Investigating the mode of action of AN5568, a novel therapeutic against African trypanosomiasis

No abstract available.

Sonic hedgehog signaling regulates mode of cell division of early cerebral cortex progenitors and increases

The morphogen Sonic Hedgehog (SHH) plays a critical role in the development of different tissues. In the central nervous system, SHH is well known to contribute to the patterning of the spinal cord and separation of the brain hemispheres. In addition, it has recently been shown that SHH signaling also contributes to the patterning of the telencephalon and establishment of adult neurogenic niches. In this work, we investigated whether SHH signaling influences the behavior of neural progenitors isolated from the dorsal telencephalon, which generate excitatory neurons and macroglial cells in vitro. We observed that SHH increases proliferation of cortical progenitors and generation of astrocytes, whereas blocking SHH signaling with cyclopamine has opposite effects. In both cases, generation of neurons did not seem to be affected. However, cell survival was broadly affected by blockade of SHH signaling. SHH effects were related to three different cell phenomena: mode of cell division, cell cycle length and cell growth. Together, our data in vitro demonstrate that SHH signaling controls cell behaviors that are important for proliferation of cerebral cortex progenitors, as well as differentiation and survival of neurons and astroglial cells.

Biosynthesis and mode of action of ribosomally synthesized and post-translationally modified antimicrobial peptides

One of the greatest sources of biologically active compounds is natural products. Often these compounds serve as platforms for the design and development of novel drugs and therapeutics. The overwhelming amount of genomic information acquired in recent years has revealed that ribosomally synthesized and post-translationally modified natural products are much more widespread than originally anticipated. Identified in nearly all forms of life, these natural products display incredible structural diversity and possess a wide range of biological functions that include antimicrobial, antiviral, anti-inflammatory, antitumor, and antiallodynic activities. The unique pathways taken to biosynthesize these compounds offer exciting opportunities for the bioengineering of these complex molecules. The studies described herein focus on both the mode of action and biosynthesis of antimicrobial peptides. In Chapter 2, it is demonstrated that haloduracin, a recently discovered two-peptide lantibiotic, possesses nanomolar antimicrobial activity against a panel of bacteria strains. The potency of haloduracin rivals that of nisin, an economically and therapeutically relevant lantibiotic, which can be attributed to a similar dual mode of action. Moreover, it was demonstrated that this lantibiotic of alkaliphile origin has better stability at physiological pH than nisin. The molecular target of haloduracin was identified as the cell wall peptidoglycan precursor lipid II. Through the in vitro biosynthesis of haloduracin, several analogues of Halα were prepared and evaluated for their ability to inhibit peptidoglycan biosynthesis as well as bacterial cell growth. In an effort to overcome the limitations of in vitro biosynthesis strategies, a novel strategy was developed resulting in a constitutively active lantibiotic synthetase enzyme. This methodology, described in Chapter 3, enabled the production of fully-modified lacticin 481 products with proteinogenic and non-proteinogenic amino acid substitutions. A number of lacticin 481 analogues were prepared and their antimicrobial activity and ability to bind lipid II was assessed. Moreover, site-directed mutagenesis of the constitutively active synthetase resulted in a kinase-like enzyme with the ability to phosphorylate a number of peptide substrates. The hunt for a lantibiotic synthetase enzyme responsible for installing the presumed dehydro amino acids and a thioether ring in the natural product sublancin, led to the identification and characterization of a unique post-translational modification. The studies described in Chapter 4, demonstrate that sublancin is not a lantibiotic, but rather an unusual S-linked glycopeptide. Its structure was revised based on extensive chemical, biochemical, and spectroscopic characterization. In addition to structural investigation, bioinformatic analysis of the sublancin gene cluster led to the identification of an S-glycosyltransferase predicted to be responsible for the post-translational modification of the sublancin precursor peptide. The unprecedented glycosyltransferase was reconstituted in vitro and demonstrated remarkable substrate promiscuity for both the NDP-sugar co-substrate as well as the precursor peptide itself. An in vitro method was developed for the production of sublancin and analogues which were subsequently evaluated in bioactivity assays. Finally, a number of putative biosynthetic gene clusters were identified that appear to harbor the necessary genes for production of an S-glycopeptide. An additional S-glycosyltransferase with more favorable intrinsic properties including better expression, stability, and solubility was reconstituted in vitro and demonstrated robust catalytic abilities.

Candidacidal potential of licorice phenolic extract: emphasis on its mode of action

Medicinal plants bave gained a special attention in the last years, due to its renowned health benefits, such as antimicrobial effects [I]. In fact, several natural matrices bave been increasingly studied, namely for its antifungal activity against opportunistic fungi [2,3]. Candida species, although commensa! microorganisms, have caused severe organic dysfunctions to the host, once current antifungal agents have lost their recognized efficiency [2]. So, numerous studies have been carried out focusing the mechanisms of acquired drug-resistance by Candida species [of an efficient multi-drug therapy (i.e. chemical drugs and also natural extracts combination), the discovery of the involved mechanisms of actions, morphological changes and related kinetic parameters are of major importance. Glycyrrhiza glabra L. (licorice) hydromethanolic extract have evidenced promissory candidacidal effects, and therefore, the involved mechanisms of action need to be clarified. Thus, in the present study these modes of action were assessed, by using flow cytometry.Overall, the licorice extract induced significant and irreversible primary damages on Candida cells, being membrane disruption and consequent unviability one of the main targets. In fact, after membrane destabilization, cells lost their proper homeostasis, their metabolic functions were blocked and, consequently cells lost functionality. The relevance and interest of the achieved results open new insights towards the upcorning use of the present phenolic matrix, being important to evaluate its in viva efficacy. Therefore, further studies are necessary to deepen knowledge on this field, aiming not only to establish therapeutic and prophylactic doses, but also to improve the clinical intervention in Candida infections.

Marine Actinomycetes as Source of Antimicrobial Compounds and as Probiotics and Single Cell Protein for Application in Penaeid Prawn Culture Systems

This thesis Entitled Marine actinomycetes as source of antimicrobial compounds and as probiotics and single cell protein for application in penaeid peawn culture systems. Ocean harbours more than 80% of all life on earth and remains our greatest untapped natural resource. The study revealed the potential of marine actinomycetes as a source of antimicrobial compounds. The selected streptomycetes were found to be capable of inhibiting most of the pathogenic vibrios, whichis a major problem both in hatcheries and grow out systems. The bioactive principle can be incorporated with commercial feeds and applied as medicated diet for the control of vibrios in culture systems.The hydrolytic potential inhibitory property against pathogens and non—pathogenicity to penaeid prawns make the selected Streptomycesspp.an effective probioic in aquaculture. Since there is considerably less inhibition to the natural in pond ecosystem the microbial diversityis being maintained and thereby the water quality. Actinomycetes was found to be a good source of single cell protein as an ingredient inaquaculture feed formulations. Large amount of mycelial waste (actinomycete biomassO is produced from antibiotic industries and this nutrient rich waste can be effectively used as a protein source in aquaculture feeds.This study reveals the importance of marine actinomycetes as a source of antimicrobial compounds and as a probiotic and single cell protein for aquaculture applications.

Application of molecular biological methods for studying probiotics and the gut flora

Increasingly, the microbiological scientific community is relying on molecular biology to define the complexity of the gut flora and to distinguish one organism from the next. This is particularly pertinent in the field of probiotics, and probiotic therapy, where identifying probiotics from the commensal flora is often warranted. Current techniques, including genetic fingerprinting, gene sequencing, oligonucleotide probes and specific primer selection, discriminate closely related bacteria with varying degrees of success. Additional molecular methods being employed to determine the constituents of complex microbiota in this area of research are community analysis, denaturing gradient gel electrophoresis (DGGE)/temperature gradient gel electrophoresis (TGGE), fluorescent in situ hybridisation (FISH) and probe grids. Certain approaches enable specific aetiological agents to be monitored, whereas others allow the effects of dietary intervention on bacterial populations to be studied. Other approaches demonstrate diversity, but may not always enable quantification of the population. At the heart of current molecular methods is sequence information gathered from culturable organisms. However, the diversity and novelty identified when applying these methods to the gut microflora demonstrates how little is known about this ecosystem. Of greater concern is the inherent bias associated with some molecular methods. As we understand more of the complexity and dynamics of this diverse microbiota we will be in a position to develop more robust molecular-based technologies to examine it. In addition to identification of the microbiota and discrimination of probiotic strains from commensal organisms, the future of molecular biology in the field of probiotics and the gut flora will, no doubt, stretch to investigations of functionality and activity of the microflora, and/or specific fractions. The quest will be to demonstrate the roles of probiotic strains in vivo and not simply their presence or absence.

Chiral HPLC analysis of venlafaxine metabolites in rat liver microsomal preparations after LPME extraction and application to an in vitro biotransformation study

A three-phase LPME (liquid-phase microextraction) method for the enantioselective analysis of venlafaxine (VF) metabolites (O-desmethylvenlafaxine (ODV) and N-desmethylvenlafaxine (NDV) in microsomal preparations is described for the first time. The assay involves the chiral HPLC separation of drug and metabolites using a Chiralpak AD column under normal-phase mode of elution and detection at 230 nm. The LPME procedure was optimized using multifactorial experiments and the following optimal condition was established: sample agitation at 1,750 rpm, 20 min of extraction, acetic acid 0.1 mol/L as acceptor phase, 1-octanol as organic phase and donor phase pH adjustment to 10.0. Under these conditions, the mean recoveries were 41% and 42% for (-)-(R)-ODV and (+)-(S)-ODV, respectively, and 47% and 48% for (-)-( R)-NDV and (+)-( S)-NDV, respectively. The method presented quantification limits of 200 ng/mL and it was linear over the concentration range of 200-5,000 ng/mL for all analytes. The validated method was employed to study the in vitro biotransformation of VF using rat liver microsomal fraction. The results demonstrated the enantioselective biotransformation of VF.

Can the durability of one-step self-etch adhesives be improved by double application or by an extra layer of hydrophobic resin?

Objectives: This study evaluated the immediate and 6-month resin-dentin mu-bond strength (mu TBS) of one-step self-etch systems (Adper Prompt L-Pop [AD] 3M ESPE; Xeno III [XE] Dentsply De Trey; iBond [iB] Heraeus Kulzer) under different application modes. Materials and methods: Dentin oclusal surfaces were exposed by grinding with 600-grit SiC paper. The adhesives were applied according to the manufacturer`s directions [MD], or with double application of the adhesive layer [DA] or following the manufacturer`s directions plus a hydrophobic resin layer coating [HL]. After applying the adhesive resins, composite crowns were built up incrementally. After 24-h water storage, the specimens were serially sectioned in ""x"" and ""y"" directions to obtain bonded sticks of about 0.8 mm 2 to be tested immediately [IM] or after 6 months of water storage [6M] at a crosshead speed of 0.5 mm/min. The data from each adhesive was analyzed by a two-way repeated measures ANOVA (mode of application vs. storage time) and Tukey`s test (alpha = 0.05). Results: The adhesives performed differently according to the application mode. The DA and HL either improved the immediate performance of the adhesive or did not differ from the MD. The resin-dentin bond strength values observed after 6 months were higher when a hydrophobic resin coat was used than compared to those values observed under the manufacturer`s directions. Conclusions: The double application of one-step self-etch system can be safety performed however the application of an additional hydrophobic resin layer can improve the immediate resin-dentin bonds and reduce the degradation of resin bonds over time. (c) 2008 Elsevier Ltd. All rights reserved.

Potentiation of polarized intestinal Caco-2 cell responsiveness to probiotics complexed with secretory IgA.

The precise mechanisms underlying the interaction between intestinal bacteria and the host epithelium lead to multiple consequences that remain poorly understood at the molecular level. Deciphering such events can provide valuable information as to the mode of action of commensal and probiotic microorganisms in the gastrointestinal environment. Potential roles of such microorganisms along the privileged target represented by the mucosal immune system include maturation prior, during and after weaning, and the reduction of inflammatory reactions in pathogenic conditions. Using human intestinal epithelial Caco-2 cell grown as polarized monolayers, we found that association of a Lactobacillus or a Bifidobacterium with nonspecific secretory IgA (SIgA) enhanced probiotic adhesion by a factor of 3.4-fold or more. Bacteria alone or in complex with SIgA reinforced transepithelial electrical resistance, a phenomenon coupled with increased phosphorylation of tight junction proteins zonula occludens-1 and occludin. In contrast, association with SIgA resulted in both enhanced level of nuclear translocation of NF-κB and production of epithelial polymeric Ig receptor as compared with bacteria alone. Moreover, thymic stromal lymphopoietin production was increased upon exposure to bacteria and further enhanced with SIgA-based complexes, whereas the level of pro-inflammatory epithelial cell mediators remained unaffected. Interestingly, SIgA-mediated potentiation of the Caco-2 cell responsiveness to the two probiotics tested involved Fab-independent interaction with the bacteria. These findings add to the multiple functions of SIgA and underscore a novel role of the antibody in interaction with intestinal bacteria.

General unknown screening procedure for the characterization of human drug metabolites: application to loratadine phase I metabolism.

This article describes the application of a recently developed general unknown screening (GUS) strategy based on LC coupled to a hybrid linear IT-triple quadrupole mass spectrometer (LC-MS/MS-LIT) for the simultaneous detection and identification of drug metabolites following in vitro incubation with human liver microsomes. The histamine H1 receptor antagonist loratadine was chosen as a model compound to demonstrate the interest of such approach, because of its previously described complex and extensive metabolism. Detection and mass spectral characterization were based on data-dependent acquisition, switching between a survey scan acquired in the ion-trapping Q3 scan mode with dynamic subtraction of background noise, and a dependent scan in the ion-trapping product ion scan mode of automatically selected parent ions. In addition, the MS(3) mode was used in a second step to confirm the structure of a few fragment ions. The sensitivity of the ion-trapping modes combined with the selectivity of the triple quadrupole modes allowed, with only one injection, the detection and identification of 17 phase I metabolites of loratadine. The GUS procedure used in this study may be applicable as a generic technique for the characterization of drug metabolites after in vitro incubation, as well as probably in vivo experiments.

Application of electrokinetic Fenton process for the remediation of soil contaminated with HCB

Electrokinetic remediation coupled with Fenton oxidation, widely called as Electrokinetic Fenton process is a potential soil remediation technique used for low permeable soil. The applicability of the process has been proved with soil contaminated with a wide range of organic compounds from phenol to the most recalcitrant ones such as PAHs and POPs. This thesis summarizes the major findings observed during an Electrokinetic Fenton Process study conducted for the remediation of low permeable soil contaminated with HCB, a typical hydrophobic organic contaminant. Model low permeable soil, kaolin, was artificially contaminated with HCB and subjected to Electrokinetic Fenton treatments in a series of laboratory scale batch experiments. The use of cyclodextrins as an enhancement agent to mobilize the sorbed contaminant through the system was investigated. Major process hindrances such as the oxidant availability and treatment duration were also addressed. The HCB degradation along with other parameters like soil pH, redox and cumulative catholyte flow were analyzed and monitored. The results of the experiments strengthen the existing knowledge on electrokinetic Fenton process as a promising technology for the treatment of soil contaminated with hydrophobic organic compounds. It has been demonstrated that HCB sorbed to kaolin can be degraded by the use of high concentrations of hydrogen peroxide during such processes. The overall system performances were observed to be influenced by the point and mode of oxidant delivery. Furthermore, the study contributes to new knowledge in shortening the treatment duration by adopting an electrode polarity reversal during the process.

"Weathering a hidden storm" : an application of Andersen's behavioral model of health, and health services use for those with diagnosable anxiety disorder

"Weathering a Hidden Storm": An App~ication of Andersen's Behaviora~ Mode~ of Hea~th, and Hea~th Services Use for Those With Diagnosab~e Anxiety Disorder Research has primarily focused on depression and mood disorders, but little research has been devoted to an examination of mental health services use amongst those with diagnosable anxiety disorder (Wittchen et al., 2002; Bergeron et al., 2005). This study examined the possible predicting factors for mental health services utilization amongst those with identifiable anxiety disorder in the Canadian population. The methods used for this study was the application of Andersen's Behavioral Model of Health Services Use, where predisposing, need and enabling 111 characteristics were regressed on the dependent variable of mental health services use. This study used the Canadian Community Health Survey (cycle 1.2: Mental Health and Well- Being) in a secondary data analysis. Several multiple logistics models predicted the likelihood to seek and use mental health services. Predisposing characteristics of gender and age, Enabling characteristics of education and geographical location, and those with co-occurring mood disorders were at the greatest increased likelihood to seek and use mental health services.

Isolation, purification, characterization and application of proteinaceous protease inhibitor from marine bacterium Pseudomonas mendocina BTMW 301

The microorganisms are recognized as important sources of protease inhibitors which are valuable in the fields of medicine, agriculture and biotechnology. The protease inhibitors of microbial origin are found to be versatile in their structure and mode of inhibition that vary from those of other sources. Although surplus of low molecular weight non-protein protease inhibitors from microorganisms have been reported, there is a dearth of reports on proteinaceous protease inhibitors. The search for new metabolites from marine organisms has resulted in the isolation of more or less 10,000 metabolites (Fuesetani and Fuesetani, 2000) many of which are gifted with pharmacodynamic properties. The existence of marine microorganisms was reported earlier, and they were found to be metabolically and physiologically dissimilar from terrestrial microorganisms. Marine microorganisms have potential as important new sources of enzyme inhibitors and consequently a detailed study of new marine microbial inhibitors will provide the basis for future research (Imada, 2004).

Penaeus monodon larvae can be protected from Vibrio harveyi infection by pre-emptive treatment of a rearing system with antagonistic or non-antagonistic bacterial probiotics

This study shows that the disease resistance and survival rate of Penaeus monodon in a larval rearing systems can be enhanced by supplementing with antagonistic or non-antagonistic probiotics. The antagonistic mode of action of Pseudomonas MCCB 102 and MCCB 103 against vibrios was demonstrated in larval mesocosm with cultures having su⁄cient concentration of antagonistic compounds in their culture supernatant. Investigations on the antagonistic properties of Bacillus MCCB 101, Pseudomonas MCCB 102 and MCCB 103 and Arthrobacter MCCB 104 against Vibrio harveyi MCCB111under in vitro conditions revealed that Pseudomonas MCCB 102 and MCCB 103 were inhibitory to the pathogen.These inhibitory propertieswere further con¢rmed in the larval rearing systems of P. monodon. All these four probionts signi¢cantly improved larval survival in long-term treatments as well as when challengedwith a pathogenic strain ofV. harveyiMCCB111. We could demonstrate that Pseudomonas MCCB 102 andMCCB103 accorded disease resistance and a higher survival rate in P. monodon larval rearing systems throughactive antagonism of vibrios,whereas Bacillus MCCB 101 and Arthrobacter MCCB 104 functioned as probiotics through immunostimulatory and digestive enzyme-supporting modes of action.

Modulation of the microbial ecology of the human colon by probiotics, prebiotics and synbiotics to enhance human health: an overview of enabling science and potential applications

The application of probiotics and prebiotics to the manipulation of the microbial ecology of the human colon has recently seen many scientific advances. The sequencing of probiotic genomes is providing a wealth of new information on the biology of these microorganisms. In addition, we are learning more about the interactions of probiotics with human cells and with pathogenic bacteria. An alternative means of modulating the colonic microbial community is by the use of prebiotic oligosaccharides. Increasing knowledge of the metabolism of prebiotics by probiotics is allowing us to consider specifically targeting such dietary intervention tools at specific populatiori groups and specific disease states. (c) 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.