The Plasmodium falciparum, Nima-related kinase Pfnek-4: a marker for asexual parasites committed to sexual differentiation

<b>Background</b> Malaria parasites undergo, in the vertebrate host, a developmental switch from asexual replication to sexual differentiation leading to the formation of gametocytes, the only form able to survive in the mosquito vector. Regulation of the onset of the sexual phase remains largely unknown and represents an important gap in the understanding of the parasite's complex biology. <b>Methods:</b> The expression and function of the Nima-related kinase Pfnek-4 during the early sexual development of the human malaria parasite Plasmodium falciparum were investigated, using three types of transgenic Plasmodium falciparum 3D7 lines: (i) episomally expressing a Pfnek-4-GFP fusion protein under the control of its cognate pfnek-4 promoter; (ii) episomally expressing negative or positive selectable markers, yeast cytosine deaminase-uridyl phosphoribosyl transferase, or human dihydrofolate reductase, under the control of the pfnek-4 promoter; and (iii) lacking a functional pfnek-4 gene. Parasite transfectants were analysed by fluorescence microscopy and flow cytometry. In vitro growth rate and gametocyte formation were determined by Giemsa-stained blood smears. <b>Results:</b> The Pfnek-4-GFP protein was found to be expressed in stage II to V gametocytes and, unexpectedly, in a subset of asexual-stage parasites undergoing schizogony. Culture conditions stimulating gametocyte formation resulted in significant increase of this schizont subpopulation. Moreover, sorted asexual parasites expressing the Pfnek-4-GFP protein displayed elevated gametocyte formation when returned to in vitro culture in presence of fresh red blood cells, when compared to GFP- parasites from the same initial population. Negative selection of asexual parasites expressing pfnek-4 showed a marginal reduction in growth rate, whereas positive selection caused a marked reduction in parasitaemia, but was not sufficient to completely abolish proliferation. Pfnek-4- clones are not affected in their asexual growth and produced normal numbers of stage V gametocytes. <b>Conclusions:</b> The results indicate that Pfnek-4 is not strictly gametocyte-specific, and is expressed in a small subset of asexual parasites displaying high rate conversion to sexual development. Pfnek-4 is not required for erythrocytic schizogony and gametocytogenesis. This is the first study to report the use of a molecular marker for the sorting of sexually-committed schizont stage P. falciparum parasites, which opens the way to molecular characterization of this pre-differentiated subpopulation

Plasmodium falciparum infection induces dynamic changes in the erythrocyte phospho-proteome.

The phosphorylation status of red blood cell proteins is strongly altered during the infection by the malaria parasite Plasmodium falciparum. We identify the key phosphorylation events that occur in the erythrocyte membrane and cytoskeleton during infection, by a comparative analysis of global phospho-proteome screens between infected (obtained at schizont stage) and uninfected RBCs. The meta-analysis of reported mass spectrometry studies revealed a novel compendium of 495 phosphorylation sites in 182 human proteins with regulatory roles in red cell morphology and stability, with about 25% of these sites specific to infected cells. A phosphorylation motif analysis detected 7 unique motifs that were largely mapped to kinase consensus sequences of casein kinase II and of protein kinase A/protein kinase C. This analysis highlighted prominent roles for PKA/PKC involving 78 phosphorylation sites. We then compared the phosphorylation status of PKA (PKC) specific sites in adducin, dematin, Band 3 and GLUT-1 in uninfected RBC stimulated or not by cAMP to their phosphorylation status in iRBC. We showed cAMP-induced phosphorylation of adducin S59 by immunoblotting and we were able to demonstrate parasite-induced phosphorylation for adducin S726, Band 3 and GLUT-1, corroborating the protein phosphorylation status in our erythrocyte phosphorylation site compendium

Street-Based Female Adolescent Puerto Rican Sex Workers: Contextual Issues and Health Needs

Adolescent sex workers potentially have many health needs but are included rarely in health assessments; therefore, little is known about their needs or how to reach them. This study examines the motivations, social context, and health needs of street-based adolescent sex workers. The results show these youths are more likely to experience negative health outcomes, such as unintended pregnancies and sexually transmitted infections, when they are using drugs or are depressed. These findings suggest the need for comprehensive programming to include individual, interpersonal, and greater environmental interventions. Examples for reaching adolescent sex workers are discussed

Nanoimprinted superlattice metallic photonic crystal as ultraselective solar absorber

A two-dimensional superlattice metallic photonic crystal (PhC) and its fabrication by nanoimprint lithography on tantalum substrates are presented. The superior tailoring capacity of the superlattice PhC geometry is used to achieve spectrally selective solar absorption optimized for high-temperature and high-efficiency solar-energy-conversion applications. The scalable fabrication route by nanoimprint lithography allows for a high-throughput and high-resolution replication of this complex pattern over large areas. Despite the high fill factor, the pattern of polygonal cavities is accurately replicated into a resist that hardens under ultraviolet radiation over an area of 10  mm². In this way, cavities of 905 nm and 340 nm width are achieved with a period of 1 μm. After pattern transfer into tantalum via a deep reactive ion-etching process, the achieved cavities are 2.2 μm deep, separated by 85–95 nm wide ridges with vertical sidewalls. The room-temperature reflectance spectra of the fabricated samples show excellent agreement with simulated results, with a high spectral absorptance approaching blackbody absorption in the range from 300 to 1900 nm and a steep cutoff. The calculated solar absorptivity of this superlattice PhC is 96% and its thermal transfer efficiency is 82.8% at an operating temperature of 1500 K and an irradiance of 1000  kW/m².United States. Army Research Office (W911NF-13-D-0001)United States. Department of Energy (DE-SC0001299

A new tool for the chemical genetic investigation of the Plasmodium falciparum Pfnek-2 NIMA-related kinase

Background: Examining essential biochemical pathways in Plasmodium falciparum presents serious challenges, as standard molecular techniques such as siRNA cannot be employed in this organism, and generating gene knock-outs of essential proteins requires specialized conditional approaches. In the study of protein kinases, pharmacological inhibition presents a feasible alternative option. However, as in mammalian systems, inhibitors often lack the desired selectivity. Described here is a chemical genetic approach to selectively inhibit Pfnek-2 in P. falciparum, a member of the NIMA-related kinase family that is essential for completion of the sexual development of the parasite. Results: Introduction of a valine to cysteine mutation at position 24 in the glycine rich loop of Pfnek-2 does not affect kinase activity but confers sensitivity to the protein kinase inhibitor 4-(6-ethynyl-9H-purin-2-ylamino) benzene sulfonamide (NCL-00016066). Using a combination of in vitro kinase assays and mass spectrometry, (including phosphoproteomics) the study shows that this compound acts as an irreversible inhibitor to the mutant Pfnek2 likely through a covalent link with the introduced cysteine residue. In particular, this was shown by analysis of total protein mass using mass spectrometry which showed a shift in molecular weight of the mutant kinase in the presence of the inhibitor to be precisely equivalent to the molecular weight of NCL-00016066. A similar molecular weight shift was not observed in the wild type kinase. Importantly, this inhibitor has little activity towards the wild type Pfnek-2 and, therefore, has all the properties of an effective chemical genetic tool that could be employed to determine the cellular targets for Pfnek-2. Conclusions: Allelic replacement of wild-type Pfnek-2 with the mutated kinase will allow for targeted inhibition of Pfnek-2 with NCL-00016066 and hence pave the way for comparative studies aimed at understanding the biological role and transmission-blocking potential of Pfnek-2. © 2016 The Author(s)

High Temperature Electron Localization in dense He Gas

We report new accurate mesasurements of the mobility of excess electrons in high density Helium gas in extended ranges of temperature $[(26\leq T\leq 77) K ]$ and density $[ (0.05\leq N\leq 12.0) {atoms} \cdot {nm}^{-3}]$ to ascertain the effect of temperature on the formation and dynamics of localized electron states. The main result of the experiment is that the formation of localized states essentially depends on the relative balance of fluid dilation energy, repulsive electron-atom interaction energy, and thermal energy. As a consequence, the onset of localization depends on the medium disorder through gas temperature and density. It appears that the transition from delocalized to localized states shifts to larger densities as the temperature is increased. This behavior can be understood in terms of a simple model of electron self-trapping in a spherically symmetric square well.Comment: 23 pages, 13 figure

SAS6-like protein in Plasmodium indicates that conoid-associated apical complex proteins persist in invasive stages within the mosquito vector

The SAS6-like (SAS6L) protein, a truncated paralogue of the ubiquitous basal body/centriole protein SAS6, has been characterised recently as a flagellum protein in trypanosomatids, but associated with the conoid in apicomplexan Toxoplasma. The conoid has been suggested to derive from flagella parts, but is thought to have been lost from some apicomplexans including the malaria-causing genus Plasmodium. Presence of SAS6L in Plasmodium, therefore, suggested a possible role in flagella assembly in male gametes, the only flagellated stage. Here, we have studied the expression and role of SAS6L throughout the Plasmodium life cycle using the rodent malaria model P. berghei. Contrary to a hypothesised role in flagella, SAS6L was absent during gamete flagellum formation. Instead, SAS6L was restricted to the apical complex in ookinetes and sporozoites, the extracellular invasive stages that develop within the mosquito vector. In these stages SAS6L forms an apical ring, as we show is also the case in Toxoplasma tachyzoites. The SAS6L ring was not apparent in blood-stage invasive merozoites, indicating that the apical complex is differentiated between the different invasive forms. Overall this study indicates that a conoid-associated apical complex protein and ring structure is persistent in Plasmodium in a stage-specific manner

Addressing the high cervical cancer rates along the Texas-Mexico border through community outreach, patient navigation, and provider training/telementoring

Objective: Cervical cancer incidence and mortality rates are 68% and 57% higher, respectively, along the Texas-Mexico border compared with the rest of the United States. This is likely due to a combination of low health literacy, limited access to affordable screening, and a lack of trained personnel to perform colposcopy, loop electrosurgical excision procedures (LEEP), and appropriate management of women with pre-invasive disease. The objective of our study was to increase cervical cancer screening, diagnosis, and treatment rates in the Rio Grande Valley (RGV). Method: We initiated a comprehensive program at two health centers and one mobile clinic in the RGV consisting of (1) a public education program designed for community health workers to teach women about cervical cancer screening and HPV vaccination coupled with patient navigation to participating clinics; (2) colposcopy and LEEP training for physicians and advanced-practice providers through locally held hands-on courses and mentoring program; and (3) implementation of Project ECHO (Extension for Community Health Outcomes), a well-established telementoring program using video conferencing to connect academic specialists with community providers for case-based learning. We compared screening, diagnosis, and treatment rates pre- and post-program implementation. Results: From November 2014 to June 2018, local providers screened 19,028 women with Pap ± HPV testing (baseline 12,460, 53% increase); performed colposcopy on 2,644 women with abnormal screening results (baseline 945, 180% increase); and performed 483 LEEP procedures for treatment of cervical dysplasia (baseline 0). Ten women were diagnosed with invasive cancer and navigated to one of the participating gynecologic oncologists for treatment (baseline N/A). Five additional providers in the RGV completed the mentoring program to be certified to perform colposcopy (100% increase from baseline of 5) and two additional providers to perform LEEP (baseline 0). ECHO telementoring video conferences have been held every two weeks for a total 94 sessions (average of 22 participants/session) with 182 patient cases presented and discussed. Conclusion: Our comprehensive approach has led to an increase in the number of women undergoing cervical cancer screening and diagnosis/treatment of dysplasia. If sustained, we anticipate these efforts will decrease cervical cancer rates in the RGV. The program is currently being expanded to additional medically underserved regions of Texas

A small CD11b+ human B1 cell subpopulation stimulates T cells and is expanded in lupus

Human B1 cells can be divided, based on surface CD11b expression, into two transcriptionally and functionally distinct subsets, one of which is more abundant in lupus patients than healthy individuals

The association between family and community social capital and health risk behaviours in young people: an integrative review

Background: Health risk behaviours known to result in poorer outcomes in adulthood are generally established in late childhood and adolescence. These ‘risky’ behaviours include smoking, alcohol and illicit drug use and sexual risk taking. While the role of social capital in the establishment of health risk behaviours in young people has been explored, to date, no attempt has been made to consolidate the evidence in the form of a review. Thus, this integrative review was undertaken to identify and synthesise research findings on the role and impact of family and community social capital on health risk behaviours in young people and provide a consolidated evidence base to inform multi-sectorial policy and practice.<p></p> Methods: Key electronic databases were searched (i.e. ASSIA, CINAHL, Cochrane Database of Systematic Reviews, Cochrane Central Register of Controlled Trials, Database of Abstracts of Reviews of Effects, Embase, Medline, PsycINFO, Sociological Abstracts) for relevant studies and this was complemented by hand searching. Inclusion/exclusion criteria were applied and data was extracted from the included studies. Heterogeneity in study design and the outcomes assessed precluded meta-analysis/meta-synthesis; the results are therefore presented in narrative form.<p></p> Results: Thirty-four papers satisfied the review inclusion criteria; most were cross-sectional surveys. The majority of the studies were conducted in North America (n=25), with three being conducted in the UK. Sample sizes ranged from 61 to 98,340. The synthesised evidence demonstrates that social capital is an important construct for understanding the establishment of health risk behaviours in young people. The different elements of family and community social capital varied in terms of their saliency within each behavioural domain, with positive parent–child relations, parental monitoring, religiosity and school quality being particularly important in reducing risk.<p></p> Conclusions: This review is the first to systematically synthesise research findings about the association between social capital and health risk behaviours in young people. While providing evidence that may inform the development of interventions framed around social capital, the review also highlights key areas where further research is required to provide a fuller account of the nature and role of social capital in influencing the uptake of health risk behaviours.<p></p&gt