Promiscuous Expression of alpha-Tubulin II in Maturing Male and Female Plasmodium falciparum Gametocytes

Background: Antimalarial interventions designed to impact on the transmissible sexual stages of Plasmodium falciparum are evaluated by measurement of peripheral gametocyte carriage in vivo and infectivity to mosquitoes. Drug or vaccine-elicited effects may differentially affect the relative abundance of mature male and female sexual forms, and this can be measured by estimation of sex ratios before and after intervention in vivo and in vitro. Measuring the impact of anti-gametocyte drugs on sexual commitment of immature gametocyte stages in vitro is not currently possible as male and female parasites cannot be distinguished by morphology alone prior to stage IV.Methodology/Principal Findings: We have modified an existing immunofluorescence-based approach for distinguishing male and female gametocytes during development in vitro, by using highly synchronised magnetically-enriched gametocyte preparations at different stages of maturity. Antibodies recognising alpha-tubulin II ( males) and Pfg377 ( females) were used to attempt to discriminate the sexes. Transcription of these two proteins was not coordinated during in vitro development, with pfg377 transcripts accumulating only late in development, immediately prior to immunofluorescent signals from the PfG377 protein appearing in stage IV gametocytes. Contrary to previous descriptions of this protein as male-specific in P. falciparum, alpha-tubulin II recognised both male and female gametocytes at stages I to IV, but evidence of differential expression levels of this protein in late stage male and female gametocytes was found. Using antibodies recognising PfG377 as the primary marker and alpha-tubulin II as a secondary marker, robust estimates of sex ratio in in vitro cultures were obtained for gametocytes at stage IV or later, and validated by light microscopic counts. However, sex ratio estimation was not possible for early stage gametocytes due to the promiscuity of alpha-tubulin II protein expression, and the relatively late accumulation of PfG377 during the development process.Conclusions/Significance: This approach is a feasible method for the evaluation of drug impacts on late-stage gametocyte sex ratio in in vitro studies. Additional sex-specific antigens need to be evaluated for sex ratio estimation in early stage gametocyte preparations

Antibody responses to surface antigens of Plasmodium falciparum gametocyte-infected erythrocytes and their relation to gametocytaemia.

: An essential element for continuing transmission of Plasmodium falciparum is the availability of mature gametocytes in human peripheral circulation for uptake by mosquitoes. Natural immune responses to circulating gametocytes may play a role in reducing transmission from humans to mosquitoes. Here, antibody recognition of the surface of mature intra-erythrocytic gametocytes produced either by a laboratory-adapted parasite, 3D7, or by a recent clinical isolate of Kenyan origin (HL1204), was evaluated longitudinally in a cohort of Ghanaian school children by flow cytometry. This showed that a proportion of children exhibited antibody responses that recognized gametocyte surface antigens on one or both parasite lines. A subset of the children maintained detectable anti-gametocyte surface antigen (GSA) antibody levels during the 5 week study period. There was indicative evidence that children with anti-GSA antibodies present at enrolment were less likely to have patent gametocytaemia at subsequent visits (odds ratio = 0·29, 95% CI 0·06-1·05; P = 0·034). Our data support the existence of antigens on the surface of gametocyte-infected erythrocytes, but further studies are needed to confirm whether antibodies against them reduce gametocyte carriage. The identification of GSA would allow their evaluation as potential anti-gametocyte vaccine candidates and/or biomarkers for gametocyte carriage.<br/

Truncated Schwinger-Dyson Equations and Gauge Covariance in QED3

We study the Landau-Khalatnikov-Fradkin transformations (LKFT) in momentum space for the dynamically generated mass function in QED3. Starting from the Landau gauge results in the rainbow approximation, we construct solutions in other covariant gauges. We confirm that the chiral condensate is gauge invariant as the structure of the LKFT predicts. We also check that the gauge dependence of the constituent fermion mass is considerably reduced as compared to the one obtained directly by solving SDE.Comment: 17 pages, 11 figures. v3. Improved and Expanded. To appear in Few Body System

Comparative population structure of <i>Plasmodium malariae</i> and <i>Plasmodium falciparum</i> under different transmission settings in Malawi

&lt;b&gt;Background:&lt;/b&gt; Described here is the first population genetic study of Plasmodium malariae, the causative agent of quartan malaria. Although not as deadly as Plasmodium falciparum, P. malariae is more common than previously thought, and is frequently in sympatry and co-infection with P. falciparum, making its study increasingly important. This study compares the population parameters of the two species in two districts of Malawi with different malaria transmission patterns - one seasonal, one perennial - to explore the effects of transmission on population structures. &lt;BR/&gt; &lt;b&gt;Methods:&lt;/b&gt; Six species-specific microsatellite markers were used to analyse 257 P. malariae samples and 257 P. falciparum samples matched for age, gender and village of residence. Allele sizes were scored to within 2 bp for each locus and haplotypes were constructed from dominant alleles in multiple infections. Analysis of multiplicity of infection (MOI), population differentiation, clustering of haplotypes and linkage disequilibrium was performed for both species. Regression analyses were used to determine association of MOI measurements with clinical malaria parameters. &lt;BR/&gt; &lt;b&gt;Results:&lt;/b&gt; Multiple-genotype infections within each species were common in both districts, accounting for 86.0% of P. falciparum and 73.2% of P. malariae infections and did not differ significantly with transmission setting. Mean MOI of P. falciparum was increased under perennial transmission compared with seasonal (3.14 vs 2.59, p = 0.008) and was greater in children compared with adults. In contrast, P. malariae mean MOI was similar between transmission settings (2.12 vs 2.11) and there was no difference between children and adults. Population differentiation showed no significant differences between villages or districts for either species. There was no evidence of geographical clustering of haplotypes. Linkage disequilibrium amongst loci was found only for P. falciparum samples from the seasonal transmission setting. &lt;BR/&gt; &lt;b&gt;Conclusions:&lt;/b&gt; The extent of similarity between P. falciparum and P. malariae population structure described by the high level of multiple infection, the lack of significant population differentiation or haplotype clustering and lack of linkage disequilibrium is surprising given the differences in the biological features of these species that suggest a reduced potential for out-crossing and transmission in P. malariae. The absence of a rise in P. malariae MOI with increased transmission or a reduction in MOI with age could be explained by differences in the duration of infection or degree of immunity compared to P. falciparum

Plasmodium knowlesi Genome Sequences from Clinical Isolates Reveal Extensive Genomic Dimorphism.

Plasmodium knowlesi is a newly described zoonosis that causes malaria in the human population that can be severe and fatal. The study of P. knowlesi parasites from human clinical isolates is relatively new and, in order to obtain maximum information from patient sample collections, we explored the possibility of generating P. knowlesi genome sequences from archived clinical isolates. Our patient sample collection consisted of frozen whole blood samples that contained excessive human DNA contamination and, in that form, were not suitable for parasite genome sequencing. We developed a method to reduce the amount of human DNA in the thawed blood samples in preparation for high throughput parasite genome sequencing using Illumina HiSeq and MiSeq sequencing platforms. Seven of fifteen samples processed had sufficiently pure P. knowlesi DNA for whole genome sequencing. The reads were mapped to the P. knowlesi H strain reference genome and an average mapping of 90% was obtained. Genes with low coverage were removed leaving 4623 genes for subsequent analyses. Previously we identified a DNA sequence dimorphism on a small fragment of the P. knowlesi normocyte binding protein xa gene on chromosome 14. We used the genome data to assemble full-length Pknbpxa sequences and discovered that the dimorphism extended along the gene. An in-house algorithm was developed to detect SNP sites co-associating with the dimorphism. More than half of the P. knowlesi genome was dimorphic, involving genes on all chromosomes and suggesting that two distinct types of P. knowlesi infect the human population in Sarawak, Malaysian Borneo. We use P. knowlesi clinical samples to demonstrate that Plasmodium DNA from archived patient samples can produce high quality genome data. We show that analyses, of even small numbers of difficult clinical malaria isolates, can generate comprehensive genomic information that will improve our understanding of malaria parasite diversity and pathobiology

A four-helix bundle stores copper for methane oxidation

Methane-oxidising bacteria (methanotrophs) require large quantities of copper for the membrane-bound (particulate) methane monooxygenase (pMMO). Certain methanotrophs are also able to switch to using the iron-containing soluble MMO (sMMO) to catalyse methane oxidation, with this switchover regulated by copper. MMOs are Nature’s primary biological mechanism for suppressing atmospheric levels of methane, a potent greenhouse gas. Furthermore, methanotrophs and MMOs have enormous potential in bioremediation and for biotransformations producing bulk and fine chemicals, and in bioenergy, particularly considering increased methane availability from renewable sources and hydraulic fracturing of shale rock. We have discovered and characterised a novel copper storage protein (Csp1) from the methanotroph Methylosinus trichosporium OB3b that is exported from the cytosol, and stores copper for pMMO. Csp1 is a tetramer of 4-helix bundles with each monomer binding up to 13 Cu(I) ions in a previously unseen manner via mainly Cys residues that point into the core of the bundle. Csp1 is the first example of a protein that stores a metal within an established protein-folding motif. This work provides a detailed insight into how methanotrophs accumulate copper for the oxidation of methane. Understanding this process is essential if the wide-ranging biotechnological applications of methanotrophs are to be realised. Cytosolic homologues of Csp1 are present in diverse bacteria thus challenging the dogma that such organisms do not use copper in this location

Analysis of the conversion of delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-alpha-aminobutyrate by active-site mutants of Aspergillus nidulans isopenicillin N synthase.

BACKGROUND: Penicillins and cephalosporins constitute a major class of clinically useful antibiotics. A key step in their biosynthesis involves the oxidative cyclisation of delta-(Lalpha-aminoadipoyl)-L-cysteinyl-D-valine to isopenicillin N by isopenicillin N synthase (IPNS). This chemically remarkable transformation has been extensively studied using substrate analogues. The conversion of an analogue in which the valine is replaced by alpha-aminobutyrate results in three products, two epimeric penams and a cepham. The ratio of these products in reactions catalysed by four different IPNS isozymes has been used previously to probe the thermicity of the chemical mechanism. But how IPNS restricts the products from the natural substrate to a single penam (isopenicillin N) has remained unknown. RESULTS: A key active-site residue, Leu223, identified according to a model of enzyme-substrate binding, has been altered to sterically less demanding residues. As the steric constraints on the upper part of the active site are reduced, the ratio of the beta-methyl penam to the cepham increases when the alpha-aminobutyrate-containing substrate analogue is used. These results suggest a mechanism for processing of the natural substrate in which IPNS uses steric control to restrict the conformational freedom of an intermediate such that the only product is the penam. CONCLUSIONS: Using steric pressure to control conformation, and hence to disfavour reactions leading to alternate products, is probably the result of evolutionary selection for a biologically active product at the expense of biologically inactive byproducts. It is likely that this sort of enzymatic catalysis is used in situations where substrate conversion is highly exothermic and a variety of products are possible

On misunderstanding Heraclitus: The justice of organisation structure

Writers on organisational change often refer to the cosmology of Heraclitus in their work. Some use these references to support arguments for the constancy and universality of organisational change and the consignment to history of organisational continuity and stability. These writers misunderstand the scope of what Heraclitus said. Other writers focus exclusively on the idea that originated with Heraclitus that the universe is composed of processes and not of things. This idea, which has been particularly associated with Heraclitus’s thought from the time of Plato, does indeed provide a rich source of insights into organisational analysis, not least the current trends towards giving proper attention to processual studies of organisational change. Yet there is some uncertainty as to whether Heraclitus actually said that the universe was composed exclusively of processes rather than things, and even if that was what he thought, he intended his ideas on flux to be understood not in isolation but in the context of other aspects of his cosmology. Writers on organisational change seldom make reference to this wider context. Heraclitus was a rational but also a religious thinker. A central element in his thought was the notion of divine Justice, which to a Greek of his era meant the order of the universe. Remote as his Olympian theology may seem today, it sets a crucial and entirely rational context for understanding his ideas about flux. It means that ideas about continuity and stability were quite as important in Heraclitus’s cosmology as his more commonly quoted ideas about change. This paper sets out an overview of Heraclitus’s philosophy, insofar as it appears to have potential relevance to organisational analysis, and discusses how far it supports or contradicts the ideas that organisational change scholars have drawn from it

Developing cardiac and skeletal muscle share fast-skeletal myosin heavy chain and cardiac troponin-I expression

Skeletal muscle derived stem cells (MDSCs) transplanted into injured myocardium can differentiate into fast skeletal muscle specific myosin heavy chain (sk-fMHC) and cardiac specific troponin-I (cTn-I) positive cells sustaining recipient myocardial function. We have recently found that MDSCs differentiate into a cardiomyocyte phenotype within a three-dimensional gel bioreactor. It is generally accepted that terminally differentiated myocardium or skeletal muscle only express cTn-I or sk-fMHC, respectively. Studies have shown the presence of non-cardiac muscle proteins in the developing myocardium or cardiac proteins in pathological skeletal muscle. In the current study, we tested the hypothesis that normal developing myocardium and skeletal muscle transiently share both sk-fMHC and cTn-I proteins. Immunohistochemistry, western blot, and RT-PCR analyses were carried out in embryonic day 13 (ED13) and 20 (ED20), neonatal day 0 (ND0) and 4 (ND4), postnatal day 10 (PND10), and 8 week-old adult female Lewis rat ventricular myocardium and gastrocnemius muscle. Confocal laser microscopy revealed that sk-fMHC was expressed as a typical striated muscle pattern within ED13 ventricular myocardium, and the striated sk-fMHC expression was lost by ND4 and became negative in adult myocardium. cTn-I was not expressed as a typical striated muscle pattern throughout the myocardium until PND10. Western blot and RT-PCR analyses revealed that gene and protein expression patterns of cardiac and skeletal muscle transcription factors and sk-fMHC within ventricular myocardium and skeletal muscle were similar at ED20, and the expression patterns became cardiac or skeletal muscle specific during postnatal development. These findings provide new insight into cardiac muscle development and highlight previously unknown common developmental features of cardiac and skeletal muscle. © 2012 Clause et al