The identification and differentiation of the Candida parapsilosis complex species by polymerase chain reaction-restriction fragment length polymorphism of the internal transcribed spacer region of the rDNA

Currently, it is accepted that there are three species that were formerly grouped under Candida parapsilosis : C. parapsilosis sensu stricto, Candida orthopsilosis , and Candida metapsilosis . In fact, the antifungal susceptibility profiles and distinct virulence attributes demonstrate the differences in these nosocomial pathogens. An accurate, fast, and economical identification of fungal species has been the main goal in mycology. In the present study, we searched sequences that were available in the GenBank database in order to identify the complete sequence for the internal transcribed spacer (ITS)1-5.8S-ITS2 region, which is comprised of the forward and reverse primers ITS1 and ITS4. Subsequently, an in silico polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to differentiate the C. parapsilosis complex species. Ninety-eight clinical isolates from patients with fungaemia were submitted for analysis, where 59 isolates were identified as C. parapsilosis sensu stricto, 37 were identified as C. orthopsilosis, and two were identified as C. metapsilosis. PCR-RFLP quickly and accurately identified C. parapsilosis complex species, making this method an alternative and routine identification system for use in clinical mycology laboratories.

Fish Biodiversity Of The Vitória-trindade Seamount Chain, Southwestern Atlantic: An Updated Database.

Despite a strong increase in research on seamounts and oceanic islands ecology and biogeography, many basic aspects of their biodiversity are still unknown. In the southwestern Atlantic, the Vitória-Trindade Seamount Chain (VTC) extends ca. 1,200 km offshore the Brazilian continental shelf, from the Vitória seamount to the oceanic islands of Trindade and Martin Vaz. For a long time, most of the biological information available regarded its islands. Our study presents and analyzes an extensive database on the VTC fish biodiversity, built on data compiled from literature and recent scientific expeditions that assessed both shallow to mesophotic environments. A total of 273 species were recorded, 211 of which occur on seamounts and 173 at the islands. New records for seamounts or islands include 191 reef fish species and 64 depth range extensions. The structure of fish assemblages was similar between islands and seamounts, not differing in species geographic distribution, trophic composition, or spawning strategies. Main differences were related to endemism, higher at the islands, and to the number of endangered species, higher at the seamounts. Since unregulated fishing activities are common in the region, and mining activities are expected to drastically increase in the near future (carbonates on seamount summits and metals on slopes), this unique biodiversity needs urgent attention and management.

Alcohol dehydrogenase activities and ethanol tolerance in Anastrepha (Diptera, Tephritidae) fruit-fly species and their hybrids

The ADH (alcohol dehydrogenase) system is one of the earliest known models of molecular evolution, and is still the most studied in Drosophila. Herein, we studied this model in the genus Anastrepha (Diptera, Tephritidae). Due to the remarkable advantages it presents, it is possible to cross species with different Adh genotypes and with different phenotype traits related to ethanol tolerance. The two species studied here each have a different number of Adh gene copies, whereby crosses generate polymorphisms in gene number and in composition of the genetic background. We measured certain traits related to ethanol metabolism and tolerance. ADH specific enzyme activity presented gene by environment interactions, and the larval protein content showed an additive pattern of inheritance, whilst ADH enzyme activity per larva presented a complex behavior that may be explained by epistatic effects. Regression models suggest that there are heritable factors acting on ethanol tolerance, which may be related to enzymatic activity of the ADHs and to larval mass, although a pronounced environmental effect on ethanol tolerance was also observed. By using these data, we speculated on the mechanisms of ethanol tolerance and its inheritance as well as of associated traits.

Reactivity, photolability, and computational studies of the ruthenium nitrosyl complex with a substituted cyclam fac-[Ru(NO)Cl(2)(kappa(3)N(4),N(8),N(11)(1-carboxypropyl)cyclam)]Cl center dot H(2)O

Chemical reactivity, photolability, and computational studies of the ruthenium nitrosyl complex with a substituted cyclam, fac-[Ru(NO)Cl(2)(kappa(3)N(4),N(8),N(11)(1-carboxypropyl)cyclam)]Cl center dot H(2)O ((1-carboxypropyl) cyclam = 3-(1,4,8,11-tetraazacyclotetradecan-1-yl) propionic acid)), (I) are described. Chloride ligands do not undergo aquation reactions (at 25 degrees C, pH 3). The rate of nitric oxide (NO) dissociation (k(obs-NO)) upon reduction of I is 2.8 s(-1) at 25 +/- 1 degrees C (in 0.5 mol L(-1) HCl), which is close to the highest value found for related complexes. The uncoordinated carboxyl of I has a pK(a) of similar to 3.3, which is close to that of the carboxyl of the non coordinated (1-carboxypropyl) cyclam (pK(a) = 3.4). Two additional pK(a) values were found for I at similar to 8.0 and similar to 11.5. Upon electrochemical reduction or under irradiation with light (lambda(irr) = 350 or 520 nm; pH 7.4), I releases NO in aqueous solution. The cyclam ring N bound to the carboxypropyl group is not coordinated, resulting in a fac configuration that affects the properties and chemical reactivities of I, especially as NO donor, compared with analogous trans complexes. Among the computational models tested, the B3LYP/ECP28MDF, cc-pVDZ resulted in smaller errors for the geometry of I. The computational data helped clarify the experimental acid-base equilibria and indicated the most favourable site for the second deprotonation, which follows that of the carboxyl group. Furthermore, it showed that by changing the pH it is possible to modulate the electron density of I with deprotonation. The calculated NO bond length and the Ru/NO charge ratio indicated that the predominant canonical structure is [Ru(III)NO], but the Ru-NO bond angles and bond index (b.i.) values were less clear; the angles suggested that [Ru(II)NO(+)] could contribute to the electronic structure of I and b.i. values indicated a contribution from [Ru(IV)NO(-)]. Considering that some experimental data are consistent with a [Ru(II)NO(+)] description, while others are in agreement with [Ru(III)NO], the best description for I would be a linear combination of the three canonical forms, with a higher weight for [Ru(II)NO(+)] and [Ru(III)NO].

Selective Decrease of Components of the Creatine Kinase System and ATP Synthase Complex in Chronic Chagas Disease Cardiomyopathy

Background: Chronic Chagas disease cardiomyopathy (CCC) is an inflammatory dilated cardiomyopathy with a worse prognosis than other cardiomyopathies. CCC occurs in 30 % of individuals infected with Trypanosoma cruzi, endemic in Latin America. Heart failure is associated with impaired energy metabolism, which may be correlated to contractile dysfunction. We thus analyzed the myocardial gene and protein expression, as well as activity, of key mitochondrial enzymes related to ATP production, in myocardial samples of end-stage CCC, idiopathic dilated (IDC) and ischemic (IC) cardiomyopathies. Methodology/Principal Findings: Myocardium homogenates from CCC (N = 5), IC (N = 5) and IDC (N = 5) patients, as well as from heart donors (N = 5) were analyzed for protein and mRNA expression of mitochondrial creatine kinase (CKMit) and muscular creatine kinase (CKM) and ATP synthase subunits aplha and beta by immunoblotting and by real-time RT-PCR. Total myocardial CK activity was also assessed. Protein levels of CKM and CK activity were reduced in all three cardiomyopathy groups. However, total CK activity, as well as ATP synthase alpha chain protein levels, were significantly lower in CCC samples than IC and IDC samples. CCC myocardium displayed selective reduction of protein levels and activity of enzymes crucial for maintaining cytoplasmic ATP levels. Conclusions/Significance: The selective impairment of the CK system may be associated to the loss of inotropic reserve observed in CCC. Reduction of ATP synthase alpha levels is consistent with a decrease in myocardial ATP generation through oxidative phosphorylation. Together, these results suggest that the energetic deficit is more intense in the myocardium of CCC patients than in the other tested dilated cardiomyopathies.

Shoulder Muscular Activity During Isometric Three-Point Kneeling Exercise on Stable and Unstable Surfaces

The purpose of this study was to determine if performing isometric 3-point kneeling exercises on a Swiss ball influenced the isometric force output and EMG activities of the shoulder muscles when compared with performing the same exercises on a stable base of support. Twenty healthy adults performed the isometric 3-point kneeling exercises with the hand placed either on a stable surface or on a Swiss ball. Surface EMG was recorded from the posterior deltoid, pectoralis major, biceps brachii, triceps brachii, upper trapezius, and serratus anterior muscles using surface differential electrodes. All EMG data were reported as percentages of the average root mean square (RMS) values obtained in maximum voluntary contractions for each muscle studied. The highest load value was obtained during exercise on a stable surface. A significant increase was observed in the activation of glenohumeral muscles during exercises on a Swiss ball. However, there were no differences in EMG activities of the scapulothoracic muscles. These results suggest that exercises performed on unstable surfaces may provide muscular activity levels similar to those performed on stable surfaces, without the need to apply greater external loads to the musculoskeletal system. Therefore, exercises on unstable surfaces may be useful during the process of tissue regeneration.

A simplified minimal residual disease polymerase chain reaction method at early treatment points can stratify children with acute lymphoblastic leukemia into good and poor outcome groups

Background Minimal residual disease is an important independent prognostic factor in childhood acute lymphoblastic leukemia. The classical detection methods such as multiparameter flow cytometry and real-time quantitative polymerase chain reaction analysis are expensive, time-consuming and complex, and require considerable technical expertise. Design and Methods We analyzed 229 consecutive children with acute lymphoblastic leukemia treated according to the GBTLI-99 protocol at three different Brazilian centers. Minimal residual disease was analyzed in bone marrow samples at diagnosis and on days 14 and 28 by conventional homo/heteroduplex polymerase chain reaction using a simplified approach with consensus primers for IG and TCR gene rearrangements. Results At least one marker was detected by polymerase chain reaction in 96.4%, of the patients. By combining the minimal residual disease results obtained on days 14 and 28, three different prognostic groups were identified: minimal residual disease negative on days 14 and 28, positive on day 14/negative on day 28, and positive on both. Five-year event-free survival rates were 85%, 75.6%,, and 27.8%, respectively (p<0.0001). The same pattern of stratification held true for the group of intensively treated children. When analyzed in other subgroups of patients such as those at standard and high risk at diagnosis, those with positive B-derived CD10, patients positive for the TEL/AML1 transcript, and patients in morphological remission on a day 28 marrow, the event-free survival rate was found to be significantly lower in patients with positive minimal residual disease on day 28. Multivariate analysis demonstrated that the detection of minimal residual disease on day 28 is the most significant prognostic factor. Conclusions This simplified strategy for detection of minimal residual disease was feasible, reproducible, cheaper and simpler when compared with other methods, and allowed powerful discrimination between children with acute lymphoblastic leukemia with a good and poor outcome.

RNA interference-mediated knockdown of CD49e (alpha 5 integrin chain) in human thymic epithelial cells modulates the expression of multiple genes and decreases thymocyte adhesion

Background: The thymus is a central lymphoid organ, in which bone marrow-derived T cell precursors undergo a complex process of maturation. Developing thymocytes interact with thymic microenvironment in a defined spatial order. A component of thymic microenvironment, the thymic epithelial cells, is crucial for the maturation of T-lymphocytes through cell-cell contact, cell matrix interactions and secretory of cytokines/chemokines. There is evidence that extracellular matrix molecules play a fundamental role in guiding differentiating thymocytes in both cortical and medullary regions of the thymic lobules. The interaction between the integrin alpha 5 beta 1 (CD49e/CD29; VLA-5) and fibronectin is relevant for thymocyte adhesion and migration within the thymic tissue. Our previous results have shown that adhesion of thymocytes to cultured TEC line is enhanced in the presence of fibronectin, and can be blocked with anti-VLA-5 antibody. Results: Herein, we studied the role of CD49e expressed by the human thymic epithelium. For this purpose we knocked down the CD49e by means of RNA interference. This procedure resulted in the modulation of more than 100 genes, some of them coding for other proteins also involved in adhesion of thymocytes; others related to signaling pathways triggered after integrin activation, or even involved in the control of F-actin stress fiber formation. Functionally, we demonstrated that disruption of VLA-5 in human TEC by CD49e-siRNA-induced gene knockdown decreased the ability of TEC to promote thymocyte adhesion. Such a decrease comprised all CD4/CD8-defined thymocyte subsets. Conclusion: Conceptually, our findings unravel the complexity of gene regulation, as regards key genes involved in the heterocellular cell adhesion between developing thymocytes and the major component of the thymic microenvironment, an interaction that is a mandatory event for proper intrathymic T cell differentiation.

Analgesic and Anti-Inflammatory Activities of Salicylaldehyde 2-Chlorobenzoyl Hydrazone (H(2)LASSBio-466), Salicylaldehyde 4-Chlorobenzoyl Hydrazone (H(2)LASSBio-1064) and Their Zinc(II) Complexes

Salicylaldehyde 2-chlorobenzoyl hydrazone (H(2)LASSBio-466), salicylaldehyde 4-chlorobenzoyl hydrazone (H(2)LASSBio-1064) and their complexes [Zn(LASSBio-466) H(2)O](2) (1) and [Zn(HLASSBio-1064) Cl](2) (2) were evaluated in animal models of peripheral and central nociception, and acute inflammation. All studied compounds significantly inhibited acetic acid-induced writhing response. Upon coordination the anti-nociceptive activity was favored in the complex 1. H(2)LASSBio-466 inhibited only the first phase of the formalin test, while 1 was active in the second phase, like indomethacin, indicating its ability to inhibit nociception associated with the inflammatory response. Hence coordination to zinc(II) altered the pharmacological profile of H(2)LASSBio-466. H(2)LASSBio-1064 inhibited both phases but this effect was not improved by coordination. The studied compounds did not increase the latency of response in the hot plate model, indicating their lack of central anti-nociceptive activity. All compounds showed levels of inhibition of zymosan-induced peritonitis comparable or superior to indomethacin, indicating an expressive anti-inflammatory profile.

MAMMALIAN TARGET OF RAPAMYCIN COMPLEX 1 IS INVOLVED IN DIFFERENTIATION OF REGENERATING MYOFIBERS IN VIVO

This work was undertaken to provide further insight into the role of mammalian target of rapamycin complex 1 (mTORC1) in skeletal muscle regeneration, focusing on myofiber size recovery. Rats were treated or not with rapamycin, an mTORC1 inhibitor. Soleus muscles were then subjected to cryolesion and analyzed 1, 10, and 21 days later. A decrease in soleus myofiber cross-section area on post-cryolesion days 10 and 21 was accentuated by rapamycin, which was also effective in reducing protein synthesis in these freeze-injured muscles. The incidence of proliferating satellite cells during regeneration was unaltered by rapamycin, although immunolabeling for neonatal myosin heavy chain (MHC) was weaker in cryolesion+rapamycin muscles than in cryolesion-only muscles. In addition, the decline in tetanic contraction of freeze-injured muscles was accentuated by rapamycin. This study indicates that mTORC1 plays a key role in the recovery of muscle mass and the differentiation of regenerating myofibers, independently of necrosis and satellite cell proliferation mechanisms. Muscle Nerve 42: 778-787,2010

Multipoint covalent immobilization of lipase on chitosan hybrid hydrogels: influence of the polyelectrolyte complex type and chemical modification on the catalytic properties of the biocatalysts

This work aimed at the production of stabilized derivatives of Thermomyces lanuginosus lipase (TLL) by multipoint covalent immobilization of the enzyme on chitosan-based matrices. The resulting biocatalysts were tested for synthesis of biodiesel by ethanolysis of palm oil. Different hydrogels were prepared: chitosan alone and in polyelectrolyte complexes (PEC) with kappa-carrageenan, gelatin, alginate, and polyvinyl alcohol (PVA). The obtained supports were chemically modified with 2,4,6-trinitrobenzene sulfonic acid (TNBS) to increase support hydrophobicity, followed by activation with different agents such as glycidol (GLY), epichlorohydrin (EPI), and glutaraldehyde (GLU). The chitosan-alginate hydrogel, chemically modified with TNBS, provided derivatives with higher apparent hydrolytic activity (HA(app)) and thermal stability, being up to 45-fold more stable than soluble lipase. The maximum load of immobilized enzyme was 17.5 mg g(-1) of gel for GLU, 7.76 mg g(-1) of gel for GLY, and 7.65 mg g(-1) of gel for EPI derivatives, the latter presenting the maximum apparent hydrolytic activity (364.8 IU g(-1) of gel). The three derivatives catalyzed conversion of palm oil to biodiesel, but chitosan-alginate-TNBS activated via GLY and EPI led to higher recovered activities of the enzyme. Thus, this is a more attractive option for both hydrolysis and transesterification of vegetable oils using immobilized TLL, although industrial application of this biocatalyst still demands further improvements in its half-life to make the enzymatic process economically attractive.

Model for facilities or vendors location in a global scale considering several echelons in the Chain

The facilities location problem for companies with global operations is very complex and not well explored in the literature. This work proposes a MILP model that solves the problem through minimization of the total logistic cost. Main contributions of the model are the pioneer carrying cost calculation, the treatment given to the take-or-pay costs and to the international tax benefits such as drawback and added value taxes in Brazil. The model was successfully applied to a real case of a chemical industry with industrial plants and sales all over the world. The model application recommended a totally new sourcing model for the company.

Microencapsulation of propolis extract by complex coacervation

The propolis has potential to be a natural food additive However its application is limited because It is alcohol-soluble and has strong flavour Microencapsulation may be an alternative for reducing these problems The aim of this study was to encapsulate propolis extract by complex coacervation using isolated soy protein and pectin as encapsulant agents The coacervation was studied as a function of pH (5 0 4 5 4 0 and 3 5) and the concentration of encapsulants and core (2 5 and 5 0 g/100 mL) Samples obtained at pH 4 0 in both concentrations were lyophilized and analyzed for hygroscopicity encapsulation efficiency particle size morphology thermal behavior stability of phenolic and flavonoids during storage as well as antioxidant and antimicrobial activities It was possible to encapsulate propolis extract by complex coacervation and to obtain it in the form of powder alcohol-free stable with antioxidant property antimicrobial activity against Staphylococcus aureus and with the possibility of controlled release in foods (C) 2010 Elsevier Ltd All rights reserved

Characterization of Rubus fruticosus mitochondria and salicylic acid inhibition of reactive oxygen species generation at Complex III/Q cycle: potential implications for hypersensitive response in plants

In addition to adenosine triphosphate (ATP) production, mitochondria have been implicated in the regulation of several physiological responses in plants, such as programmed cell death (PCD) activation. Salicylic acid (SA) and reactive oxygen species (ROS) are essential signaling molecules involved in such physiological responses; however, the mechanisms by which they act remain unknown. In non-photosynthesizing tissues, mitochondria appear to serve as the main source of ROS generation. Evidence suggests that SA and ROS could regulate plant PCD through a synergistic mechanism that involves mitochondria. Herein, we isolate and characterize the mitochondria from non-photosynthesizing cell suspension cultures of Rubus fruticosus. Furthermore, we assess the primary site of ROS generation and the effects of SA on isolated organelles. Mitochondrial Complex III was found to be the major source of ROS generation in this model. In addition, we discovered that SA inhibits the electron transport chain by inactivating the semiquinone radical during the Q cycle. Computational analyses confirmed the experimental data, and a mechanism for this action is proposed.

Classical and alternative components of the mitochondrial respiratory chain in pathogenic fungi as potential therapeutic targets

The frequency of opportunistic fungal infection has increased drastically, mainly in patients who are immunocompromised due to organ transplant, leukemia or HIV infection. In spite of this, only a few classes of drugs with a limited array of targets, are available for antifungal therapy. Therefore, more specific and less toxic drugs with new molecular targets is desirable for the treatment of fungal infections. In this context, searching for differences between mitochondrial mammalian hosts and fungi in the classical and alternative components of the mitochondrial respiratory chain may provide new potential therapeutic targets for this purpose.