Insights Gained from P. falciparum

In vitro cultivation of Plasmodium falciparum, the agent of severe human malaria, has enabled advances in basic research and accelerated the development of new therapies. Since the introduction of in vitro parasite culture nearly 40 years ago, most workers have used a medium consisting of RPMI 1640 medium supplemented with lipids and hypoxanthine. While these standardized conditions yield robust parasite growth and facilitate comparison of results from different studies, they may also lead to implicit assumptions that limit future advances. Here, I review recent studies that used modified culture conditions to challenge these assumptions and explore parasite physiology. The findings are relevant to understanding in vivo parasite phenotypes and the prioritization of antimalarial targets

Viscoelasticity as a Biomarker for High-Throughput Flow Cytometry

AbstractThe mechanical properties of living cells are a label-free biophysical marker of cell viability and health; however, their use has been greatly limited by low measurement throughput. Although examining individual cells at high rates is now commonplace with fluorescence activated cell sorters, development of comparable techniques that nondestructively probe cell mechanics remains challenging. A fundamental hurdle is the signal response time. Where light scattering and fluorescence signatures are virtually instantaneous, the cell stress relaxation, typically occurring on the order of seconds, limits the potential speed of elastic property measurement. To overcome this intrinsic barrier to rapid analysis, we show here that cell viscoelastic properties measured at frequencies far higher than those associated with cell relaxation can be used as a means of identifying significant differences in cell phenotype. In these studies, we explore changes in erythrocyte mechanical properties caused by infection with Plasmodium falciparum and find that the elastic response alone fails to detect malaria at high frequencies. At timescales associated with rapid assays, however, we observe that the inelastic response shows significant changes and can be used as a reliable indicator of infection, establishing the dynamic viscoelasticity as a basis for nondestructive mechanical analogs of current high-throughput cell classification methods

A high-throughput method to detect Plasmodium falciparum clones in limiting dilution microplates

<p>Abstract</p> <p>Background</p> <p>Molecular and cellular studies of <it>Plasmodium falciparum </it>require cloning of parasites by limiting dilution cultivation, typically performed in microplates. The parasite's slow replication rate combined with laborious methods for identification of positive wells has limited these studies. A new high-throughput method for detecting growth without compromising parasite viability is reported.</p> <p>Methods</p> <p><it>In vitro </it>parasite cultivation is associated with extracellular acidification. A survey of fluorescent pH indicators identified 5-(and-6)-carboxy SNARF-1 as a membrane-impermeant dye with a suitable <it>pK_a</it>value. Conditions for facile detection of viable parasites in 96-well microplates were optimized and used for limiting dilution cloning of genetic cross progeny and transfected parasites.</p> <p>Results</p> <p>5-(and-6)-carboxy SNARF-1 is a two-emission wavelength dye that accurately reported extracellular pH in parasite cultures. It readily detected parasite growth in microplate wells and yielded results comparable to labour-intensive examination of Giemsa-stained smears. The dye is non-toxic, allowing parasite detection without transfer of culture material to additional plates for separate assays. This dye was used with high-throughput limiting dilution culture to generate additional progeny clones from the HB3 × Dd2 genetic cross.</p> <p>Conclusions</p> <p>This fluorescence-based assay represents a low-cost, efficient method for detection of viable parasites in microplate wells; it can be easily expanded by automation.</p

Malaria Parasite clag3 Genes Determine Channel-Mediated Nutrient Uptake by Infected Red Blood Cells

SummaryDevelopment of malaria parasites within vertebrate erythrocytes requires nutrient uptake at the host cell membrane. The plasmodial surface anion channel (PSAC) mediates this transport and is an antimalarial target, but its molecular basis is unknown. We report a parasite gene family responsible for PSAC activity. We used high-throughput screening for nutrient uptake inhibitors to identify a compound highly specific for channels from the Dd2 line of the human pathogen P. falciparum. Inheritance of this compound's affinity in a Dd2 × HB3 genetic cross maps to a single parasite locus on chromosome 3. DNA transfection and in vitro selections indicate that PSAC-inhibitor interactions are encoded by two clag3 genes previously assumed to function in cytoadherence. These genes are conserved in plasmodia, exhibit expression switching, and encode an integral protein on the host membrane, as predicted by functional studies. This protein increases host cell permeability to diverse solutes.PaperFlic

In vitro effect of seed bio-priming techniques on seed germination and seedling vigour of few vegetable crops

Bio-priming can also be viewed as a new technique of seed treatment using biological agents to stimulate germination of seed and growth of the plant and further protecting the seed from soil-and seed-borne pathogens. The present investigation was carried out in vitro conditions on seed germination and seedling vigour of few vegetable crops viz.  tomato (Solanum lycopersicum L.), brinjal (Solanum melongena L.), onion (Allium cepa L.) and chilli (Capsicum annuum L.), during 2015 and 2016. The treatments comprised viz. T1: Non primed seeds (Control), T2: Seed treatment with Carbendazim 2.5g/kg seed, T3: Hydro-priming for 6 hrs, T4: Hydro-priming for 12 hrs, T5: Hydro-priming for 18 hrs, T6: Biopriming with Trichoderma  viride for 6 hrs, T7: Biopriming with T.viride for 12 hrs, T8: Biopriming with T.viride for 18 hrs, T9: Biopriming with Trichoderma harzianum for 6 hrs, T10: Biopriming with T. harzianum for  12 hrs, T11: Biopriming with T. harzianum for 18 hrs, T12: Biopriming with Pseudomonas fluorescens for 6 hrs, T13: Biopriming with P. fluorescens for 12 hrs and T14: Biopriming with P. fluorescens for 18 hrs.  The results revealed that maxiumum germination percentage (92.92, 90.77,83.00 and 86.33), seedling length (32.38 cm, 29.35 cm, 31.75 and 31.60 cm), seedling fresh weight (2.07 g, 4.01 g, 3.05 g and 2.04 g),  seedling dry weight (0.42 g, 0.86 g, 0.62 g and 0.42 g) and seedling vigour index (3008.11, 2664.00, 2635.00 and 2728.00) were recorded in T10 (bio priming with T. harzianum for 12 hrs) in tomato (S. lycopersicum L.), brinjal (S. melongena L.), onion (A. cepa L.) and chilli (C. annuum L.), respectively. Thus, it indicated that priming of seeds of these crops with T. harzianum/P. fluorescens/ T. viride  for 12 hrs was very effective with respect to their vegetative growth along with the quality yield

Study protocol: The Improving Care of Acute Lung Injury Patients (ICAP) study

INTRODUCTION: The short-term mortality benefit of lower tidal volume ventilation (LTVV) for patients with acute lung injury/acute respiratory distress syndrome (ALI/ARDS) has been demonstrated in a large, multi-center randomized trial. However, the impact of LTVV and other critical care therapies on the longer-term outcomes of ALI/ARDS survivors remains uncertain. The Improving Care of ALI Patients (ICAP) study is a multi-site, prospective cohort study that aims to evaluate the longer-term outcomes of ALI/ARDS survivors with a particular focus on the effect of LTVV and other critical care therapies. METHODS: Consecutive mechanically ventilated ALI/ARDS patients from 11 intensive care units (ICUs) at four hospitals in the city of Baltimore, MD, USA, will be enrolled in a prospective cohort study. Exposures (patient-based, clinical management, and ICU organizational) will be comprehensively collected both at baseline and throughout patients' ICU stay. Outcomes, including mortality, organ impairment, functional status, and quality of life, will be assessed with the use of standardized surveys and testing at 3, 6, 12, and 24 months after ALI/ARDS diagnosis. A multi-faceted retention strategy will be used to minimize participant loss to follow-up. RESULTS: On the basis of the historical incidence of ALI/ARDS at the study sites, we expect to enroll 520 patients over two years. This projected sample size is more than double that of any published study of long-term outcomes in ALI/ARDS survivors, providing 86% power to detect a relative mortality hazard of 0.70 in patients receiving higher versus lower exposure to LTVV. The projected sample size also provides sufficient power to evaluate the association between a variety of other exposure and outcome variables, including quality of life. CONCLUSION: The ICAP study is a novel, prospective cohort study that will build on previous critical care research to improve our understanding of the longer-term impact of ALI/ARDS, LTVV and other aspects of critical care management. Given the paucity of information about the impact of interventions on long-term outcomes for survivors of critical illness, this study can provide important information to inform clinical practice

Lung protective mechanical ventilation and two year survival in patients with acute lung injury: prospective cohort study

Objective To evaluate the association of volume limited and pressure limited (lung protective) mechanical ventilation with two year survival in patients with acute lung injury

Abnormal ECG Findings in Athletes: Clinical Evaluation and Considerations.

PURPOSE OF REVIEW: Pre-participation cardiovascular evaluation with electrocardiography is normal practice for most sporting bodies. Awareness about sudden cardiac death in athletes and recognizing how screening can help identify vulnerable athletes have empowered different sporting disciplines to invest in the wellbeing of their athletes. RECENT FINDINGS: Discerning physiological electrical alterations due to athletic training from those representing cardiac pathology may be challenging. The mode of investigation of affected athletes is dependent on the electrical anomaly and the disease(s) in question. This review will highlight specific pathological ECG patterns that warrant assessment and surveillance, together with an in-depth review of the recommended algorithm for evaluation

Microsatellite Genotyping of Plasmodium vivax Isolates from Pregnant Women in Four Malaria Endemic Countries

Plasmodium vivax is the most widely distributed human parasite and the main cause of human malaria outside the African continent. However, the knowledge about the genetic variability of P. vivax is limited when compared to the information available for P. falciparum. We present the results of a study aimed at characterizing the genetic structure of P. vivax populations obtained from pregnant women from different malaria endemic settings. Between June 2008 and October 2011 nearly 2000 pregnant women were recruited during routine antenatal care at each site and followed up until delivery. A capillary blood sample from the study participants was collected for genotyping at different time points. Seven P. vivax microsatellite markers were used for genotypic characterization on a total of 229 P. vivax isolates obtained from Brazil, Colombia, India and Papua New Guinea. In each population, the number of alleles per locus, the expected heterozygosity and the levels of multilocus linkage disequilibrium were assessed. The extent of genetic differentiation among populations was also estimated. Six microsatellite loci on 137 P. falciparum isolates from three countries were screened for comparison. The mean value of expected heterozygosity per country ranged from 0.839 to 0.874 for P. vivax and from 0.578 to 0.758 for P. falciparum. P. vivax populations were more diverse than those of P. falciparum. In some of the studied countries, the diversity of P. vivax population was very high compared to the respective level of endemicity. The level of inter-population differentiation was moderate to high in all P. vivax and P. falciparum populations studied

International criteria for electrocardiographic interpretation in athletes: Consensus statement.

Sudden cardiac death (SCD) is the leading cause of mortality in athletes during sport. A variety of mostly hereditary, structural or electrical cardiac disorders are associated with SCD in young athletes, the majority of which can be identified or suggested by abnormalities on a resting 12-lead electrocardiogram (ECG). Whether used for diagnostic or screening purposes, physicians responsible for the cardiovascular care of athletes should be knowledgeable and competent in ECG interpretation in athletes. However, in most countries a shortage of physician expertise limits wider application of the ECG in the care of the athlete. A critical need exists for physician education in modern ECG interpretation that distinguishes normal physiological adaptations in athletes from distinctly abnormal findings suggestive of underlying pathology. Since the original 2010 European Society of Cardiology recommendations for ECG interpretation in athletes, ECG standards have evolved quickly, advanced by a growing body of scientific data and investigations that both examine proposed criteria sets and establish new evidence to guide refinements. On 26-27 February 2015, an international group of experts in sports cardiology, inherited cardiac disease, and sports medicine convened in Seattle, Washington (USA), to update contemporary standards for ECG interpretation in athletes. The objective of the meeting was to define and revise ECG interpretation standards based on new and emerging research and to develop a clear guide to the proper evaluation of ECG abnormalities in athletes. This statement represents an international consensus for ECG interpretation in athletes and provides expert opinion-based recommendations linking specific ECG abnormalities and the secondary evaluation for conditions associated with SCD