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sequencing data for each isolate, and these are compared against a panel of polymorphic sites derived from published or unpublished but publicly available data.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.     

The range of axial rotation of the glenohumeral joint

There is a paucity of data in the literature on the restraining effects of the glenohumeral (GH) ligaments; cadaveric testing is one of the best methods for determining the function of these types of tissues. The aim of this work was to commission a custom-made six degrees of freedom (dof) joint loading apparatus and to establish a protocol for laxity testing of cadaveric shoulder specimens. Nine cadaveric shoulder specimens were used in this study and each specimen had all muscle resected leaving the scapula, humerus (transected at mid-shaft) and GH capsule. Specimens were mounted on the testing apparatus with the joint in the neutral position and at 30°, 60° and 90° GH abduction in the coronal, scapula and 30° forward flexion planes. For each orientation, 0–1 N m in 0.1 N m increments was applied in internal/external rotation and the angular displacement recorded. The toe-region of the moment–displacement curves ended at approximately ±0.5 N m. The highest rotational range of motion for the joint was 140° for ±1.0 N m at 30° GH abduction in the scapula plane. The range of motion shifted towards external rotation with increasing levels of abduction. The results provide the optimum loading regime to pre-condition shoulder specimens and minimise viscoelastic effects in the ligaments prior to laxity testing (>0.5 N m at 30° GH abduction in any of the three planes). Knowledge of the mechanical properties of the GH capsuloligamentous complex has implications for modelling of the shoulder as well surgical planning and intervention.     

Tyrosine kinase regulation of nuclear envelope assembly

We have described the co-localization of PLCγ and SFK1/7 in vivo and in vitro, and demonstrated their recruitment together to nuclei during membrane binding. We hypothesize that SFK1/7 is activated in response to GTP hydrolysis, phosphorylating and priming PLCγ for activation and thus producing the fusogen DAG during NE assembly. In addition to enhancing our understanding of the temporal and spatial mechanisms of NE formation, this pathway offers a novel link between protein regulation and lipid metabolism that has implications for general membrane fusion events.     

In-hospital case fatality rates for acute myocardial infarction in Romania

Background

We describe the clinical characteristics, treatments and in-hospital case-fatality rates in an unselected population of patients admitted for acute myocardial infarction.

Methods

From January 2000 to June 2007, we tracked consecutive patients who were admitted to 7 tertiary referral and 21 county hospitals in Romania for medical treatment of ST-segment elevation acute myocardial infarction. These patients were enrolled in the Romanian Registry for ST-segment Elevation Myocardial Infarction. For this prospective study, we collected data on demographic characteristics, cardiovascular risk factors, various aspects of treatment for myocardial infarction, and in-hospital death.

Results

The 9186 patients in the study group had a mean age of 63.8 years. The median time from onset of symptoms to thrombolysis was 230 (interquartile range 120–510) minutes. Of the 9186 patients, 4986 (54.3%) had hypertension, 1974 (21.5%) had diabetes mellitus, 3545 (38.6%) had lipid disorders and 4653 (50.7%) were smokers. The in-hospital mortality rate was 12.7% (1170 deaths). The study group consisted of 2893 women and 6293 men. The women were older than the men and had higher rates of hypertension and diabetes mellitus but were less likely to be smokers. A smaller proportion of women than men presented within 2 hours after onset of symptoms (23.1% v. 34.4%, p < 0.001). Smaller proportions of women received thrombolytics (40.8% v. 53.5%, p < 0.001), anticoagulants (93.4% v. 95.2%; p = 0.001), antiplatelet agents (88.3% v. 91.2%, p < 0.001) and primary percutaneous coronary interventions (1.5% v. 2.2%, p = 0.030). The risk of in-hospital death was greater for women, even after adjustment for confounders (odds ratio 1.33, 95% confidence interval 1.13–1.56; p < 0.001).

Interpretation

The rates of reperfusion therapy for patients with acute myocardial infarction were low, and in-hospital case-fatality rates were high in this study. Excess in-hospital mortality was more pronounced among women.During the past 10 years, the health of people in Eastern Europe and the former Soviet Union has undergone changes very different from the health patterns seen in their Western counterparts. For example, mortality from cardiovascular disease has been decreasing continuously in the United States and many Western European countries, but it has increased or remained unchanged in many of the states of Eastern Europe.¹Analysis of this phenomenon has been hindered by insufficient information. The World Health Organization’s MONICA project for monitoring cardiovascular mortality and risk factors considered only 6 Eastern European countries: Russia, Yugoslavia, Poland, Czechoslovakia, Hungary and the former East Germany.² In geographic terms, Romania is the largest country in southeastern Europe, with a large population and substantial natural resources,³ but in some ways it is a “forgotten country.” Few studies evaluating risks for cardiovascular disease have included Romania. Only 4 trials examining ST-segment elevation myocardial infarction have enrolled patients from Romania,^4–6 and the numbers of patients were too few for reliable subgroup analysis.Our aim was to investigate clinical characteristics, treatments and case fatality rates in an unselected population of patients admitted to hospital in Romania for ST-segment elevation myocardial infarction. In a secondary analysis, we analyzed sex differences in relation to treatment and outcome.     

Delta agonist hydroxy bioisosteres: The discovery of 3-((1-benzylpiperidin-4-yl){4-[(diethylamino)carbonyl]phenyl}amino)benzamide with improved delta agonist activity and in vitro metabolic stability

We have investigated phenol replacements in a series of diaryl amino piperidine delta opioid agonists. From this study we have demonstrated that the hydroxy functional group can be replaced with a primary amide group, giving enhanced activity at the delta receptor, increased selectivity versus mu and kappa as well as improved in vitro metabolic stability.     

Overcoming species boundaries in peptide identification with Bayesian information criterion-driven error-tolerant peptide search (BICEPS)

Currently, the reliable identification of peptides and proteins is only feasible when thoroughly annotated sequence databases are available. Although sequencing capacities continue to grow, many organisms remain without reliable, fully annotated reference genomes required for proteomic analyses. Standard database search algorithms fail to identify peptides that are not exactly contained in a protein database. De novo searches are generally hindered by their restricted reliability, and current error-tolerant search strategies are limited by global, heuristic tradeoffs between database and spectral information. We propose a Bayesian information criterion-driven error-tolerant peptide search (BICEPS) and offer an open source implementation based on this statistical criterion to automatically balance the information of each single spectrum and the database, while limiting the run time. We show that BICEPS performs as well as current database search algorithms when such algorithms are applied to sequenced organisms, whereas BICEPS only uses a remotely related organism database. For instance, we use a chicken instead of a human database corresponding to an evolutionary distance of more than 300 million years (International Chicken Genome Sequencing Consortium (2004) Sequence and comparative analysis of the chicken genome provide unique perspectives on vertebrate evolution. Nature 432, 695-716). We demonstrate the successful application to cross-species proteomics with a 33% increase in the number of identified proteins for a filarial nematode sample of Litomosoides sigmodontis.     

Tracking protein aggregation and mislocalization in cells with flow cytometry

We applied pulse-shape analysis (PulSA) to monitor protein localization changes in mammalian cells by flow cytometry. PulSA enabled high-throughput tracking of protein aggregation, translocation from the cytoplasm to the nucleus and trafficking from the plasma membrane to the Golgi as well as stress-granule formation. Combining PulSA with tetracysteine-based oligomer sensors in a cell model of Huntington's disease enabled further separation of cells enriched with monomers, oligomers and inclusion bodies.     

The baby effect and young male syndrome: social influences on cooperative risk-taking in women and men

Parental investment theory predicts differences in risk-taking for females and males as a consequence of reproductive context, with females attempting to reduce risks in relation to their own offspring (here called the baby effect) and males taking more risks in competition with one another (young male syndrome). The experiment we report tests these predictions in a cooperative context by introducing the Social Balloon Analogue Risk Task—the Balloon Analogue Risk Task modified to include a social partner (adult male, adult female, or baby)—along with a commitment device in which participants choose among several possible social partners, with whom they will share their earnings. Results were consistent with the predictions of parental investment theory. Females did not change their levels of risk-taking when paired with adult males or females, but showed a strong reduction in risk when paired with babies. Consistent with previous research, males were strongly inclined to take more risks when paired with another male of the same age, but males showed no change in risk-taking when paired with a female of the same age or a child. The current work provides the first experimental evidence of gender differences in cooperative social risk-taking, as well as the first experimental evidence of a mediator of female risk-taking, i.e., babies.