Naturally Occurring Triggers that Induce Apoptosis-Like Programmed Cell Death in Plasmodium berghei Ookinetes

Several protozoan parasites have been shown to undergo a form of programmed cell death that exhibits morphological features associated with metazoan apoptosis. These include the rodent malaria parasite, Plasmodium berghei. Malaria zygotes develop in the mosquito midgut lumen, forming motile ookinetes. Up to 50% of these exhibit phenotypic markers of apoptosis; as do those grown in culture. We hypothesised that naturally occurring signals induce many ookinetes to undergo apoptosis before midgut traversal. To determine whether nitric oxide and reactive oxygen species act as such triggers, ookinetes were cultured with donors of these molecules. Exposure to the nitric oxide donor SNP induced a significant increase in ookinetes with condensed nuclear chromatin, activated caspase-like molecules and translocation of phosphatidylserine that was dose and time related. Results from an assay that detects the potential-dependent accumulation of aggregates of JC-1 in mitochondria suggested that nitric oxide does not operate via loss of mitochondrial membrane potential. L-DOPA (reactive oxygen species donor) also caused apoptosis in a dose and time dependent manner. Removal of white blood cells significantly decreased ookinetes exhibiting a marker of apoptosis in vitro. Inhibition of the activity of nitric oxide synthase in the mosquito midgut epithelium using L-NAME significantly decreased the proportion of apoptotic ookinetes and increased the number of oocysts that developed. Introduction of a nitric oxide donor into the blood meal had no effect on mosquito longevity but did reduce prevalence and intensity of infection. Thus, nitric oxide and reactive oxygen species are triggers of apoptosis in Plasmodium ookinetes. They occur naturally in the mosquito midgut lumen, sourced from infected blood and mosquito tissue. Up regulation of mosquito nitric oxide synthase activity has potential as a transmission blocking strategy.     

Pathogenicity of Different Rabies Virus Variants Inversely Correlates with Apoptosis and Rabies Virus Glycoprotein Expression in Infected Primary Neuron Cultures

The mouse-adapted rabies virus strain CVS-24 has stable variants, CVS-B2c and CVS-N2c, which differ greatly in their pathogenicity for normal adult mice and in their ability to infect nonneuronal cells. The glycoprotein (G protein), which has previously been implicated in rabies virus pathogenicity, shows substantial structural differences between these variants. Although prior studies have identified antigenic site III of the G protein as the major pathogenicity determinant, CVS-B2c and CVS-N2c do not vary at this site. The possibility that pathogenicity is inversely related to G protein expression levels is suggested by the finding that CVS-B2c, the less pathogenic variant, expresses at least fourfold-higher levels of G protein than CVS-N2c in infected neurons. Although there is some difference between CVS-B2c- and CVS-N2c-infected neurons in G protein mRNA expression levels, the differential expression of G protein appears to be largely determined by posttranslational mechanisms that affect G protein stability. Pulse-chase experiments indicated that the G protein of CVS-B2c is degraded more slowly than that of CVS-N2c. The accumulation of G protein correlated with the induction of programmed cell death in CVS-B2c-infected neurons. The extent of apoptosis was considerably lower in CVS-N2c-infected neurons, where G protein expression was minimal. While nucleoprotein (N protein) expression levels were similar in neurons infected with either variant, the transport of N protein into neuronal processes was strongly inhibited in CVS-B2c-infected cells. Thus, downregulation of G protein expression in neuronal cells evidently contributes to rabies virus pathogenesis by preventing apoptosis and the apparently associated failure of the axonal transport of N protein.     

Microtubule nucleation at non-spindle pole body microtubule-organizing centers requires fission yeast centrosomin-related protein mod20p

BACKGROUND: Many types of differentiated eukaryotic cells display microtubule distributions consistent with nucleation from noncentrosomal intracellular microtubule organizing centers (MTOCs), although such structures remain poorly characterized. In fission yeast, two types of MTOCs exist in addition to the spindle pole body, the yeast centrosome equivalent. These are the equatorial MTOC, which nucleates microtubules from the cell division site at the end of mitosis, and interphase MTOCs, which nucleate microtubules from multiple sites near the cell nucleus during interphase. RESULTS: From an insertional mutagenesis screen we identified a novel gene, mod20+, which is required for microtubule nucleation from non-spindle pole body MTOCs in fission yeast. Mod20p is not required for intranuclear mitotic spindle assembly, although it is required for cytoplasmic astral microtubule growth during mitosis. Mod20p localizes to MTOCs throughout the cell cycle and is also dynamically distributed along microtubules themselves. We find that mod20p is required for the localization of components of the gamma-tubulin complex to non-spindle pole body MTOCs and physically interacts with the gamma-tubulin complex in vivo. Database searches reveal a family of eukaryotic proteins distantly related to mod20p; these are found in organisms ranging from fungi to mammals and include Drosophila centrosomin. CONCLUSIONS: Mod20p appears to act by recruiting components of the gamma-tubulin complex to non-spindle pole body MTOCs. The identification of mod20p-related proteins in higher eukaryotes suggests that this may represent a general mechanism for the organization of noncentrosomal MTOCs in eukaryotic cells.     

The Revised Classification of Eukaryotes

This revision of the classification of eukaryotes, which updates that of Adl et al. [J. Eukaryot. Microbiol. 52 (2005) 399], retains an emphasis on the protists and incorporates changes since 2005 that have resolved nodes and branches in phylogenetic trees. Whereas the previous revision was successful in re‐introducing name stability to the classification, this revision provides a classification for lineages that were then still unresolved. The supergroups have withstood phylogenetic hypothesis testing with some modifications, but despite some progress, problematic nodes at the base of the eukaryotic tree still remain to be statistically resolved. Looking forward, subsequent transformations to our understanding of the diversity of life will be from the discovery of novel lineages in previously under‐sampled areas and from environmental genomic information.     

Decline of FoxP3+ Regulatory CD4 T Cells in Peripheral Blood of Children Heavily Exposed to Malaria

FoxP3+ regulatory CD4 T cells (T_regs) help to maintain the delicate balance between pathogen-specific immunity and immune-mediated pathology. Prior studies suggest that T_regs are induced by P. falciparum both in vivo and in vitro; however, the factors influencing T_reg homeostasis during acute and chronic infections, and their role in malaria immunopathogenesis, remain unclear. We assessed the frequency and phenotype of T_regs in well-characterized cohorts of children residing in a region of high malaria endemicity in Uganda. We found that both the frequency and absolute numbers of FoxP3+ T_regs in peripheral blood declined markedly with increasing prior malaria incidence. Longitudinal measurements confirmed that this decline occurred only among highly malaria-exposed children. The decline of T_regs from peripheral blood was accompanied by reduced in vitro induction of T_regs by parasite antigen and decreased expression of TNFR2 on T_regs among children who had intense prior exposure to malaria. While T_reg frequencies were not associated with protection from malaria, there was a trend toward reduced risk of symptomatic malaria once infected with P. falciparum among children with lower T_reg frequencies. These data demonstrate that chronic malaria exposure results in altered T_reg homeostasis, which may impact the development of antimalarial immunity in naturally exposed populations.     

Genomewide Multipoint Linkage Analysis of Seven Extended Palauan Pedigrees with Schizophrenia, by a Markov-Chain Monte Carlo Method

Palauans are an isolated population in Micronesia with lifetime prevalence of schizophrenia (SCZD) of 2%, compared to the world rate of approximately 1%. The possible enrichment for SCZD genes, in conjunction with the potential for reduced etiological heterogeneity and the opportunity to ascertain statistically powerful extended pedigrees, makes Palauans a population of choice for the mapping of SCZD genes. We have used a Markov-chain Monte Carlo method to perform a genomewide multipoint analysis in seven extended pedigrees from Palau. Robust multipoint parametric and nonparametric linkage (NPL) analyses were performed under three nested diagnostic classifications-core, spectrum, and broad. We observed four regions of interest across the genome. Two of these regions-on chromosomes 2p13-14 (for which, under core diagnostic classification, NPL=6.5 and parametric LOD=4.8) and 13q12-22 (for which, under broad diagnostic classification, parametric LOD=3.6, and, under spectrum diagnostic classification, parametric LOD=3.5)-had evidence for linkage with genomewide significance, after correction for multiple testing; with the current pedigree resource and genotyping, these regions are estimated to be 4.3 cM and 19.75 cM in size, respectively. A third region, with intermediate evidence for linkage, was identified on chromosome 5q22-qter (for which, under broad diagnostic classification, parametric LOD=2.5). The fourth region of interest had only borderline suggestive evidence for linkage (on 3q24-28; for this region, under broad diagnostic classification, parametric LOD=2.0). All regions exhibited evidence for genetic heterogeneity. Our findings provide significant evidence for susceptibility loci on chromosomes 2p13-14 and 13q12-22 and support both a model of genetic heterogeneity and the utility of a broader set of diagnostic classifications in the population from Palau.     

Cadmium Accumulation and DNA Homology with Metal Resistance Genes in Sulfate-Reducing Bacteria

Cadmium resistance (0.1 to 1.0 mM) was studied in four pure and one mixed culture of sulfate-reducing bacteria (SRB). The growth of the bacteria was monitored with respect to carbon source (lactate) oxidation and sulfate reduction in the presence of various concentrations of cadmium chloride. Two strains Desulfovibrio desulfuricans DSM 1926 and Desulfococcus multivorans DSM 2059 showed the highest resistance to cadmium (0.5 mM). Transmission electron microscopy of the two strains showed intracellular and periplasmic accumulation of cadmium. Dot blot DNA hybridization using the probes for the smtAB, cadAC, and cadD genes indicated the presence of similar genetic determinants of heavy metal resistance in the SRB tested. DNA sequencing of the amplified DNA showed strong nucleotide homology in all the SRB strains with the known smtAB genes encoding synechococcal metallothioneins. Protein homology with the known heavy metal-translocating ATPases was also detected in the cloned amplified DNA of Desulfomicrobium norvegicum I1 and Desulfovibrio desulfuricans DSM 1926, suggesting the presence of multiple genetic mechanisms of metal resistance in the two strains.     

Whole-genome sequencing to detect mutations associated with resistance to insecticides and Bt proteins in Spodoptera frugiperda

The fall armyworm (FAW), Spodoptera frugiperda, is a major pest native to the Americas that has recently invaded the Old World. Point mutations in the target-site proteins acetylcholinesterase-1 (ace-1), voltage-gated sodium channel (VGSC) and ryanodine receptor (RyR) have been identified in S. frugiperda as major resistance mechanisms to organophosphate, pyrethroid and diamide insecticides respectively. Mutations in the adenosine triphosphate-binding cassette transporter C2 gene (ABCC2) have also been identified to confer resistance to Cry1F protein. In this study, we applied a whole-genome sequencing (WGS) approach to identify point mutations in the target-site genes in 150 FAW individuals collected from China, Malawi, Uganda and Brazil. This approach revealed three amino acid substitutions (A201S, G227A and F290V) of S. frugiperda ace-1, which are known to be associated with organophosphate resistance. The Brazilian population had all three ace-1 point mutations and the 227A allele (mean frequency = 0.54) was the most common. Populations from China, Malawi and Uganda harbored two of the three ace-1 point mutations (A201S and F290V) with the 290V allele (0.47–0.58) as the dominant allele. Point mutations in VGSC (T929I, L932F and L1014F) and RyR (I4790M and G4946E) were not detected in any of the 150 individuals. A novel 12-bp insertion mutation in exon 15 of the ABCC2 gene was identified in some of the Brazilian individuals but absent in the invasive populations. Our results not only demonstrate robustness of the WGS-based genomic approach for detection of resistance mutations, but also provide insights for improvement of resistance management tactics in S. frugiperda.     

Growing up aspen: ontogeny and trade-offs shape growth,defence and reproduction in a foundation species

Background and AimsIntraspecific variation in foundation species of forest ecosystems can shape community and ecosystem properties, particularly when that variation has a genetic basis. Traits mediating interactions with other species are predicted by simple allocation models to follow ontogenetic patterns that are rarely studied in trees. The aim of this research was to identify the roles of genotype, ontogeny and genotypic trade-offs shaping growth, defence and reproduction in aspen.MethodsWe established a common garden replicating >500 aspen genets in Wisconsin, USA. Trees were measured through the juvenile period into the onset of reproduction, for growth, defence chemistry (phenolic glycosides and condensed tannins), nitrogen, extrafloral nectaries, leaf morphology (specific leaf area), flower production and foliar herbivory and disease. We also assayed the TOZ19 sex marker and heterozygosity at ten microsatellite loci.Key ResultsWe found high levels of genotypic variation for all traits, and high heritabilities for both the traits and their ontogenetic trajectories. Ontogeny strongly shaped intraspecific variation, and trade-offs among growth, defence and reproduction supported some predictions while contradicting others. Both direct resistance (chemical defence) and indirect defence (extrafloral nectaries) declined during the juvenile stage, prior to the onset of reproduction. Reproduction was higher in trees that were larger, male and had higher individual heterozygosity. Growth was diminished by genotypic allocation to both direct and indirect defence as well as to reproduction, but we found no evidence of trade-offs between defence and reproduction.ConclusionsKey traits affecting the ecological communities of aspen have high levels of genotypic variation and heritability, strong patterns of ontogeny and clear trade-offs among growth, defence and reproduction. The architecture of aspen’s community genetics – its ontogeny, trade-offs and especially its great variability – is shaped by both its broad range and the diverse community of associates, and in turn further fosters that diversity.