Malaria vaccines - The long synthetic peptide approach: Technical and conceptual advancements.

This review describes the advances in malaria antigen discovery and vaccine development using the long synthetic peptide platforms that have been made available during the past 5 years. The most recent technical developments regarding peptide synthesis with the optimized production of large synthetic fragments are discussed. Clinical trials of long synthetic peptides are also reviewed. These trials demonstrated that long synthetic peptides are safe and immunogenic when formulated with various adjuvants. In addition, long synthetic peptides can elicit an antibody response in humans and have demonstrated inhibitory activity against parasite growth in vitro. Finally, new approaches to exploit the abundance of genomic data and the flexibility and speed of peptide synthesis are proposed.

Determination of Vasopressin and Desmopressin in urine by means of liquid chromatography coupled to quadrupole time-of-flight mass spectrometry for doping control purposes.

The anti-diuretic neurohypophysial hormone Vasopressin (Vp) and its synthetic analogue Desmopressin (Dp, 1-desamino-vasopressin) have received considerable attention from doping control authorities due to their impact on physiological blood parameters. Accordingly, the illicit use of Desmopressin in elite sport is sanctioned by the World Anti-Doping Agency (WADA) and the drug is classified as masking agent. Vp and Dp are small (8-9 amino acids) peptides administered orally as well as intranasally. Within the present study a method to determine Dp and Vp in urinary doping control samples by means of liquid chromatography coupled to quadrupole high resolution time-of-flight mass spectrometry was developed. After addition of Lys-Vasopressin as internal standard and efficient sample clean up with a mixed mode solid phase extraction (weak cation exchange), the samples were directly injected into the LC-MS system. The method was validated considering the parameters specificity, linearity, recovery (80-100%), accuracy, robustness, limit of detection/quantification (20/50 pg mL(-1)), precision (inter/intra-day<10%), ion suppression and stability. The analysis of administration study urine samples collected after a single intranasal or oral application of Dp yielded in detection windows for the unchanged target analyte for up to 20 h at concentrations between 50 and 600 pg mL(-1). Endogenous Vp was detected in concentrations of approximately 20-200 pg mL(-1) in spontaneous urine samples obtained from healthy volunteers. The general requirements of the developed method provide the characteristics for an easy transfer to other anti-doping laboratories and support closing another potential gap for cheating athletes.

Phase I safety and immunogenicity trial of Plasmodium vivax CS derived long synthetic peptides adjuvanted with montanide ISA 720 or montanide ISA 51.

We assessed the safety, tolerability, and immunogenicity of a mixture of three synthetic peptides derived from the Plasmodium vivax circumsporozoite protein formulated in Montanide ISA 720 or Montanide ISA 51. Forty healthy malaria-naive volunteers were allocated to five experimental groups (A-E): four groups (A-D) were immunized intramuscularly with 50 and 100 μg/dose injections of a mixture of N, R, and C peptides formulated in the two different adjuvants at 0, 2, and 4 months and one group was administered placebo. Vaccines were immunogenic, safe, well tolerated, and no serious adverse events related to the vaccine occurred. Seroconversion occurred in > 90% of the vaccines and antibodies recognized the sporozoite protein on immunofluorescent antibody test. Vaccines in Montanide ISA 51 showed a higher sporozoite protein recognition and interferon production. Results encourage further testing of the vaccine protective efficacy.

Metabolic control in cancer cells.

An important hallmark of cancer cells is a profound change in metabolism. Indeed, most tumor cells are characterized by higher rates of glycolysis, lactate production, and biosynthesis of lipids and other macromolecules. Our group, among others, has previously demonstrated a close relationship between metabolic responses and proliferative stimuli, showing that cell cycle regulators have a major role in the control of metabolism. Changes in this coordinated response might lead to abnormal metabolic changes during tumor development and cancer progression. In this paper we review the dual role of cell cycle regulators in the control of both proliferation and metabolism in normal and in cancer cells. We show participation of the E2F1-CDK4 axis in the modulation of oxidative metabolism, in the positive regulation of lipid synthesis, and the regulation glycolysis. These three metabolic pathways are, interestingly fundamental in providing synthetic processes, energy production and cell signaling events, which are crucial factors for cancer cell survival.

Intranasal administration of the synthetic polypeptide from the C-terminus of the circumsporozoite protein of Plasmodium berghei with the modified heat-labile toxin of Escherichia coli (LTK63) induces a complete protection against malaria challenge.

Needle-free procedures are very attractive ways to deliver vaccines because they diminish the risk of contamination and may reduce local reactions, pain or pain fear especially in young children with a consequence of increasing the vaccination coverage for the whole population. For this purpose, the possible development of a mucosal malaria vaccine was investigated. Intranasal immunization was performed in BALB/c mice using a well-studied Plasmodium berghei model antigen derived from the circumsporozoite protein with the modified heat-labile toxin of Escherichia coli (LTK63), which is devoid of any enzymatic activity compared to the wild type form. Here, we show that intranasal administration of the two compounds activates the T and B cell immune response locally and systemically. In addition, a total protection of mice is obtained upon a challenge with live sporozoites.

Testosterone and doping control.

BACKGROUND AND OBJECTIVES: Anabolic steroids are synthetic derivatives of testosterone, modified to enhance its anabolic actions (promotion of protein synthesis and muscle growth). They have numerous side effects, and are on the International Olympic Committee's list of banned substances. Gas chromatography-mass spectrometry allows identification and characterisation of steroids and their metabolites in the urine but may not distinguish between pharmaceutical and natural testosterone. Indirect methods to detect doping include determination of the testosterone/epitestosterone glucuronide ratio with suitable cut-off values. Direct evidence may be obtained with a method based on the determination of the carbon isotope ratio of the urinary steroids. This paper aims to give an overview of the use of anabolic-androgenic steroids in sport and methods used in anti-doping laboratories for their detection in urine, with special emphasis on doping with testosterone. METHODS: Review of the recent literature of anabolic steroid testing, athletic use, and adverse effects of anabolic-androgenic steroids. RESULTS: Procedures used for detection of doping with endogenous steroids are outlined. The World Anti-Doping Agency provided a guide in August 2004 to ensure that laboratories can report, in a uniform way, the presence of abnormal profiles of urinary steroids resulting from the administration of testosterone or its precursors, androstenediol, androstenedione, dehydroepiandrosterone or a testosterone metabolite, dihydrotestosterone, or a masking agent, epitestosterone. CONCLUSIONS: Technology developed for detection of testosterone in urine samples appears suitable when the substance has been administered intramuscularly. Oral administration leads to rapid pharmacokinetics, so urine samples need to be collected in the initial hours after intake. Thus there is a need to find specific biomarkers in urine or plasma to enable detection of long term oral administration of testosterone.

Fatty acids and retinoids control lipid metabolism through activation of peroxisome proliferator-activated receptor-retinoid X receptor heterodimers.

The nuclear hormone receptors called PPARs (peroxisome proliferator-activated receptors alpha, beta, and gamma) regulate the peroxisomal beta-oxidation of fatty acids by induction of the acyl-CoA oxidase gene that encodes the rate-limiting enzyme of the pathway. Gel retardation and cotransfection assays revealed that PPAR alpha heterodimerizes with retinoid X receptor beta (RXR beta; RXR is the receptor for 9-cis-retinoic acid) and that the two receptors cooperate for the activation of the acyl-CoA oxidase gene promoter. The strongest stimulation of this promoter was obtained when both receptors were exposed simultaneously to their cognate activators. Furthermore, we show that natural fatty acids, and especially polyunsaturated fatty acids, activate PPARs as potently as does the hypolipidemic drug Wy 14,643, the most effective activator known so far. Moreover, we discovered that the synthetic arachidonic acid analogue 5,8,11,14-eicosatetraynoic acid is 100 times more effective than Wy 14,643 in the activation of PPAR alpha. In conclusion, our data demonstrate a convergence of the PPAR and RXR signaling pathways in the regulation of the peroxisomal beta-oxidation of fatty acids by fatty acids and retinoids.

Preclinical vaccine study of Plasmodium vivax circumsporozoite protein derived-synthetic polypeptides formulated in montanide ISA 720 and montanide ISA 51 adjuvants.

Plasmodium vivax circumsporozoite (CS) protein is a leading malaria vaccine candidate previously assessed in animals and humans. Here, combinations of three synthetic polypeptides corresponding to amino (N), central repeat (R), and carboxyl (C) regions of the CS protein formulated in Montanide ISA 720 or Montanide ISA 51 adjuvants were assessed for immunogenicity in rodents and primates. BALB/c mice and Aotus monkeys were divided into test and control groups and were immunized three times with doses of 50 and 100 μg of vaccine or placebo. Antigen-specific antimalarial antibodies were determined by enzyme-linked immunosorbent assay, immunofluorescent antibody test, and IFN-γ responses by enzyme-linked immunosorbent spot (ELIspot). Both vaccine formulations were highly immunogenic in both species. Mice developed better antibody responses against C and R polypeptides, whereas the N polypeptide was more immunogenic in monkeys. Anti-peptide antibodies remained detectable for several months and recognized native proteins on sporozoites. Differences between Montanide ISA 720 and Montanide ISA 51 formulations were not significant.

Antibody-mediated and cellular immune responses induced in naive volunteers by vaccination with long synthetic peptides derived from the Plasmodium vivax circumsporozoite protein.

Plasmodium vivax circumsporozoite (CS) protein is a leading malaria vaccine candidate. We describe the characterization of specific immune responses induced in 21 malaria-naive volunteers vaccinated with long synthetic peptides derived from the CS protein formulated in Montanide ISA 720. Both antibody- and cell-mediated immune responses were analyzed. Antibodies were predominantly of IgG1 and IgG3 isotypes, recognized parasite proteins on the immunofluorescent antibody test, and partially blocked sporozoite invasion of hepatoma cell lines in vitro. Peripheral blood mononuclear cells from most volunteers (94%) showed IFN-γ production in vitro upon stimulation with both long signal peptide and short peptides containing CD8+ T-cell epitopes. The relatively limited sample size did not allow conclusions about HLA associations with the immune responses observed. In summary, the inherent safety and tolerability together with strong antibody responses, invasion blocking activity, and the IFN-γ production induced by these vaccine candidates warrants further testing in a phase II clinical trial.

Statistical modeling of bowing control applied to violin sound synthesis

Excitation-continuous music instrument control patterns are often not explicitly represented in current sound synthesis techniques when applied to automatic performance. Both physical model-based and sample-based synthesis paradigmswould benefit from a flexible and accurate instrument control model, enabling the improvement of naturalness and realism. Wepresent a framework for modeling bowing control parameters inviolin performance. Nearly non-intrusive sensing techniques allow for accurate acquisition of relevant timbre-related bowing control parameter signals.We model the temporal contour of bow velocity, bow pressing force, and bow-bridge distance as sequences of short Bézier cubic curve segments. Considering different articulations, dynamics, and performance contexts, a number of note classes are defined. Contours of bowing parameters in a performance database are analyzed at note-level by following a predefined grammar that dictates characteristics of curve segment sequences for each of the classes in consideration. As a result, contour analysis of bowing parameters of each note yields an optimal representation vector that is sufficient for reconstructing original contours with significant fidelity. From the resulting representation vectors, we construct a statistical model based on Gaussian mixtures suitable for both the analysis and synthesis of bowing parameter contours. By using the estimated models, synthetic contours can be generated through a bow planning algorithm able to reproduce possible constraints caused by the finite length of the bow. Rendered contours are successfully used in two preliminary synthesis frameworks: digital waveguide-based bowed stringphysical modeling and sample-based spectral-domain synthesis.

Premenopausal serum androgens and breast cancer risk: a nested case-control study.

ABSTRACT: INTRODUCTION: Prospective epidemiologic studies have consistently shown that levels of circulating androgens in postmenopausal women are positively associated with breast cancer risk. However, data in premenopausal women are limited. METHODS: A case-control study nested within the New York University Women's Health Study was conducted. A total of 356 cases (276 invasive and 80 in situ) and 683 individually-matched controls were included. Matching variables included age and date, phase, and day of menstrual cycle at blood donation. Testosterone, androstenedione, dehydroandrosterone sulfate (DHEAS) and sex hormone-binding globulin (SHBG) were measured using direct immunoassays. Free testosterone was calculated. RESULTS: Premenopausal serum testosterone and free testosterone concentrations were positively associated with breast cancer risk. In models adjusted for known risk factors of breast cancer, the odds ratios for increasing quintiles of testosterone were 1.0 (reference), 1.5 (95% confidence interval (CI), 0.9 to 2.3), 1.2 (95% CI, 0.7 to 1.9), 1.4 (95% CI, 0.9 to 2.3) and 1.8 (95% CI, 1.1 to 2.9; Ptrend = 0.04), and for free testosterone were 1.0 (reference), 1.2 (95% CI, 0.7 to 1.8), 1.5 (95% CI, 0.9 to 2.3), 1.5 (95% CI, 0.9 to 2.3), and 1.8 (95% CI, 1.1 to 2.8, Ptrend = 0.01). A marginally significant positive association was observed with androstenedione (P = 0.07), but no association with DHEAS or SHBG. Results were consistent in analyses stratified by tumor type (invasive, in situ), estrogen receptor status, age at blood donation, and menopausal status at diagnosis. Intra-class correlation coefficients for samples collected from 0.8 to 5.3 years apart (median 2 years) in 138 cases and 268 controls were greater than 0.7 for all biomarkers except for androstenedione (0.57 in controls). CONCLUSIONS: Premenopausal concentrations of testosterone and free testosterone are associated with breast cancer risk. Testosterone and free testosterone measurements are also highly reliable (that is, a single measurement is reflective of a woman's average level over time). Results from other prospective studies are consistent with our results. The impact of including testosterone or free testosterone in breast cancer risk prediction models for women between the ages of 40 and 50 years should be assessed. Improving risk prediction models for this age group could help decision making regarding both screening and chemoprevention of breast cancer.

Pose-based SLAM with probabilistic scan matching algorithm using a mechanical scanned imaging sonar

This paper proposes a pose-based algorithm to solve the full SLAM problem for an autonomous underwater vehicle (AUV), navigating in an unknown and possibly unstructured environment. The technique incorporate probabilistic scan matching with range scans gathered from a mechanical scanning imaging sonar (MSIS) and the robot dead-reckoning displacements estimated from a Doppler velocity log (DVL) and a motion reference unit (MRU). The proposed method utilizes two extended Kalman filters (EKF). The first, estimates the local path travelled by the robot while grabbing the scan as well as its uncertainty and provides position estimates for correcting the distortions that the vehicle motion produces in the acoustic images. The second is an augment state EKF that estimates and keeps the registered scans poses. The raw data from the sensors are processed and fused in-line. No priory structural information or initial pose are considered. The algorithm has been tested on an AUV guided along a 600 m path within a marina environment, showing the viability of the proposed approach

The International Distribution of Energy Intensities: some synthetic results

The paper examines the international distribution of energy intensities as a conventional proxy indicator of energy efficiency and sustainability in the consumption of resources, by employing some descriptive tools from the analysis of inequality and polarization. The analysis specifically focuses on the following points: firstly, inequalities are evaluated synthetically based on diverse summary measures and Lorenz curves; secondly, different factorial decompositions are undertaken that assist in investigating some explanatory factors (weighting factors, multiplicative factors and decomposition by groups); and thirdly, an analysis is made of the polarization of intensities when groups of countries are defined endogenously and exogenously. The results obtained have significant implications from both academic and political perspectives.

Green tea in transdermal formulation: HPLC method for quality control and in vitro drug release assays

RP-HPLC based analytical method for use in both quality control of green tea in a semisolid formulation and for in vitro drug release assays was developed and validated. The method was precise (CV < 5%), accurate (recovery between 98% and 102%), linear (R² > 0.99), robust, and specific for the determination of epigallocatechin 3-gallate (EGCG), caffeine (CAF), and gallic acid (GA). In a diffusion cell chamber, the release rate of EGCG was 8896.01 µg cm-2. This data showed that EGCG will be able to exert its systemic activity when delivered though the transdermal formulation, due to its good flux rates with the synthetic membrane.

Efficacy, safety, quality control, marketing and regulatory guidelines for herbal medicines (phytotherapeutic agents)

This review highlights the current advances in knowledge about the safety, efficacy, quality control, marketing and regulatory aspects of botanical medicines. Phytotherapeutic agents are standardized herbal preparations consisting of complex mixtures of one or more plants which contain as active ingredients plant parts or plant material in the crude or processed state. A marked growth in the worldwide phytotherapeutic market has occurred over the last 15 years. For the European and USA markets alone, this will reach about $7 billion and $5 billion per annum, respectively, in 1999, and has thus attracted the interest of most large pharmaceutical companies. Insufficient data exist for most plants to guarantee their quality, efficacy and safety. The idea that herbal drugs are safe and free from side effects is false. Plants contain hundreds of constituents and some of them are very toxic, such as the most cytotoxic anti-cancer plant-derived drugs, digitalis and the pyrrolizidine alkaloids, etc. However, the adverse effects of phytotherapeutic agents are less frequent compared with synthetic drugs, but well-controlled clinical trials have now confirmed that such effects really exist. Several regulatory models for herbal medicines are currently available including prescription drugs, over-the-counter substances, traditional medicines and dietary supplements. Harmonization and improvement in the processes of regulation is needed, and the general tendency is to perpetuate the German Commission E experience, which combines scientific studies and traditional knowledge (monographs). Finally, the trend in the domestication, production and biotechnological studies and genetic improvement of medicinal plants, instead of the use of plants harvested in the wild, will offer great advantages, since it will be possible to obtain uniform and high quality raw materials which are fundamental to the efficacy and safety of herbal drugs.