falcipain 1 minus clones was not overtly affected, and they produced morphologically normal gametocytes and gametes. However, when falcipain 1 minus parasites were fed to a mosquito, oocyst production was reduced by 70-90%, suggesting an important role for falcipain 1 during parasite development in the mosquito midgut.

A glycosylphosphatidylinositol anchor signal sequence enhances the immunogenicity of a DNA vaccine encoding Plasmodium falciparum sexual-stage antigen, Pfs230.

Mammalian expression vectors encoding region C of malaria transmission-blocking vaccine candidate Pfs230 (aa 443-1132) with and without a 3' glycosylphosphatidylinositol (GPI) anchor signal sequence were tested for their immunogenicity in mice. The plasmid containing the GPI anchor signal sequence consistently induced higher titers of anti-Pfs230 antibodies using three delivery systems: intramuscular (i.m.), intradermal (i.d.), and gene gun (g.g.). In contrast, the isotype profile elicited varied depending on the delivery system and was not effected by the presence of the GPI anchor sequence. Both gene gun and intradermal administration induced primarily an IgG1 response, while intramuscular injection induced both IgG1 and IgG2a antibodies. Regardless of the mode of delivery, all the plasmids encoding Pfs230 region C primed for a mixed IgG1/IgG2a response to an intraperitoneal (i.p.) injection of E. coli-produced recombinant Pfs230 region C. None of these vaccination strategies were more effective than r230/MBP.C alone in generating malaria transmission-blocking immunity.