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/

Drug-resistant genotypes and multi-clonality in Plasmodium falciparum analysed by direct genome sequencing from peripheral blood of malaria patients.

Naturally acquired blood-stage infections of the malaria parasite Plasmodium falciparum typically harbour multiple haploid clones. The apparent number of clones observed in any single infection depends on the diversity of the polymorphic markers used for the analysis, and the relative abundance of rare clones, which frequently fail to be detected among PCR products derived from numerically dominant clones. However, minority clones are of clinical interest as they may harbour genes conferring drug resistance, leading to enhanced survival after treatment and the possibility of subsequent therapeutic failure. We deployed new generation sequencing to derive genome data for five non-propagated parasite isolates taken directly from 4 different patients treated for clinical malaria in a UK hospital. Analysis of depth of coverage and length of sequence intervals between paired reads identified both previously described and novel gene deletions and amplifications. Full-length sequence data was extracted for 6 loci considered to be under selection by antimalarial drugs, and both known and previously unknown amino acid substitutions were identified. Full mitochondrial genomes were extracted from the sequencing data for each isolate, and these are compared against a panel of polymorphic sites derived from published or unpublished but publicly available data. Finally, genome-wide analysis of clone multiplicity was performed, and the number of infecting parasite clones estimated for each isolate. Each patient harboured at least 3 clones of P. falciparum by this analysis, consistent with results obtained with conventional PCR analysis of polymorphic merozoite antigen loci. We conclude that genome sequencing of peripheral blood P. falciparum taken directly from malaria patients provides high quality data useful for drug resistance studies, genomic structural analyses and population genetics, and also robustly represents clonal multiplicity

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

Evaluation of growth methods for the heteroepitaxy of non-polar (1120) GAN on sapphire by MOVPE

Non-polar a-plane gallium nitride (GaN) lms have been grown on r-plane (1102) sapphire by metal organic vapour phase epitaxy (MOVPE). A total of ve in-situ defect reduction techniques for a-plane GaN are compared, including two variants with a low temperature GaN nucleation layer (LTNL) and three variants without LTNL, in which the high- temperature growth of GaN is performed directly on the sapphire using various crystallite sizes. The material quality is investigated by photoluminescence (PL), x-ray di raction, cathodoluminescence, atomic force and optical microscopy. It is found that all layers are anisotropically strained with threading dislocation densities over 109 cm2. The PL spectrum is typically dominated by emission from basal plane stacking faults. Overall, growth techniques without LTNL do not yield any particular improvement and even result in the creation of new defects, ie. inversion domains, which are seldom observed if a low temperature GaN nucleation layer is used. The best growth method uses a LTNL combined with a single silicon nitride interlayer.This work is supported by the Engineering and Physical Sciences Research Council (United Kingdom) under EP/J003603/1 and EP/H0495331. The European Research Council has also provided nancial support under the European Community's Seventh Framework Programme (FP7/2007-2013) / ERC grant agreement no 279361 (MACONS).This is the final published version, also available from Elsevier at: http://dx.doi.org/10.1016/j.jcrysgro.2014.09.00

Theoretical and experimental analysis of the photoluminescence and photoluminescence excitation spectroscopy spectra of m\textit{m}-plane InGaN/GaN quantum wells

We present a combined theoretical and experimental analysis of the optical properties of $\textit{m}$-plane InGaN/GaN quantum wells. The sample was studied by photoluminescence and photoluminescence excitation spectroscopy at low temperature. The spectra show a large Stokes shift between the lowest exciton peak in the excitation spectra and the peak of the photoluminescence spectrum. This behavior is indicative of strong carrier localization effects. These experimental results are complemented by tight-binding calculations, accounting for random alloy fluctuations and Coulomb effects. The theoretical data explain the main features of the experimental spectra. Moreover, by comparison with calculations based on a virtual crystal approximation, the importance of carrier localization effects due to random alloy fluctuations is explicitly shown.This work was supported by Science Foundation Ireland (Project No. 13/SIRG/2210) and the United Kingdom Engineering and Physical Sciences Research Council (Grant Agreement Nos. EP\J001627\1 and EP\J003603\1). S.S. acknowledges computing resources provided by the SFI/HEA Irish Centre for High-End Computing. R.A.O. and F.T. acknowledge the support of the European Research Council under the European Community's 7th Framework Programme (FP7/2007–2013)/ERC Grant Agreement No. 279361 (MACONS)

Increased sensitivity for detecting malaria parasites in human umbilical cord blood using scaled-up DNA preparation

BACKGROUND: All mothers donating umbilical cord blood units to the NHS cord blood bank undergo an assessment for the likelihood of prior exposure to malaria infection. Those deemed at risk due to a history of travel to, or residence in, malaria endemic regions are screened serologically to detect anti-malaria antibodies. A positive result excludes the use of the cord blood for transplant therapy unless a risk assessment can ensure that malaria transmission is extremely unlikely. This paper details the screening of cord blood units from malaria serology positive mothers to detect malaria parasite DNA using a highly sensitive nested PCR. METHODS: Uninfected blood from a healthy volunteer was spiked with known quantities of malaria parasites and 5 millilitre and 200 microlitre aliquots were subjected to DNA extraction using QIAamp DNA maxi and DNA mini kits respectively. Nested PCR, to detect malarial SSU rRNA sequences, was performed on the purified DNA samples to determine the limit of detection for this assay with both extraction methodologies. Following assay validation, 54 cord blood units donated by mothers who were positive for anti-malaria antibodies were screened by this approach. RESULTS: When DNA was purified from 5 millilitres of blood it was possible to routinely detect as few as 50 malaria parasites per millilitre using nested PCR. This equates to a significant increase in the sensitivity of the current gold standard nucleic acid amplification technique used to detect malaria parasites (routinely performed from > 200 microlitre volumes of blood). None of the 54 donated cord blood units from serology positive mothers tested positive for malaria parasites using this scaled up DNA preparation method. CONCLUSION: Serological testing for malaria parasites may be overly conservative, leading to unnecessary rejection of cord blood donations that lack malaria parasites and which are, therefore, safe for use in stem cell therapy

Identifying the science and technology dimensions of emerging public policy issues through horizon scanning

Public policy requires public support, which in turn implies a need to enable the public not just to understand policy but also to be engaged in its development. Where complex science and technology issues are involved in policy making, this takes time, so it is important to identify emerging issues of this type and prepare engagement plans. In our horizon scanning exercise, we used a modified Delphi technique [1]. A wide group of people with interests in the science and policy interface (drawn from policy makers, policy adviser, practitioners, the private sector and academics) elicited a long list of emergent policy issues in which science and technology would feature strongly and which would also necessitate public engagement as policies are developed. This was then refined to a short list of top priorities for policy makers. Thirty issues were identified within broad areas of business and technology; energy and environment; government, politics and education; health, healthcare, population and aging; information, communication, infrastructure and transport; and public safety and national security.Public policy requires public support, which in turn implies a need to enable the public not just to understand policy but also to be engaged in its development. Where complex science and technology issues are involved in policy making, this takes time, so it is important to identify emerging issues of this type and prepare engagement plans. In our horizon scanning exercise, we used a modified Delphi technique [1]. A wide group of people with interests in the science and policy interface (drawn from policy makers, policy adviser, practitioners, the private sector and academics) elicited a long list of emergent policy issues in which science and technology would feature strongly and which would also necessitate public engagement as policies are developed. This was then refined to a short list of top priorities for policy makers. Thirty issues were identified within broad areas of business and technology; energy and environment; government, politics and education; health, healthcare, population and aging; information, communication, infrastructure and transport; and public safety and national security

Reactive oxygen species initiate luminal but not basal cell death in cultured human mammary alveolar structures: a potential regulator of involution

Post-lactational involution of the mammary gland is initiated within days of weaning. Clearing of cells occurs by apoptosis of the milk-secreting luminal cells in the alveoli and through stromal tissue remodeling to return the gland almost completely to its pre-pregnant state. The pathways that specifically target involution of the luminal cells in the alveoli but not the basal and ductal cells are poorly understood. In this study we show in cultured human mammary alveolar structures that the involution process is initiated by fresh media withdrawal, and is characterized by cellular oxidative stress, expression of activated macrophage marker CD68 and finally complete clearing of the luminal but not basal epithelial layer. This process can be simulated by ectopic addition of reactive oxygen species (ROS) in cultures without media withdrawal. Cells isolated from post-involution alveoli were enriched for the CD49f+ mammary stem cell (MaSC) phenotype and were able to reproduce a complete alveolar structure in subcultures without any significant loss in viability. We propose that the ROS produced by accumulated milk breakdown post-weaning may be the mechanism underlying the selective involution of secretory alveolar luminal cells, and that our culture model represents an useful means to investigate this and other mechanisms further