Epigenetic studies in insects and the valproic acid perspective.

Valproic acid in association with sodium valproate (VPA) is an important anticonvulsant drug used for decades to treat neurological disorders. VPA also acts as an epigenetic modulator by inhibiting histone deacetylases, permitting histone acetylation, affecting the DNA and histone methylation status and gene expression, and inducing chromatin remodeling. Insects represent an important animal model for studies in several areas of science. Their high phenotypic plasticity makes them alternative models for epigenetic studies. This brief review emphasizes recent reports on insect epigenetics and the contribution of studies on the VPA action in insects, including effects on epigenetic markers, extending the pharmacological understanding of the potential of this drug, and demonstrating the usefulness of insects as an alternative animal model to drug studies.

Validation of Mycobacterium tuberculosis dihydroneopterin aldolase as a molecular target for anti-tuberculosis drug development.

An early step of target validation in antimicrobial drug discovery is to prove that a gene coding for a putative target is essential for pathogen's viability. However, little attention has been paid to demonstrate the causal links between gene essentiality and a particular protein function that will be the focus of a drug discovery effort. This should be considered an important step in target validation since a growing number of proteins are found to exhibit multiple and unrelated tasks. Here, we show that the Mycobacterium tuberculosis (Mtb) folB gene is essential and that this essentiality depends on the dihydroneopterin aldolase/epimerase activities of its protein product, the FolB protein from the folate biosynthesis pathway. The wild-type (WT) MtFolB and point mutants K99A and Y54F were cloned, expressed, purified and monitored for the aldolase, epimerase and oxygenase activities using HPLC. In contrast to the WT MtFolB, both mutants have neither aldolase nor epimerase activities in the conditions assayed. We then performed gene knockout experiments and showed that folB gene is essential for Mtb survival under the conditions tested. Moreover, only the WT folB sequence could be used as a rescue copy in gene complementation studies. When the sequences of mutants K99A or Y54F were used for complementation, no viable colonies were obtained, indicating that aldolase and/or epimerase activities are crucial for Mtb survival. These results provide a solid basis for further work aiming to develop new anti-TB agents acting as inhibitors of the aldolase/epimerase activities of MtFolB.

Efficacy of oral administration of lactic acid bacteria isolated from cocoa in a fermented milk preparation: reduction of colitis in an experimental rat model.

We investigated the probiotic potential of lactic acid bacteria (LAB) obtained from cocoa fermentation using an experimental rat model of colitis. Cocoa beans were collected from fermentation boxes every 12 h for 5 days to isolate the microorganisms. Strains were isolated by serial dilution and plating on MRS agar. Gram-positive and catalase-negative rods were subjected to DNA extraction, polymerase chain reaction, and sequencing. Ten strains were randomly pooled and used to prepare a fermented milk drink that was used to treat the experimental colitis. A parallel group was treated with a single strain drink. Serum concentrations of cytokines and IgA, total and differential counts of blood leukocytes, and histological appearance were compared with the untreated control colitis group. Eighty strains of LAB were identified as Lactobacillus fermentum (68) and Lactobacillus plantarum (12). The multi-strain LAB pool significantly reduced the total number of leukocytes. There was a significant reduction in the percentage of neutrophils and monocytes compared with the control colitis group. IFN-γ concentration was downregulated in animals treated with the LAB pool. IL-10 and IgA increased significantly in the group treated with the strains. Histological analysis showed that the LAB pool reduced the inflammatory infiltrate and restored tissue architecture. The group treated with the single strain LAB drink (L. fermentum) showed no signs of inflammation remission. The results confirm the probiotic action of cocoa-derived LAB in the treatment of experimental colitis. Studies using isogenic models and humans will clarify the mechanisms of immune response modulation in inflammatory bowel disease.

Performance of Eriopis connexa (Coleoptera: Coccinellidae) resistant to lambda-cyhalothrin after extended recovery from knockdown.

A population of the predatory lady beetle Eriopis connexa (Germar) (Coleoptera: Coccinellidae) was recorded as resistant to lambda-cyhalothrin. Adults exposed to this insecticide have recovered from knockdown after 72 h. Thus, the performance of resistant (R) and susceptible (S) populations of E. connexa not exposed to insecticide (R0 and S0) and R adults recovering from knockdown 24, 48, and 72 h after exposure (R24, R48, and R72) was studied. In addition, the fertility life table parameters were calculated for one generation considering the progenies from R0, S0, and R24 populations. The recovery rate from knockdown was 69.4% for R-adults, and greater recovery rate was observed within 48 h following lambda-cyhalothrin exposure. The S-females produced about 50% more eggs and lived longer, when compared with R-females irrespective of the recovery periods after knockdown. The R-females produced similar number of eggs and exhibited similar longevity across all treatments (R0, R24, R48, and R72). Progenies produced by R- and S-populations did not exhibit consistent differences in development and survival. The fertility life table parameters showed higher intrinsic rate of population growth (rm) and lower mean generation time (T) for R0- and R24-females, when compared with those for S0-females. Thus, the time interval needed to recover from knockdown is not related to the adaptive cost of resistance in E. connexa.

Lambda-Cyhalothrin Resistance in the Lady Beetle Eriopis connexa (Coleoptera: Coccinellidae) Confers Tolerance to Other Pyrethroids.

Pyrethroid insecticides are widely recommended to control insect defoliators but lack efficacy against most aphid species. Thus, conserving aphid predators such as the lady beetle Eriopis connexa (Germar) is important to pest management in crop ecosystems that require pyrethroid sprays. In a greenhouse, early fourth-instar larvae and 5-day-old adults from susceptible (S) and resistant (R) E. connexa populations were caged on lambda-cyhalothrin-treated cotton plants, after which survival and egg production (for those caged at adult stage) were assessed. In the laboratory, similar groups were subjected to dried residues and topical treatment with one of eight pyrethroids (alpha-cypermethrin, bifenthrin, deltamethrin, esfenvalerate, fenpropathrin, permethrin, zeta-cypermethrin, and lambda-cyhalothrin), the organophosphate methidathion, or water and wetting agent. After caging on treated cotton terminals, 66% of the R-population larvae survived to adulthood, compared with 2% of those from the S-population. At 12 d after caging at adult stage under the same conditions, 64% of the females from the R-population survived and laid eggs, compared with 100% mortality and no oviposition for the S-females. In trials involving dried insecticide residues, gain in survival based on the survival difference (percentage for R-population minus percentage for S-population) across all tested pyrethroids varied from 3 to 63% for larvae and from 3 to 70% for adults. In trials involving topical sprays of the tested pyrethroids, survival differences ranged from 36 to 96% for larvae and from 21 to 82% for adults. Fenpropathrin and bifenthrin were the least and most toxic, respectively.

Conformação de úbere de caprinos da raça saanen: parâmetros estéticos ou funcionais? / Udder conformation of saanen goats: aesthetic or functional parameters?

Diversos fatores predisponentes são descritos para as afecções mamárias ou distúrbios secundários que comprometem a qualidade e produtividade de leite de fêmeas nas diferentes espécies. As características fenotípicas do úbere são consideradas na avaliação econômica de uma fêmea caprina, tanto pelo potencial de produção como pelo registro genealógico dessa fêmea. A limitação de estudos correlacionando essas características com a saúde do úbere gera a dúvida a respeito do significado da conformação do mesmo à saúde e produtividade da glândula mamária. Sendo assim, este estudo teve como objetivo relacionar os parâmetros da conformação do úbere com a celularidade da glândula mamária aferida pelo teste California Mastitis Test (CMT) e contagem de células somáticas (CCS) em 80 cabras da raça Saanen sem alterações no exame clínico da glândula mamária nem no teste de Tamis. Observou-se que a maioria dos parâmetros fenotípicos de úbere não influenciou a CCS, sendo considerados puramente estéticos. A circunferência e profundidade de úbere demonstraram correlação negativa com a celularidade e, por serem características de herdabilidade moderada a alta, podem ser parâmetros considerados para seleção genética de caprinos.

Several predisposing factors are described for mammary diseases or secondary disorders that compromise the quality and productivity of milk from females in different species. The phenotypic characteristics of the udder are considered in the economic evaluation of a female goat, whether for production potential, or as the genealogical record for these females. The limitation of studies correlating these features with the udder health raises doubt about the meaning of the conformation to the health and productivity of the mammary gland. Therefore, this study aimed to list the parameters of the udder conformation with the cellularity of the mammary gland checked by California Mastitis Test (CMT) and somatic cell count (SCC) in 80 Saanen goats without alterations in the clinical examination of the mammary gland or the Tamis test. It was observed that most of the phenotypic parameters of the udder did not influence the CCS, being considered purely esthetic. The circumference and udder depth showed negative correlation with the cellularity and moderate to high heritability traits can be considered parameters for genetic selection of goats.

A low cost, safe, disposable, rapid and self-sustainable paper-based platform for diagnostic testing: lab-on-paper.

There is a strong interest in the use of biopolymers in the electronic and biomedical industries, mainly towards low-cost applications. The possibility of developing entirely new kinds of products based on cellulose is of current interest, in order to enhance and to add new functionalities to conventional paper-based products. We present our results towards the development of paper-based microfluidics for molecular diagnostic testing. Paper properties were evaluated and compared to nitrocellulose, the most commonly used material in lateral flow and other rapid tests. Focusing on the use of paper as a substrate for microfluidic applications, through an eco-friendly wax-printing technology, we present three main and distinct colorimetric approaches: (i) enzymatic reactions (glucose detection); (ii) immunoassays (antibodies anti-Leishmania detection); (iii) nucleic acid sequence identification (Mycobacterium tuberculosis complex detection). Colorimetric glucose quantification was achieved through enzymatic reactions performed within specific zones of the paper-based device. The colouration achieved increased with growing glucose concentration and was highly homogeneous, covering all the surface of the paper reaction zones in a 3D sensor format. These devices showed a major advantage when compared to the 2D lateral flow glucose sensors, where some carryover of the coloured products usually occurs. The detection of anti-Leishmania antibodies in canine sera was conceptually achieved using a paper-based 96-well enzyme-linked immunosorbent assay format. However, optimization is still needed for this test, regarding the efficiency of the immobilization of antigens on the cellulose fibres. The detection of Mycobacterium tuberculosis nucleic acids integrated with a non-cross-linking gold nanoprobe detection scheme was also achieved in a wax-printed 384-well paper-based microplate, by the hybridization with a species-specific probe. The obtained results with the above-mentioned proof-of-concept sensors are thus promising towards the future development of simple and cost-effective paper-based diagnostic devices.

Maturation of mononuclear phagocytes in the lungs of young calves--in vitro study.

The influences of age in calves' immune system are described in their first phase of life. We hypothesized that variations that occur in the main mechanisms of lung innate response can help to identify periods of greater susceptibility to the respiratory diseases that affect calves in the first stage of their life. This study aimed to evaluate the innate immune system. Nine healthy calves were monitored for 3 mo and 8 immunologic evaluations were performed. Bronchoalveolar lavage samples were recovered by bronchoscopy. The alveolar macrophages in samples were identified by protein expression of cluster of differentiation 14 (CD14) and underwent functional evaluation of phagocytosis (Staphylococcus aureus stained with propidium iodide and Escherichia coli). Data was assessed by one-way ANOVA (unstacked and parametric) and the Mann-Whitney test (nonparametric). Functional alterations in CD14-positive phagocytes were observed, with punctual higher intensity of phagocytosis in the third week and its decrease starting at 45 d of life. A gradual increase in phagocytosis rate was observed starting at this date. It is concluded that from 45 d of life on, alveolar macrophages have less phagocytic capacity but more cells perform this function. We suggest that this occurs because lung macrophages of calves start to maintain their immune response without passive immunity influence. Until 90 d of life, calves did not achieve the stability to conclude the maturation of local innate immune response.

Femtosecond laser induced synthesis of Au nanoparticles mediated by chitosan.

This paper reports the synthesis of Au nanoparticles by 30-fs pulses irradiation of a sample containing HAuCl4 and chitosan, a biopolymer used as reducing agent and stabilizer. We observed that it is a multi-photon induced process, with a threshold irradiance of 3.8 × 10(11) W/cm2 at 790 nm. By transmission electron microscopy we observed nanoparticles from 8 to 50 nm with distinct shapes. Infrared spectroscopy indicated that the reduction of gold and consequent production of nanoparticles is related to the fs-pulse induced oxidation of hydroxyl to carbonyl groups in chitosan.

Shikimate kinase (EC 2.7.1.71) from Mycobacterium tuberculosis: kinetics and structural dynamics of a potential molecular target for drug development.

The enzymes of the shikimate pathway represent potential molecular targets for the development of non-toxic antimicrobial agents and anti-parasite drugs. One of the most promising of these enzymes is shikimate kinase (EC 2.7.1.71), which is responsible for the fifth step in the shikimate pathway. This enzyme phosphorylates shikimic acid to yield shikimate-3-phosphate, using ATP as a substrate. In this work, the conformational dynamics of the shikimate kinase from Mycobacterium tuberculosis was investigated in its apostate in solution. For this study, the enzyme was subjected to a gradient of temperatures from 15°C to 45°C in the presence or absence of deuterium oxide, and the amide H/D exchange was monitored using ESI-mass spectrometry. We observed: i) the phosphate binding domain in the apo-enzyme is fairly rigid and largely protected from solvent access, even at relatively high temperatures; ii) the shikimate binding domain is highly flexible, as indicated by the tendency of the apo-enzyme to exhibit large conformational changes to permit LID closure after the shikimate binding; iii) the nucleotide binding domain is initially conformationally rigid, which seems to favour the initial orientation of ADP/ATP, but becomes highly flexible at temperatures above 30°C, which may permit domain rotation; iv) part of the LID domain, including the phosphate binding site, is partially rigid, while another part is highly flexible and accessible to the solvent.

Pyrimidine salvage pathway in Mycobacterium tuberculosis.

The causative agent of tuberculosis (TB), Mycobacterium tuberculosis, infects one-third of the world population. TB remains the leading cause of mortality due to a single bacterial pathogen. The worldwide increase in incidence of M. tuberculosis has been attributed to the high proliferation rates of multi and extensively drug-resistant strains, and to co-infection with the human immunodeficiency virus. There is thus a continuous requirement for studies on mycobacterial metabolism to identify promising targets for the development of new agents to combat TB. Singular characteristics of this pathogen, such as functional and structural features of enzymes involved in fundamental metabolic pathways, can be evaluated to identify possible targets for drug development. Enzymes involved in the pyrimidine salvage pathway might be attractive targets for rational drug design against TB, since this pathway is vital for all bacterial cells, and is composed of enzymes considerably different from those present in humans. Moreover, the enzymes of the pyrimidine salvage pathway might have an important role in the mycobacterial latent state, since M. tuberculosis has to recycle bases and/or nucleosides to survive in the hostile environment imposed by the host. The present review describes the enzymes of M. tuberculosis pyrimidine salvage pathway as attractive targets for the development of new antimycobacterial agents. Enzyme functional and structural data have been included to provide a broader knowledge on which to base the search for compounds with selective biological activity.

Purine Salvage Pathway in Mycobacterium tuberculosis.

Millions of deaths worldwide are caused by the aetiological agent of tuberculosis, Mycobacterium tuberculosis. The increasing prevalence of this disease, the emergence of drug-resistant strains, and the devastating effect of human immunodeficiency virus coinfection have led to an urgent need for the development of new and more efficient antimycobacterial drugs. The modern approach to the development of new chemical compounds against complex diseases, especially the neglected endemic ones, such as tuberculosis, is based on the use of defined molecular targets. Among the advantages, this approach allows (i) the search and identification of lead compounds with defined molecular mechanisms against a specific target (e.g. enzymes from defined pathways), (ii) the analysis of a great number of compounds with a favorable cost/benefit ratio, and (iii) the development of compounds with selective toxicity. The present review describes the enzymes of the purine salvage pathway in M. tuberculosis as attractive targets for the development of new antimycobacterial agents. Enzyme kinetics and structural data have been included to provide a thorough knowledge on which to base the search for compounds with biological activity. We have focused on the mycobacterial homologues of this pathway as potential targets for the development of new antitubercular agents.

IAEA regional intercomparison of in vivo measurements of 131I in the thyroid: the Latin American and Caribbean experience.

An in vivo intercomparison exercise, organised in the scope of the IAEA Regional Latin American (RLA) Project 9066, was carried out in 2009 aimed to harmonise measurement procedures on the measurement of (131)I in the thyroid among Latin American and Caribbean countries. The exercise consisted in the measurement of an anthropomorphic thyroid phantom spiked with a certified source of (133)Ba. The phantom was supplied by the In Vivo Monitoring Laboratory of Institute for Radiation Protection and Dosimetry (IRD) to 17 Institutions from 12 countries of the regions. Among these, 13 Institutions from 10 countries returned their measurement results as well as a standard report form containing detailed information about their respective counting facilities. All participants reported activities within an acceptable range, considering American National Standard Institute (ANSI) limits. Uncertainties varied from 0.04 to 12.9 %. Although results show that the general performance was acceptable in terms of accuracy, the need for additional action towards the standardisation of uncertainty estimation in this type of measurement in the region should be highlighted.

Latin American and Caribbean intercomparison of surface contamination monitoring equipment.

In October 2009, the International Atomic Energy Agency (IAEA) sponsored an intercomparison exercise of surface contamination monitoring equipment, which was held at the Laboratório Nacional de Metrologia das Radiações Ionizantes, from the Instituto de Radioproteção e Dosimetria, IRD/CNEN, Rio de Janeiro. This intercomparison was performed to evaluate the calibration accessibility in Latin America and the Caribbean. Thirteen countries within the region and IAEA have sent instruments to be compared, but only five countries and IAEA were considered apt to participate. Analysis of instruments, results and discussions are presented and recommendations are drawn.

Purine nucleoside phosphorylase activity and expression are upregulated in sites affected by periodontal disease.

BACKGROUND AND OBJECTIVE: Purine nucleoside phosphorylase (PNP) is an enzyme that catalyzes the reversible phosphorolysis of purine nucleosides, playing a key role in the purine salvage pathway. Activated T cells seem to rely heavily on PNP to remain functionally active and are particularly sensitive to PNP deficiency. The role of PNP in periodontal tissues has not been characterized thus far. The aim of this study therefore was to assess the activity and expression of PNP in the gingival tissues of periodontitis patients. MATERIAL AND METHODS: Ten patients consecutively admitted for treatment had their periodontal clinical variables recorded and their gingival crevicular fluid collected. After periodontal treatment the patients were seen once a month for plaque and bleeding control, and had their periodontal variables recorded and gingival crevicular fluid collected at 90 and 180 d. Purine nucleoside phosphorylase-specific activity was assessed using a spectrophotometer through the addition of the PNP substrate analog 2-amino-6mercapto-7-methyl purine riboside to the gingival crevicular fluid. In parallel, PNP expression was assessed by immunohistochemistry and real-time PCR in gingival biopsies and cell culture. RESULTS: Purine nucleoside phosphorylase activity was higher in the gingival crevicular fluid of periodontally diseased sites, which was positively correlated with improvements of the clinical variables. Treatment of periodontal disease induced a striking decrease of PNP activity in periodontally diseased sites. Expression of PNP was more pronounced in mononuclear cells and endothelial cells of the gingiva, and the mRNA levels were 5.7-fold higher in inflamed tissues compared with control samples. CONCLUSION: Purine nucleoside phosphorylase activity and expression are upregulated in periodontally diseased sites and can be detected in the gingival crevicular fluid.

Two-photon polymerization for fabricating structures containing the biopolymer chitosan.

Two-photon polymerization is a powerful tool for fabricating three-dimensional micro/nano structures for applications ranging from nanophotonics to biology. To tailor such structure for specific purposes it is often important to dope them. In this paper we report on the fabrication of structures, with nanometric surface features (resolution of approximately 700 nm), using two-photon polymerization of an acrylic resin doped with the biocompatible polymer chitosan using a guest-host scheme. The fluorescence background in the Raman spectrum indicates the presence of chitosan throughout the structure. Mechanical characterization reveals that chitosan does not affect the mechanical properties of the host acrylic resin and, consequently, the structures exhibit excellent integrity. The approach presented in this work can be used in the fabrication of micro- and nanostructures containing biopolymers for biomedical applications.

Structural bioinformatics study of PNP from Listeria monocytogenes.

This work describes for the first time a model of Purine Nucleoside Phosphorylase from Listeria monocytogenes (LmPNP). We modeled the complexes of LmPNP with ligands in order to determine the structural basis for specificity. Comparative analysis of the model of LmPNP allowed identification of structural features responsible for ligand affinities.

Ab initio 3-D structure prediction of an artificially designed three-alpha-helix bundle via all-atom molecular dynamics simulations.

The rate at which knowledge about genomic sequences and their protein products is produced is increasing much faster than the rate of 3-dimensional protein structure determination by experimental methods, such as X-ray diffraction and nuclear magnetic resonance. One of the major challenges in structural bioinformatics is the conversion of genomic sequences into useful information, such as characterization of protein structure and function. Using molecular dynamics (MD) simulations, we predicted the 3-dimensional structure of an artificially designed three- alpha -helix bundle, called A3, from a fully extended initial conformation, based on its amino acid sequence. The MD protocol enabled us to obtain the secondary, in 1.0 ns, as well as the supersecondary and tertiary structures, in 4.0-10.0 ns, of A3, much faster than previously described for a similar protein system. The structure obtained at the end of the 10.0-ns MD simulation was topologically a three-alpha-helix bundle.

Excited state absorption spectrum of chlorophyll a obtained with white-light continuum.

The study of excited state properties of chlorophyll a is a subject of foremost interest, given that it plays important roles in biological process and has also been proposed for applications in photonics. This work reports on the excited state absorption spectrum of chlorophyll a solution from 460 to 700 nm, obtained through the white-light continuum Z-scan technique. Saturation of absorption was observed due to the ground state depletion, induced by the white-light continuum region that is resonant with the Q band of chlorophyll a. The authors also observed reverse saturation of absorption related to the excitation from the first excited state to a higher energy level for wavelengths below 640 nm. An energy-level diagram, based on the electronic states of chlorophyll a, was employed to interpret their results, revealing that more states than the ones related to the Q and B bands participate in the excited state absorption of this molecule.

Enoyl reductases as targets for the development of anti-tubercular and anti-malarial agents.

Tuberculosis (TB) and Malaria are neglected diseases, which continue to be major causes of morbidity and mortality worldwide, killing together around 5 million people each year. Mycolic acids, the hallmark of mycobacteria, are high-molecular-weight alpha-alkyl, beta-hydroxy fatty acids. Biochemical and genetic experimental data have shown that the product of the M. tuberculosis inhA structural gene (InhA) is the primary target of isoniazid mode of action, the most prescribed anti-tubercular agent. InhA was identified as an NADH-dependent enoyl-ACP(CoA) reductase specific for long-chain enoyl thioesters and is a member of the Type II fatty acid biosynthesis system, which elongates acyl fatty acid precursors of mycolic acids. M. tuberculosis and P. falciparum enoyl reductases are targets for the development of anti-tubercular and antimalarial agents. Here we present a brief description of the mechanism of action of, and resistance to, isoniazid. In addition, data on inhibition of mycobacterial and plasmodial enoyl reductases by triclosan are presented. We also describe recent efforts to develop inhibitors of M. tuberculosis and P. falciparum enoyl reductase enzyme activity.