Aryl amino acetamides prevent Plasmodium falciparum ring development via targeting the lipid-transfer protein PfSTART1.

With resistance to most antimalarials increasing, it is imperative that new drugs are developed. We previously identified an aryl acetamide compound, MMV006833 (M-833), that inhibited the ring-stage development of newly invaded merozoites. Here, we select parasites resistant to M-833 and identify mutations in the START lipid transfer protein (PF3D7_0104200, PfSTART1). Introducing PfSTART1 mutations into wildtype parasites reproduces resistance to M-833 as well as to more potent analogues. PfSTART1 binding to the analogues is validated using organic solvent-based Proteome Integral Solubility Alteration (Solvent PISA) assays. Imaging of invading merozoites shows the inhibitors prevent the development of ring-stage parasites potentially by inhibiting the expansion of the encasing parasitophorous vacuole membrane. The PfSTART1-targeting compounds also block transmission to mosquitoes and with multiple stages of the parasite's lifecycle being affected, PfSTART1 represents a drug target with a new mechanism of action.

The associations of opioid and benzodiazepine prescriptions with injuries among US military service members.

ABSTRACT: Given the high rates of physical trauma and pain among service members, opioid-prescribing practices and use patterns have significant implications for the well-being of service members and can affect military medicine and personnel readiness. This study measured the association between prescribed opioid and benzodiazepine medications and subsequently reported injuries (accidental, alcohol and drug related, self-inflicted, and violence related) among active duty military members. Participants were service members who entered the military between January 1, 2005, and June 30, 2010. In a nested case-control design, we compared individuals with injuries to individuals without injuries with respect to their opioid and benzodiazepine prescriptions in the 30 days before the injury of an index case. We used a multiintercept, logistic regression model to compare coefficient estimates by injury type. Overall, approximately 17% of individuals with an injury and 4% of individuals without an injury had a recorded opioid prescription. Individuals with an injury of any type had greater odds of prior exposure to opioid prescriptions than controls. Although a dose-response effect was observed for all injury types, it reached a plateau sooner for natural or environmental accidents and self-inflicted injuries relative to alcohol-related and drug-related injuries, violence-related injuries, vehicle accidents, accidental falls, and other accidents. Benzodiazepine prescriptions were found in 3.5% of individuals with an injury and 0.5% of individuals without an injury. The association between benzodiazepine prescriptions and injuries was strongest for natural and environmental accidents.

The Relationship Between Athlete Perceptions of Coaching Leadership Behaviors and Athlete Grit.

Coach leadership style has long been positively correlated with athlete experiences such as motivation, health (i.e., burnout), and performance outcomes (i.e., enhanced execution time to complete tasks) (24). More recently, grit (18) has been positively correlated with athlete experiences such as engagement (39) and decreased burnout (32). Given the impact coaches have on their athletes and the positive psychological benefits of grit, it is reasonable to explore the intersections of coaching behaviors and grit. As such, the purpose of the current study was to examine the relationship between athlete perceptions of coach leadership behaviors and athlete grit. Intercollegiate athletes completed measures of grit and the leadership behaviors of their coach. A significant positive relationship was observed between the grit perseverance subscale and the leadership behavior of training and instruction (r =.30, p < .05). Additional analyses revealed that athletes' perceptions of coach positive feedback significantly predicted their perseverance. Taken together, these findings suggest a link between positive coach feedback and athlete perseverance. Implications of these results for professional practice and future research are discussed.

Expression of the MSPDBL2 antigen in a discrete subset of Plasmodium falciparum schizonts is regulated by GDV1 but may not be linked to sexual commitment.

The Plasmodium falciparum merozoite surface protein MSPDBL2 is a polymorphic antigen targeted by acquired immune responses, and normally expressed in only a minority of mature schizonts. The potential relationship of MSPDBL2 to sexual commitment is examined, as variable mspdbl2 transcript levels and proportions of MSPDBL2-positive mature schizonts in clinical isolates have previously correlated with levels of many sexual stage parasite gene transcripts, although not with the master regulator ap2-g. It is demonstrated that conditional overexpression of the gametocyte development protein GDV1, which promotes sexual commitment, also substantially increases the proportion of MSPDBL2-positive schizonts in culture. Conversely, truncation of the gdv1 gene is shown to prevent any expression of MSPDBL2. However, across diverse P. falciparum cultured lines, the variable proportions of MSPDBL2 positivity in schizonts do not correlate significantly with variable gametocyte conversion rates, indicating it is not involved in sexual commitment. Confirming this, examining a line with endogenous hemagglutinin-tagged AP2-G showed that the individual schizonts expressing MSPDBL2 are mostly different from those expressing AP2-G. Using a selection-linked integration system, modified P. falciparum lines were engineered to express an intact or disrupted version of MSPDBL2, showing the protein is not required for sexual commitment or early gametocyte development. Asexual parasite multiplication rates were also not affected by expression of either intact or disrupted MSPDBL2 in a majority of schizonts. Occurring alongside sexual commitment, the role of the discrete MSPDBL2-positive schizont subpopulation requires further investigation in natural infections where it is under immune selection. IMPORTANCE: Malaria parasites in the blood are remarkably variable, able to switch antigenic targets so they may survive within humans who have already developed specific immune responses. This is one of the challenges in developing vaccines against malaria. MSPDBL2 is a target of naturally acquired immunity expressed in minority proportions of schizonts, the end stages of each 2-day replication cycle in red blood cells which contain merozoites prepared to invade new red blood cells. Results show that the proportion of schizonts expressing MSPDBL2 is positively controlled by the expression of the regulatory gametocyte development protein GDV1. It was previously known that expression of GDV1 leads to increased expression of AP2-G which causes parasites to switch to sexual development, so a surprising finding here is that MSPDBL2-positive parasites are mostly distinct from those that express AP2-G. This discrete antigenic subpopulation of mostly asexual parasites is regulated alongside sexually committed parasites, potentially enabling survival under stress conditions.

Treatment Failure in a UK Malaria Patient Harboring Genetically Variant Plasmodium falciparum From Uganda With Reduced In Vitro Susceptibility to Artemisinin and Lumefantrine.

BACKGROUND: Recent cases of clinical failure in malaria patients in the United Kingdom (UK) treated with artemether-lumefantrine have implications for malaria chemotherapy worldwide. METHODS: Parasites were isolated from an index case of confirmed Plasmodium falciparum treatment failure after standard treatment, and from comparable travel-acquired UK malaria cases. Drug susceptibility in vitro and genotypes at 6 resistance-associated loci were determined for all parasite isolates and compared with clinical outcomes for each parasite donor. RESULTS: A traveler, who returned to the UK from Uganda in 2022 with Plasmodium falciparum malaria, twice failed treatment with full courses of artemether-lumefantrine. Parasites from the patient exhibited significantly reduced susceptibility to artemisinin (ring-stage survival, 17.3% [95% confidence interval {CI}, 13.6%-21.1%]; P < .0001) and lumefantrine (effective concentration preventing 50% of growth = 259.4 nM [95% CI, 130.6-388.2 nM]; P = .001). Parasite genotyping identified an allele of pfk13 encoding both the A675V variant in the Pfk13 propeller domain and a novel L145V nonpropeller variant. In vitro susceptibility testing of 6 other P. falciparum lines of Ugandan origin identified reduced susceptibility to artemisinin and lumefantrine in 1 additional line, also from a 2022 treatment failure case. These parasites did not harbor a pfk13 propeller domain variant but rather the novel nonpropeller variant T349I. Variant alleles of pfubp1, pfap2mu, and pfcoronin were also identified among the 7 parasite lines. CONCLUSIONS: We confirm, in a documented case of artemether-lumefantrine treatment failure imported from Uganda, the presence of pfk13 mutations encoding L145V and A675V. Parasites with reduced susceptibility to both artemisinin and lumefantrine may be emerging in Uganda.

The global transcriptome of Plasmodium falciparum mid-stage gametocytes (stages II-IV) appears largely conserved and gametocyte-specific gene expression patterns vary in clinical isolates.

Our overall understanding of the developmental biology of malaria parasites has been greatly enhanced by recent advances in transcriptomic analysis. However, most of these investigations rely on laboratory strains (LS) that were adapted into in vitro culture many years ago, and the transcriptomes of clinical isolates (CI) circulating in human populations have not been assessed. In this study, RNA-seq was used to compare the global transcriptome of mid-stage gametocytes derived from three short-term cultured CI, with gametocytes derived from the NF54 reference laboratory strain. The core transcriptome appeared to be consistent between CI- and LS-derived gametocyte preparations, but some important differences were also observed. A majority of gametocyte-specific genes (43/53) appear to have relatively higher expression in CI-derived gametocytes than in LS-derived gametocytes, but a K-means clustering analysis showed that genes involved in flagellum- and microtubule-based processes (movement/motility) were more abundant in both groups, albeit with some differences between them. In addition, gametocytes from one CI described as CI group II gametocytes (CI:GGII) showed gene expression variation in the form of reduced gametocyte-specific gene expression compared to the other two CI-derived gametocytes (CI gametocyte group I, CI:GGI), although the mixed developmental stages used in our study is a potential confounder, only partially mitigated by the inclusion of multiple replicates for each CI. Overall, our study suggests that there may be subtle differences in the gene expression profiles of mid-stage gametocytes from CI relative to the NF54 reference strain of Plasmodium falciparum. Thus, it is necessary to deploy gametocyte-producing clinical parasite isolates to fully understand the diversity of gene expression strategies that may occur during the sequestered development of parasite sexual stages. IMPORTANCE Maturing gametocytes of Plasmodium falciparum are known to sequester away from peripheral circulation into the bone marrow until they are mature. Blocking gametocyte sequestration can prevent malaria transmission from humans to mosquitoes, but most studies aim to understand gametocyte development utilizing long-term adapted laboratory lines instead of clinical isolates. This is a particular issue for our understanding of the sexual stages, which are known to decrease rapidly during adaptation to long-term culture, meaning that many LS are unable to produce transmissible gametocytes. Using RNA-seq, we investigated the global transcriptome of mid-stage gametocytes derived from three clinical isolates and a reference strain (NF54). This identified important differences in gene expression profiles between immature gametocytes of CI and the NF54 reference strain of P. falciparum, suggesting increased investment in gametocytogenesis in clinical isolates. Our transcriptomic data highlight the use of clinical isolates in studying the morphological, cellular features and molecular biology of gametocytes.

Genomic variation during culture adaptation of genetically complex Plasmodium falciparum clinical isolates.

Experimental studies on the biology of malaria parasites have mostly been based on laboratory-adapted lines, but there is limited understanding of how these may differ from parasites in natural infections. Loss-of-function mutants have previously been shown to emerge during culture of some Plasmodium falciparum clinical isolates in analyses focusing on single-genotype infections. The present study included a broader array of isolates, mostly representing multiple-genotype infections, which are more typical in areas where malaria is highly endemic. Genome sequence data from multiple time points over several months of culture adaptation of 28 West African isolates were analysed, including previously available sequences along with new genome sequences from additional isolates and time points. Some genetically complex isolates eventually became fixed over time to single surviving genotypes in culture, whereas others retained diversity, although proportions of genotypes varied over time. Drug resistance allele frequencies did not show overall directional changes, suggesting that resistance-associated costs are not the main causes of fitness differences among parasites in culture. Loss-of-function mutants emerged during culture in several of the multiple-genotype isolates, affecting genes (including AP2-HS, EPAC and SRPK1) for which loss-of-function mutants were previously seen to emerge in single-genotype isolates. Parasite clones were derived by limiting dilution from six of the isolates, and sequencing identified de novo variants not detected in the bulk isolate sequences. Interestingly, several of these were nonsense mutants and frameshifts disrupting the coding sequence of EPAC, the gene with the largest number of independent nonsense mutants previously identified in laboratory-adapted lines. Analysis of genomic identity by descent to explore relatedness among clones revealed co-occurring non-identical sibling parasites, illustrative of the natural genetic structure within endemic populations.

Plasmodium falciparum Sexual Commitment Rate Variation among Clinical Isolates and Diverse Laboratory-Adapted Lines.

Asexual blood-stage malaria parasites must produce sexual progeny to infect mosquitoes. It is important to understand the scope and causes of intraspecific variation in sexual commitment rates, particularly for the major human parasite P. falciparum. First, two alternative assay methods of measuring sexual commitment were compared to test a genetically modified P. falciparum line with elevated commitment rates inducible by overexpression of GDV1. The methods yielded correlated measurements with higher sensitivity and precision being achieved by one employing detection of the early gametocyte differentiation marker Pfs16. Thus, this was used to survey a diverse range of parasite lines and test each in multiple biological replicate assays in a serum-free medium supplemented with Albumax. There were differences among six recent clinical isolates from Ghana in their mean rates of sexual commitment per cycle, ranging from 3.3% to 12.2%. Among 13 diverse long-term laboratory-adapted lines, mean sexual commitment rates for most ranged from 4.7% to 13.4%, a few had lower rates with means from 0.3 to 1.6%, and one with a nonfunctional ap2-g gene always showed zero commitment. Among a subset of lines tested for the effects of exogenous choline to suppress commitment, there were significant differences. As expected, there was no effect in a line that had lost the gdv1 gene and that had generally low commitment, whereas the others showed quantitatively variable but significant responses to choline, suggesting potential trait variation. The results indicated the value of performing multiple replicate assays for understanding the variation of this key reproductive trait that likely affects transmission. IMPORTANCE Only sexual-stage malaria parasites are transmitted from human blood to mosquitoes. Thus, it is vital to understand variations in sexual commitment rates because these may be modifiable or susceptible to blocking. Two different methods of commitment rate measurement were first compared, demonstrating higher sensitivity and precision by the detection of an early differentiation marker, which was subsequently used to survey diverse lines. Clinical isolates from Ghana showed significant variation in mean per-cycle commitment rates and variation among biological replicates. Laboratory-adapted lines of diverse origins had a wider range with most being within the range observed for the clinical isolates, while a minority consistently had lower or zero rates. There was quantitative variation in the effects when adding choline to suppress commitment, indicating differing responsiveness of parasites to this environmental modification. Performing multiple assay replicates and comparisons of diverse isolates was important to understand this trait and its potential effects on transmission.

Highly Variable Expression of Merozoite Surface Protein MSPDBL2 in Diverse Plasmodium falciparum Clinical Isolates and Transcriptome Scans for Correlating Genes.

The merozoite surface protein MSPDBL2 of Plasmodium falciparum is under strong balancing selection and is a target of naturally acquired antibodies. Remarkably, MSPDBL2 is expressed in only a minority of mature schizonts of any cultured parasite line, and mspdbl2 gene transcription increases in response to overexpression of the gametocyte development inducer GDV1, so it is important to understand its natural expression. Here, MSPDBL2 in mature schizonts was analyzed in the first ex vivo culture cycle of 96 clinical isolates from 4 populations with various levels of infection endemicity in different West African countries, by immunofluorescence microscopy with antibodies against a conserved region of the protein. In most isolates, less than 1% of mature schizonts were positive for MSPDBL2, but the frequency distribution was highly skewed, as nine isolates had more than 3% schizonts positive and one had 73% positive. To investigate whether the expression of other gene loci correlated with MSPDBL2 expression, whole-transcriptome sequencing was performed on schizont-enriched material from 17 of the isolates with a wide range of proportions of schizonts positive. Transcripts of particular genes were highly significantly positively correlated with MSPDBL2 positivity in schizonts as well as with mspdbl2 gene transcript levels, showing overrepresentation of genes implicated previously as involved in gametocytogenesis but not including the gametocytogenesis master regulator ap2-g. Single-cell transcriptome analysis of a laboratory-adapted clone showed that most individual parasites expressing mspdbl2 did not express ap2-g, consistent with MSPDBL2 marking a developmental subpopulation that is distinct but likely to co-occur alongside sexual commitment. IMPORTANCE These findings contribute to understanding malaria parasite antigenic and developmental variation, focusing on the merozoite surface protein encoded by the single locus under strongest balancing selection. Analyzing the initial ex vivo generation of parasites grown from a wide sample of clinical infections, we show a unique and highly skewed pattern of natural expression frequencies of MSPDBL2, distinct from that of any other antigen. Bulk transcriptome analysis of a range of clinical isolates showed significant overrepresentation of sexual development genes among those positively correlated with MSPDBL2 protein and mspdbl2 gene expression, indicating the MSPDBL2-positive subpopulation to be often coincident with parasites developing sexually in preparation for transmission. Single-cell transcriptome data confirm the absence of a direct correlation with the ap2-g master regulator of sexual development, indicating that the MSPDBL2-positive subpopulation has a separate function in asexual survival and replication under conditions that promote terminal sexual differentiation.

Generation of Plasmodium falciparum Gametocytes In Vitro with Specific Considerations for Field Isolates.

Reproducible induction of gametocytes of Plasmodium falciparum in vitro is crucial for performing various experimental analyses to understand gametocyte cellular and molecular biology and immunology, and for the evaluation of antigametocidal agents and vaccine development. In this protocol, we present specific procedures for the enrichment, synchronous production and separation of developmental stages of P. falciparum gametocytes from culture-adapted field isolates.

Failure of rapid diagnostic tests in Plasmodium falciparum malaria cases among travelers to the UK and Ireland: Identification and characterisation of the parasites.

OBJECTIVES: Our objective was to systematically investigate false-negative histidine-rich protein 2 rapid diagnostic tests (HRP2-RDT) in imported Plasmodium falciparum malaria cases from travelers to the UK and the Republic of Ireland (RoI). METHODS: Five imported malaria cases in travellers returning to the UK and RoI from East Africa were reported to the PHE Malaria Reference Laboratory as negative according to histidine-rich protein (HRP2)-RDT. The cases were systematically investigated using microscopic, RDT, molecular, genomic, and in in vitro approaches. RESULTS: In each case, HRP2-RDT was negative, whereas microscopy confirmed the presence of P. falciparum. Further analysis revealed that the genes encoding HRP2 and HRP3 were deleted in three of the five cases. Whole-genome sequencing in one of these isolates confirmed deletions in P. falciparum chromosomes 8 and 13. Our study produced evidence that the fourth case, which had high parasitemia at clinical presentation, was a rare example of antigen saturation ('prozone-like effect'), leading to a false negative in the HRP2-RDT, while the fifth case was due to low parasitemia. CONCLUSIONS: False-negative HRP2-RDT results with P. falciparum are concerning. Our findings emphasise the necessity of supporting the interpretation of RDT results with microscopy, in conjunction with clinical observations, and sets out a systematic approach to identifying parasites carrying pfhrp2 and pfhrp3 deletions.

Explosive ordnance disposal personnel in the U.S. military have higher risk of insomnia and post-traumatic stress disorder: a large retrospective cohort study.

PURPOSE: We examined the risk of post-traumatic stress disorder (PTSD), affective disorders, alcohol/substance-related disorders, traumatic brain injury (TBI) and insomnia, among explosive ordnance disposal (EOD) technicians compared to the general population of active-duty non-EOD personnel in the U.S. military. METHODS: We conducted a retrospective cohort study using administrative and healthcare utilization data from fiscal year 2004 (FY2004) to FY2015 for this comparison. We used propensity score matching to balance baseline covariates, and discrete-time hazard models to compare the odds of occurrence of the outcomes. RESULTS: EOD personnel had higher odds of having a new diagnosis of insomnia (odds ratio [OR] = 1.33; 95% confidence interval [CI]:1.22-1.45) and PTSD (OR = 1.23; 95% CI:1.08-1.41) than did non-EOD personnel. EOD technicians had lower odds of having a new diagnosis of affective disorders (OR = 0.83; 95% CI:0.79-0.87) and alcohol/substance-related disorders (OR = 0.59; 95% CI:0.54-0.64) than did non-EOD personnel. There was little evidence of a difference in the odds of a TBI diagnosis (OR = 1.07; 95% CI:0.99-1.16). CONCLUSIONS: As reliance on EOD forces continues, ongoing vigilance of the stressors, health sequelae and disincentives to access mental health care among this military occupation should be monitored and mitigated wherever possible.

Longitudinal Relationship of Combat Exposure With Mental Health Diagnoses in the Military Health System.

INTRODUCTION: Combat deployment is associated with mental and physical health disorders and functional impairment. Mental health (MH) diagnoses such as adjustment and anxiety disorders have received little research attention but may reflect important postdeployment sequelae. The purpose of this study was to investigate the association of combat exposure with the acquisition of a wide range of mental health diagnoses over 2 years. MATERIALS AND METHODS: This retrospective longitudinal study utilized multiple administrative Military Health System datasets compiled for all individuals who entered active duty in the U.S. Army from FY2005 to FY2011. A total eligible cohort of 289,922 Service members was stratified into three mutually exclusive groups according to their deployment status after 2 years in service: Deployed, Combat-Exposed; Deployed, Not-Combat-Exposed; and Not Deployed. Outcomes of interest were new mental health diagnoses grouped into six categories-posttraumatic stress disorder, anxiety, adjustment, mood, substance use disorders, and any MH diagnosis. Survival analyses over 2 years were conducted and adjusted hazard ratios were calculated. RESULTS: Combat exposure in the first 2 years of military service was associated with significantly higher rates of a wide range of mental health diagnoses over a two-year follow-up period, compared with deployment with no combat exposure and no deployment. Adjusted cumulative failure proportions demonstrated that approximately a third of the Combat-Exposed group, a quarter of the Not-Combat-Exposed, and a fifth of the Not Deployed groups received a MH diagnosis over 2 years. For all groups, cumulative failure proportions and incidence rates were highest for adjustment disorder and lowest for posttraumatic stress disorder diagnoses. CONCLUSIONS: Researchers and providers should be alerted to the impact of combat exposure and the wide range of MH conditions and diagnoses that may represent important postdeployment sequelae.

Women in combat: The effects of combat exposure and gender on the incidence and persistence of posttraumatic stress disorder diagnosis.

Recent expansions in the roles of women in combat have prompted increased interest in the psychological toll combat exposure may have on female service members as compared to males. This study examined the interactive effects of gender and combat exposure on transitions in posttraumatic stress disorder (PTSD) diagnostic status (presence or absence of PTSD diagnosis). We used administrative data of 20,000 U.S. Army soldiers whose combat exposure was assessed after return from deployment between January 1, 2008 and June 30, 2014; soldiers' PTSD diagnostic status was determined using International Classification of Diseases-9 diagnoses at four time points separated by 12 months. We used a mixed-effects logit transition model to examine the effects of combat and gender on incidence, persistence, and prevalence of PTSD diagnosis. Incidence and prevalence of PTSD diagnosis were higher among women, but persistence of PTSD diagnosis was higher in men. Higher rates of new PTSD diagnosis among women were not dependent on combat exposure, suggesting that other types of trauma may be responsible for increased rates among women. Gender differences in prevalence and persistence of PTSD diagnosis were greater among combat-exposed soldiers than among those not exposed to combat. Men maintained a PTSD diagnosis over longer periods of time than women suggesting greater PTSD persistence, and this pattern was particularly pronounced among soldiers exposed to combat. These results have implications for the recent policy changes and gender-based prevention strategies, and suggest that women in combat roles may be no more vulnerable to PTSD than are their male counterparts. Though the gender differences were small, they are indicative of healthcare utilization patterns that may be important for prevention and that warrant further exploration.

Multi-locus genotyping reveals established endemicity of a geographically distinct Plasmodium vivax population in Mauritania, West Africa.

BACKGROUND: Plasmodium vivax has been recently discovered as a significant cause of malaria in Mauritania, although very rare elsewhere in West Africa. It has not been known if this is a recently introduced or locally remnant parasite population, nor whether the genetic structure reflects epidemic or endemic transmission. METHODOLOGY/PRINCIPAL FINDINGS: To investigate the P. vivax population genetic structure in Mauritania and compare with populations previously analysed elsewhere, multi-locus genotyping was undertaken on 100 clinical isolates, using a genome-wide panel of 38 single nucleotide polymorphisms (SNPs), plus seven SNPs in drug resistance genes. The Mauritanian P. vivax population is shown to be genetically diverse and divergent from populations elsewhere, indicated consistently by genetic distance matrix analysis, principal components analyses, and fixation indices. Only one isolate had a genotype clearly indicating recent importation, from a southeast Asian source. There was no linkage disequilibrium in the local parasite population, and only a small number of infections appeared to be closely genetically related, indicating that there is ongoing genetic recombination consistent with endemic transmission. The P. vivax diversity in a remote mining town was similar to that in the capital Nouakchott, with no indication of local substructure or of epidemic population structure. Drug resistance alleles were virtually absent in Mauritania, in contrast with P. vivax in other areas of the world. CONCLUSIONS/SIGNIFICANCE: The molecular epidemiology indicates that there is long-standing endemic transmission that will be very challenging to eliminate. The virtual absence of drug resistance alleles suggests that most infections have been untreated, and that this endemic infection has been more neglected in comparison to P. vivax elsewhere.

Intrinsic multiplication rate variation and plasticity of human blood stage malaria parasites.

Pathogen multiplication rate is theoretically an important determinant of virulence, although often poorly understood and difficult to measure accurately. We show intrinsic asexual blood stage multiplication rate variation of the major human malaria parasite Plasmodium falciparum to be associated with blood-stage infection intensity in patients. A panel of clinical isolates from a highly endemic West African population was analysed repeatedly during five months of continuous laboratory culture, showing a range of exponential multiplication rates at all timepoints tested, mean rates increasing over time. All isolates had different genome sequences, many containing within-isolate diversity that decreased over time in culture, but increases in multiplication rates were not primarily attributable to genomic selection. New mutants, including premature stop codons emerging in a few isolates, did not attain sufficiently high frequencies to substantially affect overall multiplication rates. Significantly, multiplication rate variation among the isolates at each of the assayed culture timepoints robustly correlated with parasite levels seen in patients at clinical presentation, indicating innate parasite control of multiplication rate that contributes to virulence.

Investigating a Plasmodium falciparum erythrocyte invasion phenotype switch at the whole transcriptome level.

The central role that erythrocyte invasion plays in Plasmodium falciparum survival and reproduction makes this process an attractive target for therapeutic or vaccine development. However, multiple invasion-related genes with complementary and overlapping functions afford the parasite the plasticity to vary ligands used for invasion, leading to phenotypic variation and immune evasion. Overcoming the challenge posed by redundant ligands requires a deeper understanding of conditions that select for variant phenotypes and the molecular mediators. While host factors including receptor heterogeneity and acquired immune responses may drive parasite phenotypic variation, we have previously shown that host-independent changes in invasion phenotype can be achieved by continuous culturing of the W2mef and Dd2 P. falciparum strains in moving suspension as opposed to static conditions. Here, we have used a highly biologically replicated whole transcriptome sequencing approach to identify the molecular signatures of variation associated with the phenotype switch. The data show increased expression of particular invasion-related genes in switched parasites, as well as a large number of genes encoding proteins that are either exported or form part of the export machinery. The genes with most markedly increased expression included members of the erythrocyte binding antigens (EBA), reticulocyte binding homologues (RH), surface associated interspersed proteins (SURFIN), exported protein family 1 (EPF1) and Plasmodium Helical Interspersed Sub-Telomeric (PHIST) gene families. The data indicate changes in expression of a repertoire of genes not previously associated with erythrocyte invasion phenotypes, suggesting the possibility that moving suspension culture may also select for other traits.

Treatment of anxiety in older adolescents and young adults with autism spectrum disorders: A pilot study.

Anxiety disorders are commonly comorbid in adolescents and young adults with high-functioning autism. Cognitive-behavioral treatments (CBT) for anxiety, when adapted and expanded to target autism spectrum disorder (ASD) characteristics, may be beneficial, but there is minimal evidence to guide clinicians in their application. This multiple-baseline design study evaluated the initial efficacy of a CBT protocol adapted to address anxiety symptoms and adaptive functioning in this population. Anxiety and ASD symptoms were assessed for six participants at intake, after baseline, posttreatment, and at 1-month follow-up. Parent- and child-reported anxiety was also assessed during baseline and treatment. Visual inspection and reliable change index scores were used to evaluate change. All participants improved on clinician-rated measures of disorder severity, and gains were maintained at follow-up. Results were more equivocal for parent- and self-rated anxiety and parent-rated ASD, partly because of spontaneous changes during baseline.

Establishing an Evidence Synthesis Capability For Psychological Health Topics in the Military Health System.

BACKGROUND: To promote evidence-based health care, clinical providers and decision makers rely on scientific evidence to inform best practices. Evidence synthesis (ES) is a key component of this process that serves to inform health care decisions by integrating and contextualizing research findings across studies. OBJECTIVE: This paper describes the process of establishing an ES capability in the Military Health System dedicated to psychological health topics. RESEARCH DESIGNS: The goal of establishing the current ES capability was to facilitate evidence-based decision-making among clinicians, clinic managers, research funders, and policymakers, through the production and dissemination of trustworthy ES reports. We describe how we developed this capability, provide an overview of the types of evidence syntheses products we use to respond to different stakeholders, and detail the procedures established for selecting and prioritizing synthesis topics. RESULTS: We report on the productivity, acceptability, and impact of our efforts. Our reports were used by a variety of stakeholders and working groups, briefed to major committees, included in official reports and policies, and cited in clinical practice guidelines and the peer-reviewed literature. CONCLUSIONS: Our experiences thus far suggest that the current ES capability offers a needed service within our health system. Our framework may help inform other agencies interested in developing or sponsoring a similar capability.

Potent bicyclic inhibitors of malarial cGMP-dependent protein kinase: approaches to combining improvements in cell potency, selectivity and structural novelty.

Focussed studies on imidazopyridine inhibitors of Plasmodium falciparum cyclic GMP-dependent protein kinase (PfPKG) have significantly advanced the series towards desirable in vitro property space. LLE-based approaches towards combining improvements in cell potency, key physicochemical parameters and structural novelty are described, and a structure-based design hypothesis relating to substituent regiochemistry has directed efforts towards key examples with well-balanced potency, ADME and kinase selectivity profiles.