Improved methods for magnetic purification of malaria parasites and haemozoin

<p>Abstract</p> <p>Background</p> <p>Malaria parasites generate free haem upon catabolism of host haemoglobin during their intraerythrocytic growth cycle. In order to minimize oxidative toxicity of the ferric iron, the free haem molecules are polymerized into the biomineral beta-haematin (commonly referred to as haemozoin). Haemozoin crystals are paramagnetic, and this property can be exploited for the purification of late stage parasites as they contain larger haemozoin crystals than early stage parasites and uninfected cells. Commercially available magnets that were originally developed for the purpose of antibody-mediated cell purification are widely used for this purpose. As these methods are not necessarily optimized for parasite purification, the relationship between magnetic field strength and the quantity and quality of yield during parasite purification was explored.</p> <p>Methods</p> <p>Inexpensive rare-earth neodymium magnets with commercially available disposable columns were employed to explore the relationship between magnetic field strength and recovery of free haemozoin and infected erythrocytes (iRBCs).</p> <p>Results</p> <p>Yields of free haemozoin increased nearly linearly with increasing magnetic field strength to the strongest fields tested (8,500 Gauss). Stronger magnetic fields also improved the recovery of iRBCs with no detrimental effects on parasite viability. An in-house constructed magnetic stand, built for $75 in materials, produced superior results when compared with much more expensive commercial products.</p> <p>Conclusions</p> <p>Existing protocols for the magnetic purification of free haemozoin and iRBCs result in sub-optimal yields. Inexpensive high-strength neodymium magnets offer a better option, resulting in higher yields with no detrimental effects on parasite viability.</p

Methods of Higher Alcohol Synthesis

Systems, catalysts, and methods are provided for transforming carbon based material into synthetic mixed alcohol fuel

Androgen receptor regulation by histone methyltransferase Suppressor of variegation 3-9 homolog 2 and Melanoma antigen-A11

Androgen receptor (AR) transcriptional activity depends on interactions between the AR NH2-terminal region and transcriptional coregulators. A yeast two-hybrid screen of a human testis library using predicted α-helical NH2-terminal fragment AR-(370–420) as bait identified suppressor of variegation 3–9 homolog 2 (SUV39H2) histone methyltransferase as an AR interacting protein. SUV39H2 interaction with AR and the AR coregulator, melanoma antigen-A11 (MAGE-A11), was verified in two-hybrid, in vitro glutathione S-transferase affinity matrix and coimmunoprecipitation assays. Fluorescent immunocytochemistry colocalized SUV39H2 and AR in the cytoplasm without androgen, in the nucleus with androgen, and with MAGE-A11 in the nucleus independent of androgen. Chromatin immunoprecipitation using antibodies raised against SUV39H2 demonstrated androgen-dependent recruitment of AR and SUV39H2 to the androgen-responsive upstream enhancer of the prostate-specific antigen gene. SUV39H2 functioned cooperatively with MAGE-A11 to increase androgen-dependent AR transcriptional activity. SUV39H2 histone methyltransferase is an AR coactivator that increases androgen-dependent transcriptional activity through interactions with AR and MAGE-A11

Travelers’ diarrhea and other gastrointestinal symptoms among Boston-area international travelers

INTRODUCTION: Travelers' diarrhea (TD) and non-TD gastrointestinal (GI) symptoms are common among international travelers. In a study of short-term travelers from Switzerland to developing countries, the most common symptom experienced was severe diarrhea (8.5%) followed by vomiting or abdominal cramps (4%).1 GI illnesses were the most frequently reported diagnoses (34%) among ill-returned travelers to GeoSentinel clinics.2 Of those returning to U.S. GeoSentinel clinics, acute diarrhea (30%) was the most common diagnosis.3 In one cohort of U.S. travelers, 46% reported diarrhea.4 GI illnesses can last from 2 days to weeks or longer,5 disrupting plans during travel or after returning home. Eighty percent of those who experienced diarrhea during travel treated themselves with medication and 6% sought medical care. METHODS: The Boston Area Travel Medicine Network (BATMN) is a research collaboration of travel clinics in the greater Boston area representing urban-, suburban-, academic-, and university-affiliated facilities. A convenience sample of travelers ≥ 18 years of age attending three BATMN clinics between 2009 and 2011 for pre-travel consultations completed pre-travel surveys, at least one survey weekly during travel, and a post-travel survey 2–4 weeks after return. Travelers were asked to complete a survey at the end of each week of their trip. Institutional review board approvals were obtained at all sites and the Centers for Disease Control and Prevention, and participants provided written informed consent. Information collected included demographic and trip characteristics, vaccines and medications recommended/prescribed before travel, medications taken during travel, dietary practices during travel (consumption of tap water, ice in drinks, unpasteurized dairy products, and salads), symptoms experienced, and impact of illness during and after travel. Vaccinations, prescriptions, and travel health advice given during the pre-travel consultation were recorded by a clinician, and the remainder of the surveys were completed by the traveler. Data were entered into a password-protected database (CS Pro, U.S. Census Bureau, Washington, DC). RESULTS: We enrolled 987 travelers; 628 (64%) completed all three parts (pre-, during, and post-travel) and were included in the study. Comparison of the 628 to the 359 who did not complete all three parts (noncompleters) revealed no differences, except that completion rates were higher for white travelers than all other racial/ethnic groups (P < 0.001) and for older travelers (median age 47 years versus 32 years in noncompleters, P < 0.001).11 Of those 628 travelers, 208 (33%) experienced TD, 45 (7%) experienced non-TD GI symptoms, 147 (23%) experienced non-GI symptoms, and 228 (36%) did not experience any symptoms during or after travel. Of the 208 with TD, 140 (67%) reported diarrhea as their only symptom, whereas 33 (16%) also experienced nausea/vomiting, 23 (11%) abdominal pain, and 27 (13%) fever (Table 1). Of the 45 who reported non-TD GI symptoms, 21 (47%) experienced nausea/vomiting, 19 (42%) experienced constipation, and 10 (22%) experienced abdominal pain during or after travel (Table 2). Almost all travelers (99%) received advice about food and water precautions and diarrhea management during pre-travel consultation

Field Results from Three Campaigns to Validate the Performance of the Miniaturized Laser Heterodyne Radiometer (MiniLHR) for Measuring Carbon Dioxide and Methane in the Atmospheric Column

In a collaboration between NASA GSFC and GWU, a low-cost, surface instrument is being developed that can continuously monitor key carbon cycle gases in the atmospheric column: carbon dioxide (CO2) and methane (CH4). The instrument is based on a miniaturized, laser heterodyne radiometer (LHR) using near infrared (NIR) telecom lasers. Despite relatively weak absorption line strengths in this spectral region, spectrallyresolved atmospheric column absorptions for these two molecules fall in the range of 60-80% and thus sensitive and precise measurements of column concentrations are possible. In the last year, the instrument was deployed for field measurements at Park Falls, Wisconsin; Castle Airport near Atwater, California; and at the NOAA Mauna Loa Observatory in Hawaii. For each subsequent campaign, improvement in the figures of merit for the instrument has been observed. In the latest work the absorbance noise is approaching 0.002 optical density (OD) noise on a 1.8 OD signal. An overview of the measurement campaigns and the data retrieval algorithm for the calculation of column concentrations will be presented. For light transmission through the atmosphere, it is necessary to account for variation of pressure, temperature, composition, and refractive index through the atmosphere that are all functions of latitude, longitude, time of day, altitude, etc. For temperature, pressure, and humidity profiles with altitude we use the Modern-Era Retrospective Analysis for Research and Applications (MERRA) data. Spectral simulation is accomplished by integrating short-path segments along the trajectory using the SpecSyn spectral simulation suite developed at GW. Column concentrations are extracted by minimizing residuals between observed and modeled spectrum using the Nelder-Mead simplex algorithm. We will also present an assessment of uncertainty in the reported concentrations from assumptions made in the meteorological data, LHR instrument and tracker noise, and radio frequency bandwidth and describe additional future goals in instrument development and deployment targe

Efficient measurement of opsonising antibodies to Plasmodium falciparum merozoites

Background: Antibodies targeting merozoites are important in protection from malaria. Therefore, merozoite surface proteins are attractive vaccine candidates. There is a need for robust functional assays to investigate mechanisms of acquired immunity and vaccine efficacy. To date, the study of merozoite phagocytosis has been confounded by the complexity and variability of in vitro assays. Methodology/Principal findings: We have developed a new flow cytometry-based merozoite phagocytosis assay. An optimized merozoite preparation technique produced high yields of merozoites separated from haemozoin. Phagocytosis by the undifferentiated THP-1 monocytic cell line was mediated only by Fc Receptors, and was therefore ideal for studying opsonising antibody responses. The assay showed robust phagocytosis with highly diluted immune sera and strong inter-assay correlation. The assay effectively measured differences in opsonisation-dependent phagocytosis among individuals. Conclusions/Significance: This highly reproducible assay has potential applications in assessing the role of opsonic phagocytosis in naturally acquired immunity and vaccine trials

Laser Sounder Approach for Global Measurement of Tropospheric CO2 Mixing Ratio from Space

We report progress in assessing the feasibility of a new satellite-based laser-sounding instrument to measure CO2 and other trace gas abundances in the lower troposphere from space

Structural features discriminate androgen receptor N/C terminal and coactivator interactions

Human androgen receptor (AR) transcriptional activity involves interdomain and coactivator interactions with the agonist-bound AR ligand binding domain (LBD). Structural determinants of the AR NH2- and carboxyl-terminal interaction between the AR NH2-terminal FXXLF motif and activation function 2 (AF2) in the LBD were shown previously by crystallography. In this report, we provide evidence for a region in AR LBD helix 12 outside the AF2 binding cleft that facilitates interactions with the FXXLF and LXXLL motifs. Mutagenesis of glutamine 902 to alanine in AR LBD helix 12 (Q902A) disrupted AR FXXLF motif binding to AF2, but enhanced coactivator LXXLL motif binding. Functional compensation for defective FXXLF motif binding by AR-Q902A was suggested by the slower dissociation rate of bound androgen. Functional importance of glutamine 902 was indicated by the charged residue germline mutation Q902R that caused partial androgen insensitivity, and a similar somatic mutation Q902K reported in prostate cancer, both of which increased the androgen dissociation rate and decreased AR transcriptional activity. High affinity equilibrium androgen binding was retained by alanine substitution mutations at Tyr-739 in AR LBD helix 5 or Lys-905 in helix 12 structurally adjacent to AF2, whereas transcriptional activity decreased and the androgen dissociation increased. Deleterious effects of these loss of function mutations were rescued by the helix stabilizing AR prostate cancer somatic mutation H874Y. Sequence NH2-terminal to the AR FXXLF motif contributed to the AR NH2- and carboxyl-terminal interaction based on greater AR-2-30 FXXLF motif peptide binding to the agonist-bound AR LBD than a shorter AR-20-30 FXXLF motif peptide. We conclude that helix 12 residues outside the AF2 binding cleft modulate AR transcriptional activity by providing flexibility to accommodate FXXLF or LXXLL motif binding

Multiple-University Extension Program Addresses Postdisaster Oil Spill Needs Through Private Funding Partnership

In response to the Deepwater Horizon Oil Spill, the Gulf of Mexico Research Initiative (GoMRI) was formed to answer oil spill–related scientific questions. However, peer-reviewed scientific discoveries were not reaching people whose livelihoods depended on a healthy Gulf of Mexico. GoMRI and the four Gulf of Mexico Sea Grant programs partnered to develop a regional Extension program with a team of multidisciplinary specialists and a regional manager embedded within the Sea Grant programs. The team answered oil spill science questions from target audiences. The program leaders also identified the value of adding a regional Extension communicator to enhance their Extension products

Laser Sounder for Global Measurement of CO2 Concentrations in the Troposphere from Space

We report progress in assessing the feasibility of a new satellite-based laser-sounding instrument to measure CO2 concentrations in the lower troposphere from space