Roles of Virtual Screening and Molecular Dynamics Simulations in Discovering and Understanding Antimalarial Drugs.

Malaria continues to be a global health threat, with approximately 247 million cases worldwide. Despite therapeutic interventions being available, patient compliance is a problem due to the length of treatment. Moreover, drug-resistant strains have emerged over the years, necessitating urgent identification of novel and more potent treatments. Given that traditional drug discovery often requires a great deal of time and resources, most drug discovery efforts now use computational methods. In silico techniques such as quantitative structure-activity relationship (QSAR), docking, and molecular dynamics (MD) can be used to study protein-ligand interactions and determine the potency and safety profile of a set of candidate compounds to help prioritize those tested using assays and animal models. This paper provides an overview of antimalarial drug discovery and the application of computational methods in identifying candidate inhibitors and elucidating their potential mechanisms of action. We conclude with the continued challenges and future perspectives in the field of antimalarial drug discovery.

Antimicrobial Peptides and Copper(II) Ions: Novel Therapeutic Opportunities.

The emergence of new pathogens and multidrug resistant bacteria is an important public health issue that requires the development of novel classes of antibiotics. Antimicrobial peptides (AMPs) are a promising platform with great potential for the identification of new lead compounds that can combat the aforementioned pathogens due to their broad-spectrum antimicrobial activity and relatively low rate of resistance emergence. AMPs of multicellular organisms made their debut four decades ago thanks to ingenious researchers who asked simple questions about the resistance to bacterial infections of insects. Questions such as "Do fruit flies ever get sick?", combined with pioneering studies, have led to an understanding of AMPs as universal weapons of the immune system. This review focuses on a subclass of AMPs that feature a metal binding motif known as the amino terminal copper and nickel (ATCUN) motif. One of the metal-based strategies of hosts facing a pathogen, it includes wielding the inherent toxicity of copper and deliberately trafficking this metal ion into sites of infection. The sudden increase in the concentration of copper ions in the presence of ATCUN-containing AMPs (ATCUN-AMPs) likely results in a synergistic interaction. Herein, we examine common structural features in ATCUN-AMPs that exist across species, and we highlight unique features that deserve additional attention. We also present the current state of knowledge about the molecular mechanisms behind their antimicrobial activity and the methods available to study this promising class of AMPs.

Antimicrobial Susceptibility Testing of Antimicrobial Peptides to Better Predict Efficacy.

During the development of antimicrobial peptides (AMP) as potential therapeutics, antimicrobial susceptibility testing (AST) stands as an essential part of the process in identification and optimisation of candidate AMP. Standard methods for AST, developed almost 60 years ago for testing conventional antibiotics, are not necessarily fit for purpose when it comes to determining the susceptibility of microorganisms to AMP. Without careful consideration of the parameters comprising AST there is a risk of failing to identify novel antimicrobials at a time when antimicrobial resistance (AMR) is leading the planet toward a post-antibiotic era. More physiologically/clinically relevant AST will allow better determination of the preclinical activity of drug candidates and allow the identification of lead compounds. An important consideration is the efficacy of AMP in biological matrices replicating sites of infection, e.g., blood/plasma/serum, lung bronchiolar lavage fluid/sputum, urine, biofilms, etc., as this will likely be more predictive of clinical efficacy. Additionally, specific AST for different target microorganisms may help to better predict efficacy of AMP in specific infections. In this manuscript, we describe what we believe are the key considerations for AST of AMP and hope that this information can better guide the preclinical development of AMP toward becoming a new generation of urgently needed antimicrobials.

A Potent Host Defense Peptide Triggers DNA Damage and Is Active against Multidrug-Resistant Gram-Negative Pathogens.

Gram-negative bacteria are some of the biggest threats to public health due to a large prevalence of antibiotic resistance. The difficulty in treating bacterial infections, stemming from their double membrane structure combined with efflux pumps in the outer membrane, has resulted in a much greater need for antimicrobials with activity against these pathogens. Tunicate host defense peptide (HDP), Clavanin A, is capable of not only inhibiting Gram-negative growth but also potentiating activity in the presence of Zn(II). Here, we provide evidence that the improvements of Clavanin A activity in the presence of Zn(II) are due to its novel mechanism of action. We employed E. coli TD172 (ΔrecA::kan) and the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay to show in cellulae that DNA damage occurs upon treatment with Clavanin A. In vitro assays demonstrated that Zn(II) ions are required for the nuclease activity of the peptide. The quantum mechanics/molecular mechanics (QM/MM) calculations were used to investigate the mechanism of DNA damage. In the rate-determining step of the proposed mechanism, due to its Lewis acidity, the Zn(II) ion activates the scissile P-O bond of DNA and creates a hydroxyl nucleophile from a water molecule. A subsequent attack by this group to the electrophilic phosphorus cleaves the scissile phosphoester bond. Additionally, we utilized bacterial cytological profiling (BCP), circular dichroism (CD) spectroscopy in the presence of lipid vesicles, and surface plasmon resonance combined with electrical impedance spectroscopy in order to address the apparent discrepancies between our results and the previous studies regarding the mechanism of action of Clavanin A. Finally, our approach may lead to the identification of additional Clavanin A like HDPs and promote the development of antimicrobial peptide based therapeutics.

Functional and structural basis of E. coli enolase inhibition by SF2312: a mimic of the carbanion intermediate.

Many years ago, the natural secondary metabolite SF2312, produced by the actinomycete Micromonospora, was reported to display broad spectrum antibacterial properties against both Gram-positive and Gram-negative bacteria. Recent studies have revealed that SF2312, a natural phosphonic acid, functions as a potent inhibitor of human enolase. The mechanism of SF2312 inhibition of bacterial enolase and its role in bacterial growth and reproduction, however, have remained elusive. In this work, we detail a structural analysis of E. coli enolase bound to both SF2312 and its oxidized imide-form. Our studies support a model in which SF2312 acts as an analog of a high energy intermediate formed during the catalytic process. Biochemical, biophysical, computational and kinetic characterization of these compounds confirm that altering features characteristic of a putative carbanion (enolate) intermediate significantly reduces the potency of enzyme inhibition. When SF2312 is combined with fosfomycin in the presence of glucose-6 phosphate, significant synergy is observed. This suggests the two agents could be used as a potent combination, targeting distinct cellular mechanism for the treatment of bacterial infections. Together, our studies rationalize the structure-activity relationships for these phosphonates and validate enolase as a promising target for antibiotic discovery.

Molecular Dynamics Investigation into the Effect of Zinc(II) on the Structure and Membrane Interactions of the Antimicrobial Peptide Clavanin A.

Clavanin A (ClavA) is an antimicrobial peptide (AMP) whose antimicrobial activity is enhanced in the presence of Zn(II) ions. The antimicrobial activity of ClavA has been shown to increase 16-fold in the presence of Zn(II) ions. In this study, we investigate the potential sources of this enhancement, namely, the effect of Zn(II) binding on the helical conformation of ClavA and on the ClavA interaction with a model for gram-negative bacterial membranes. In addition, we investigate the effect of Zn(II) on the membrane mechanical properties. We employed all-atom equilibrium molecular dynamics simulations initiated from both fully helical and random coil structures of ClavA. We observe that Zn(II) can stabilize an existing helical conformation in the Zn(II)-binding region, but we do not observe induction of helical conformations in systems initiated in random coil configurations. Zn(II) binding to ClavA provides more favorable electrostatics for membrane association in the C-terminal region. This is evidenced by longer and stronger C-terminal-lipid interactions. Zn(II) is also capable of modulating the membrane properties in a manner which favors ClavA insertion and the potential for enhanced translocation into the cell. This work provides insights into the role of divalent metal cations in the antimicrobial activity of ClavA. This information can be used for the development of synthetic AMPs containing motifs that can bind metals (metalloAMPs) for therapeutic and medical purposes.

Single Chain Polymeric Nanoparticles to Promote Selective Hydroxylation Reactions of Phenol Catalyzed by Copper.

Metal-containing single chain polymeric nanoparticles (SCPNs) can be used as synthetic mimics of metalloenzymes. Currently, the role of the folded polymer backbones on the activity and selectivity of metal sites is not clear. Herein, we report our findings on how polymeric frameworks modulate the coordination of Cu sites and the catalytic activity/selectivity of Cu-containing SCPNs mimicking monophenol hydroxylation reactions. Imidazole-functionalized copolymers of poly(methyl methacrylate-co-3-imidazolyl-2-hydroxy propyl methacrylate) were used for intramolecular Cu-imidazole binding that triggered the self-folding of polymers. Polymer chains imposed steric hindrance which yielded unsaturated Cu sites with an average coordination number of 3.3. Cu-containing SCPNs showed a high selectivity for the hydroxylation reaction of phenol to catechol, >80%, with a turnover frequency of >870 h-1 at 60 °C. The selectivity was largely influenced by the flexibility of the folded polymer backbone where a more flexible polymer backbone allows the cooperative catalysis of two Cu sites. The second coordination sphere provided by the folded polymer that has been less studied is therefore critical in the design of active mimics of metalloenzymes.

The synthesis and characterisation of strontium and calcium folates with potential osteogenic activity.

Compounds having the general formula MFO·4H2O where M = Ca or Sr and FO = a folate anion were prepared and their structure and physico-chemical properties were determined by elemental and thermal TGA, DSC analysis, FTIR, and EDAX spectroscopies and DRX. The results indicate that the two compounds form stable structures where folic acid acts as a self-bridging ligand via two bidentate carboxylate groups. Moreover the two compounds showed a low toxicity in vitro response as h-osteoblast cell viability was not negatively affected by the presence of folate derivatives within the range of 0.063-0.5 mg ml-1. The results also indicate that the folate derivatives that are formed overcome the toxic effects related to free Sr2+ ions. The range of maximum cell viability corresponding with a concentration of SrFO falls within the in vitro physiologically active range for strontium while within the same range the strontium derivative showed a potential osteogenic activity as indicated by the overexpression of ALP activity.

Corrección de cicatrices postraqueostomía mediante doble colgajo adipofascial opuesto / Double opposite adipofascial flap for the correction of post-tracheostomy scars

La traqueostomía es un procedimiento frecuentemente para asegurar una vía aérea permeable en casos de intubación prolongada, trauma facial severo y neoplasias de cabeza y cuello. Tras la extubación, el defecto se deja cerrar por segunda intención con el resultado de una cicatriz defectuosa, de pobre resultado estético y con frecuentes alteraciones funcionales asociadas, como por ejemplo disfagia. Se han descrito muchas técnicas para la corrección de estas cicatrices, sin embargo, algunas no corrigen los defectos funcionales y otras requieren una gran disección y movilización de los tejidos. Proponemos una técnica basada en dos colgajos adipofasciales de base opuesta y dependientes de circulación random realizada en 7 pacientes con cicatriz defectuosa postraqueostomía con buenos resultados estéticos y funcionales (AU)

Tracheostomy is a frequently used procedure to secure a viable airway in cases such as prolonged intubation, facial trauma and head and neck cancer. After decannulation, the defect is left to heal by secondary intention resulting in a poor aesthetic and functional outcome. Many techniques have been described to correct this defects, however they don't correct functional defect and some of them require large and complex dissections and manipulation of subdermal tissues. In order to correct these defects we present a technique using a doble opposite adipofascial flap based on random irrigation. This technique has been used in 7 patients with good aesthetical and functional results (AU)

Clinical trial: endoscopic evaluation of naproxen etemesil, a naproxen prodrug, vs. naproxen - a proof-of-concept, randomized, double-blind, active-comparator study.

BACKGROUND: Nonsteroidal anti-inflammatory drugs are associated with upper gastrointestinal mucosal injury. Naproxen etemesil is a lipophilic, non-acidic, inactive prodrug of naproxen that is hydrolysed to pharmacologically active naproxen once absorbed. We hypothesized that with lesser topical exposure to naproxen from the prodrug, there would be reduced gastroduodenal mucosal injury compared with naproxen. AIM: To compare the degree of endoscopic mucosal damage of naproxen etemesil vs. naproxen. METHODS: This multicentre, randomized, double-blind, double-dummy trial compared oral naproxen etemesil 1200 mg twice daily (n = 61) with naproxen 500 mg twice daily (n = 59) for 7.5 days in 120 healthy subjects (45-70 years; mean 51 years; 58% female) with baseline total modified gastroduodenal Lanza score ≤ 2 (no erosions/ulcers) on endoscopy. The primary endpoint was mean total modified gastroduodenal Lanza score on day 7. A secondary endpoint was incidence of gastric ulcers. RESULTS: The day 7 mean total modified gastroduodenal Lanza score was 2.8 ± 1.7 for naproxen etemesil vs. 3.5 ± 2.0 for naproxen (P = 0.03), and significantly fewer naproxen etemesil-treated subjects (3.3%) developed gastric ulcers compared with naproxen-treated subjects (15.8%) (P = 0.02). CONCLUSION: In this first proof-of-concept study, naproxen etemesil was associated with significantly lower gastroduodenal mucosal injury compared with naproxen after 7 days of exposure ( CLINICAL TRIAL NUMBER: NCT00750243).

Comparison of two frailty measures in the Conselice Study of Brain Ageing.

OBJECTIVES: Uncertainty about the definition of frailty is reflected by the development of many ways to identify frail people. We aimed to compare the validity of two frailty measures in participants of the Conselice Study of Brain Aging. DESIGN: Prospective population-based study with 4 year follow up. PARTICIPANTS/SETTING: 1,016 subjects aged 65 and over in a rural Italian population. METHODS: For each participant, a Frailty Index (FI) and a Conselice Study of Brain Aging Score (CSBAS) were determined. The FI was created from 43 deficits according to a standardized methodology; 7 variables derived from a previously validated Easy Prognostic Score comprised the CSBAS. RESULTS: The FI had characteristic properties described in other population samples, with a gamma distribution, a 99% limit of about 0.64 and higher values in women than men. CSBAS and FI were strongly correlated with each other (r = 0.72) and both correlated with age (r = 0.32, r = 0.27, respectively). Each was independently predictive of death in a multivariate model, with greater specificity and sensitivity than age alone. CONCLUSIONS: Frailty can be measured by different tools and facilitates a more direct quantification of individual vulnerability than chronological age alone. Though the Frailty Index and the Conselice Study of Brain Aging Score are underpinned by different rationales, clinical utility will continue to motivate their development.

Genome sequence, comparative analysis, and population genetics of the domestic horse.

We report a high-quality draft sequence of the genome of the horse (Equus caballus). The genome is relatively repetitive but has little segmental duplication. Chromosomes appear to have undergone few historical rearrangements: 53% of equine chromosomes show conserved synteny to a single human chromosome. Equine chromosome 11 is shown to have an evolutionary new centromere devoid of centromeric satellite DNA, suggesting that centromeric function may arise before satellite repeat accumulation. Linkage disequilibrium, showing the influences of early domestication of large herds of female horses, is intermediate in length between dog and human, and there is long-range haplotype sharing among breeds.

Effect of smoking on the accumulation of deficits, frailty and survival in older adults: a secondary analysis from the Canadian Study of Health and Aging.

OBJECTIVES: Smoking has adverse effects on a variety of organ systems but little is known about the relationship between smoking and frailty. We aimed to investigate differences in health status between smoking and non smoking older adults. DESIGN AND SETTING: The Canadian Study of Health and Aging, a nationally representative cohort study. PARTICIPANTS: Nine thousand and eight community-dwelling men and women age 65 years and over at baseline. MEASUREMENTS: Smoking status was determined using a Self-Assessed Risk Factor Questionnaire. Comparisons were made between never smokers, light smokers and heavy smokers with heavy smokers defined as those who smoked >or= 1 pack per day for 20 years or more. A frailty index (FI) generated from 40 self-reported health deficits was also modified to exclude 5 variables that could be directly attributed to smoking (e.g. cough). Decedent information was collected over 10 years. RESULTS: Average FI values increased exponentially with age. For both men and women, heavy smokers were the most frail, light smokers had intermediate frailty status and never smokers were fittest. Modification of the FI did not impact these differences. Heavy smokers had significantly worse mortality than non smokers and higher rates of death in smokers persisted in the oldest old. 120 month survival curves, grouped for age, sex and smoking status showed that male smokers > 75 years had the highest mortality rates. CONCLUSIONS: Smoking causes poorer health status at older ages which can be captured by the frailty index. Higher rates of death in smokers persist in the oldest old, with no emergence of "survivors" with fitness or longevity advantages.

Comparación del dolor secundario a lipoaspiración tradicional versus lipolisis láser. Estudio prospectivo / Postoperative pain in suction assited lipoplasty versus laser lipolisis. Comparative prospective study

La lipoaspiración, técnica tradicional usada para el tratamiento de las lipodistrofias con cicatrices mínimas, ha sido a través de los años mejorada con los avances anestésicos, farmacológicos y de instrumental. A su vez han aparecido otras técnicas que han publicitado ventajas respecto al tiempo de recuperación, sintomatología y resultados estéticos. Una de estas técnicas es la laserlipolisis a la que se atribuye menos dolor, menos equimosis y rápido postoperatorio. No se encuentran trabajos que acrediten estas ventajas, por lo que decidimos estudiar si existen ventajas de laserlipolisis en comparación con la lipoaspiración tradicional respecto del dolor, síntoma importante a considerar en el postoperatorio. Para esto se diseñó un trabajo prospectivo randomizado con una muestra de 60 pacientes a los cuales se aplicaron las dos técnicas en diferentes áreas y al mismo paciente. Se evaluó el dolor con escala numérica analógica a las 4 horas, 48 horas y 5 días de postoperatorio. Los resultados mostraron que la laserlipolisis presenta menos dolor en los muslos medido a las 48 horas y 5 días de la intervención. No hay diferencias entre estas dos técnicas en las otras zonas medidas como abdomen, áreas pretrocantéreas (“alforjas”), zona lumbar y pectoral. Considerando todos los pacientes sin diferenciar la técnica usada, las “alforjas” a las 4 horas duelen menos que el abdomen y a los 5 días duelen menos los muslos que el abdomen. No encontramos relación entre volumen aspirado e intensidad del dolor (AU)

The traditional suction-assisted lipoplasty, is a well established and commonly performed technique used for the treatment of lipodystrophias, which has undergone improvements with advances in anesthetics, pharmacology and introduction of new instruments. New techniques have appeared, promising advantages in recovery periods, symptomathology and aesthetic results when compared to the traditional suction- assisted lipoplasty. One of these newly introduced techniques is the laser-assisted lipoplasty (laserlipolysis), which claims less pain, less bruising and faster postoperative recovery. No evidence has been published to support these advantages when comparing laser-assisted lipoplasty to the traditional method, specifically when refering to pain, an important symptom to be taken into consideration in the postoperative period. For this reason we designed a prospective, randomized clinical study comprising 60 patients to which the two techniques were applied to different body areas. Pain was assessed using the numeric analog scale at 4 and 48 hours, and at 5 days after surgery. Evidence showed that laserlipolysis results in less pain of the thighs measured at 48 hours and 5 days after surgery. No difference in pain was found between both techniques on the other body areas evaluated (abdomen, trochanteric bulge, lumbar and pectoral areas). Assesing all patients, independent of the technique used, trochanteric bulge at 4 hours was found to be less painful than the abdominal area, and at 5 day evaluation the thighs were less painful than the abdominal area. No relationship was found between pain intensity and lipoaspirated volume

The DNA sequence and biological annotation of human chromosome 1.

The reference sequence for each human chromosome provides the framework for understanding genome function, variation and evolution. Here we report the finished sequence and biological annotation of human chromosome 1. Chromosome 1 is gene-dense, with 3,141 genes and 991 pseudogenes, and many coding sequences overlap. Rearrangements and mutations of chromosome 1 are prevalent in cancer and many other diseases. Patterns of sequence variation reveal signals of recent selection in specific genes that may contribute to human fitness, and also in regions where no function is evident. Fine-scale recombination occurs in hotspots of varying intensity along the sequence, and is enriched near genes. These and other studies of human biology and disease encoded within chromosome 1 are made possible with the highly accurate annotated sequence, as part of the completed set of chromosome sequences that comprise the reference human genome.

Ensembl 2006.

The Ensembl (http://www.ensembl.org/) project provides a comprehensive and integrated source of annotation of large genome sequences. Over the last year the number of genomes available from the Ensembl site has increased from 4 to 19, with the addition of the mammalian genomes of Rhesus macaque and Opossum, the chordate genome of Ciona intestinalis and the import and integration of the yeast genome. The year has also seen extensive improvements to both data analysis and presentation, with the introduction of a redesigned website, the addition of RNA gene and regulatory annotation and substantial improvements to the integration of human genome variation data.

Ensembl 2005.

The Ensembl (http://www.ensembl.org/) project provides a comprehensive and integrated source of annotation of large genome sequences. Over the last year the number of genomes available from the Ensembl site has increased by 7 to 16, with the addition of the six vertebrate genomes of chimpanzee, dog, cow, chicken, tetraodon and frog and the insect genome of honeybee. The majority have been annotated automatically using the Ensembl gene build system, showing its flexibility to reliably annotate a wide variety of genomes. With the increased number of vertebrate genomes, the comparative analysis provided to users has been greatly improved, with new website interfaces allowing annotation of different genomes to be directly compared. The Ensembl software system is being increasingly widely reused in different projects showing the benefits of a completely open approach to software development and distribution.