Functional and structural studies of the disulfide isomerase DsbC from the plant pathogen Xylella fastidiosa reveals a redox-dependent oligomeric modulation in vitro

Xylella fastidiosa is a Gram-negative bacterium that grows as a biofilm inside the xylem vessels of susceptible plants and causes several economically relevant crop diseases. In the present study, we report the functional and low-resolution structural characterization of the X. fastidiosa disulfide isomerase DsbC (XfDsbC). DsbC is part of the disulfide bond reduction/isomerization pathway in the bacterial periplasm and plays an important role in oxidative protein folding. In the present study, we demonstrate the presence of XfDsbC during different stages of X. fastidiosa biofilm development. XfDsbC was not detected during X. fastidiosa planktonic growth; however, after administering a sublethal copper shock, we observed an overexpression of XfDsbC that also occurred during planktonic growth. These results suggest that X. fastidiosa can use XfDsbC in vivo under oxidative stress conditions similar to those induced by copper. In addition, using dynamic light scattering and small-angle X-ray scattering, we observed that the oligomeric state of XfDsbC in vitro may be dependent on the redox environment. Under reducing conditions, XfDsbC is present as a dimer, whereas a putative tetrameric form was observed under nonreducing conditions. Taken together, our findings demonstrate the overexpression of XfDsbC during biofilm formation and provide the first structural model of a bacterial disulfide isomerase in solution. Structured digital abstract XfDsbC and XfDsbC bind by x ray scattering (View Interaction: 1, 2) XfDsbC and XfDsbC bind by molecular sieving (View interaction) XfDsbC and XfDsbC bind by comigration in non denaturing gel electrophoresis (View interaction) XfDsbC and XfDsbC bind by cross-linking study (View Interaction: 1, 2) XfDsbC and XfDsbC bind by dynamic light scattering (View Interaction: 1, 2)

Oxidative Stress and Modification of Renal Vascular Permeability Are Associated with Acute Kidney Injury during P. berghei ANKA Infection

Malaria associated-acute kidney injury (AKI) is associated with 45% of mortality in adult patients hospitalized with severe form of the disease. However, the causes that lead to a framework of malaria-associated AKI are still poorly characterized. Some clinical studies speculate that oxidative stress products, a characteristic of Plasmodium infection, as well as proinflammatory response induced by the parasite are involved in its pathophysiology. Therefore, we aimed to investigate the development of malaria-associated AKI during infection by P. berghei ANKA, with special attention to the role played by the inflammatory response and the involvement of oxidative stress. For that, we took advantage of an experimental model of severe malaria that showed significant changes in the renal pathophysiology to investigate the role of malaria infection in the renal microvascular permeability and tissue injury. Therefore, BALB/c mice were infected with P. berghei ANKA. To assess renal function, creatinine, blood urea nitrogen, and ratio of proteinuria and creatininuria were evaluated. The products of oxidative stress, as well as cytokine profile were quantified in plasma and renal tissue. The change of renal microvascular permeability, tissue hypoxia and cellular apoptosis were also evaluated. Parasite infection resulted in renal dysfunction. Furthermore, we observed increased expression of adhesion molecule, proinflammatory cytokines and products of oxidative stress, associated with a decrease mRNA expression of HO-1 in kidney tissue of infected mice. The measurement of lipoprotein oxidizability also showed a significant increase in plasma of infected animals. Together, our findings support the idea that products of oxidative stress, as well as the immune response against the parasite are crucial to changes in kidney architecture and microvascular endothelial permeability of BALB/c mice infected with P. berghei ANKA.

Respiratory rehabilitation: a physiotherapy approach to the control of asthma symptoms and anxiety

OBJECTIVES: The objectives of this study were to verify the degree of anxiety, respiratory distress, and health-related quality of life in a group of asthmatic patients who have experienced previous panic attacks. Additionally, we evaluated if a respiratory physiotherapy program (breathing retraining) improved both asthma and panic disorder symptoms, resulting in an improvement in the health-related quality of life of asthmatics. METHODS: Asthmatic individuals were assigned to a chest physiotherapy group that included a breathing retraining program held once a week for three months or a paired control group that included a Subtle Touch program. All patients were assessed using the Diagnostic and Statistical Manual of Mental Disorders IV, the Sheehan Anxiety Scale, the Quality of Life Questionnaire, and spirometry parameter measurements. RESULTS: Both groups had high marks for panic disorder and agoraphobia, which limited their quality of life. The Breathing Retraining Group program improved the clinical control of asthma, reduced panic symptoms and agoraphobia, decreased patient scores on the Sheehan Anxiety Scale, and improved their quality of life. Spirometry parameters were unchanged. CONCLUSION: Breathing retraining improves the clinical control of asthma and anxiety symptoms and the health-related quality of life in asthmatic patients.

Physicochemical properties of endodontic sealers of different bases

Objective: To assess the setting time (ST), flow (FL), radiopacity (RD), solubility (SB) and dimensional change following setting (DC) of different sealers (AH Plus (R), Polifil, Apexit Plus (R), Sealapex (R), Endomethasone (R) and Endofill (R)) according to American National Standards Institute/American Dental Association (ANSI/ADA) Specification 57. Material and methods: Five samples of each material were used for each test. For ST, cast rings were filled with sealers and tested with a Gillmore needle. For FL, the sealer was placed on a glass plate. After 180 s, another plate with 20 g and a load of 100 g were applied on the material, and the diameters of the discs formed were measured. In RD, circular molds were filled with the sealers, radiographed and analyzed using Digora software. For SB, circular molds were filled with the sealers, a nylon thread was placed inside the material and another glass plate was positioned on the set, pressed and stored at 37 degrees C. Samples were weighed, placed in water, dried and reweighed. The water used for SB was analyzed by atomic absorption spectrometry. For DC, circular molds were filled with the sealers, covered by glass plates and stored at 37 degrees C. Samples were measured and stored in water for 30 days. After this period, they were dryed and measured again. Results: Regarding ST, AH Plus (R), Apexit (R) and Endofil (R) sealers are in accordance with ANSI/ADA standards. Endomethasone's manufacturer did not mention the ST; Polifil is an experimental sealer and Sealapex (R) did not set. Considering RD, SB and DC, all sealers were in accordance with ANSI/ADA. The spectrometric analysis showed that a significant amount of K+ and Zn2(+) ions was released from Apexit Plus (R) and Endofill (R), respectively. Conclusion: Except for DC, all other physicochemical properties of the tested sealers conformed to ANSI/ADA requirements.

Molecular detection of in-vivo microbial contamination of metallic orthodontic brackets by checkerboard DNA-DNA hybridization

Introduction: Knowing the microbiota that colonizes orthodontic appliances is important for planning strategies and implementing specific preventive measures during treatment. The purpose of this clinical trial was to evaluate in vivo the contamination of metallic orthodontic brackets with 40 DNA probes for different bacterial species by using the checkerboard DNA-DNA hybridization (CDDH) technique. Methods: Eighteen patients, 11 to 29 years of age having fixed orthodontic treatment, were enrolled in the study. Each subject had 2 new metallic brackets bonded to different premolars in a randomized manner. After 30 days, the brackets were removed and processed for analysis by CDDH. Data on bacterial contamination were analyzed descriptively and with the Kruskal-Wallis and Dunn post tests (alpha = 0.05). Forty microbial species (cariogenic microorganisms, bacteria of the purple, yellow, green, orange complexes, "red complex + Treponema socranskii," and the cluster of Actinomyces) were assessed. Results: Most bacterial species were present in all subjects, except for Streptococcus constellatus, Campylobacter rectus, Tannerella forsythia, T socranskii, and Lactobacillus acidophillus (94.4%), Propionibacterium acnes I and Eubacterium nodatum (88.9%), and Treponema denticola (77.8%). Among the cariogenic microorganisms, Streptococcus mutans and Streptococcus sobrinus were found in larger numbers than L acidophillus and Lactobacillus casei (P < 0.001). The periodontal pathogens of the orange complex were detected in larger numbers than those of the "red complex + T socranskii" (P < 0.0001). Among the bacteria not associated with specific pathologies, Veillonella parvula (purple complex) was the most frequently detected strain (P < 0.0001). The numbers of yellow and green complex bacteria and the cluster of Actinomyces were similar (P > 0.05). Conclusions: Metallic brackets in use for 1 month were multi-colonized by several bacterial species, including cariogenic microorganisms and periodontal pathogens, reinforcing the need for meticulous oral hygiene and additional preventive measures to maintain oral health in orthodontic patients. (Am J Orthod Dentofacial Orthop 2012;141:24-9)

DEVELOPMENT AND VALIDATION OF A HPLC METHOD FOR QUANTIFICATION OF URSOLIC ACID IN SOLID DISPERSIONS

DEVELOPMENT AND VALIDATION OF A HPLC METHOD FOR QUANTIFICATION OF URSOLIC ACID IN SOLID DISPERSIONS. Ursolic acid is a natural molecule that presents several pharmacological properties. In this work, an analytical method by RP-HPLC has been developed and validated for quantification of this drug in the solid dispersions, using PEG 6000 and Poloxamer 407 as polymers. The method was specific, linear in the range of 1.0-50.0 mu g mL(-1) (r<0.99), precise (CV < 5% for both inter-and intra-assays), accurate (maximum deviation of +/- 13%), and robust to the parameters evaluated. This method has proved to be simple and useful for ursolic acid determination in solid dispersions, enabling its determination in pharmaceutical dosage form.

Nuclear abnormalities in erythrocytes and morphometric indexes in the catfish Cathorops spixii (Ariidae) from different sites on the southeastern Brazilian coast

Nuclear abnormalities in erythrocytes (NAE) were taken as biomarkers in the catfish Cathorops spixii (Ariidae) sampled in an estuary little affected by human activity (Cananeia) and in three regions (Santos Channel: SC, Santos Bay: SB and Sao Vicente Channel: SVC) of the Santos-Sao Vicente estuary impacted by various anthropogenic activities. Increases in NAE were observed in fish from SC and SVC sampled in the summer period as compared with specimens from the Cananeia estuary. These results suggest the presence of genotoxic compounds in these regions. However, the absence of significant differences in micronuclei frequency reflects slight mutagenic effects in these individuals. It is possible that the lower NAE frequency in specimens from SB might be associated with the greater remobilization and dilution of chemicals in this region. The low frequency of NAE in C. spixii from the Cananeia estuary is in accordance with the slight anthropogenic influence in this system, and may be suggestive of the absence of genotoxic and mutagenic effects in these organisms.

Transcriptome Analysis of Renal Ischemia/Reperfusion Injury and Its Modulation by Ischemic Pre-Conditioning or Hemin Treatment

Ischemia/reperfusion injury (IRI) is a leading cause of acute renal failure. The definition of the molecular mechanisms involved in renal IRI and counter protection promoted by ischemic pre-conditioning (IPC) or Hemin treatment is an important milestone that needs to be accomplished in this research area. We examined, through an oligonucleotide microarray protocol, the renal differential transcriptome profiles of mice submitted to IRI, IPC and Hemin treatment. After identifying the profiles of differentially expressed genes observed for each comparison, we carried out functional enrichment analysis to reveal transcripts putatively involved in potential relevant biological processes and signaling pathways. The most relevant processes found in these comparisons were stress, apoptosis, cell differentiation, angiogenesis, focal adhesion, ECM-receptor interaction, ion transport, angiogenesis, mitosis and cell cycle, inflammatory response, olfactory transduction and regulation of actin cytoskeleton. In addition, the most important overrepresented pathways were MAPK, ErbB, JAK/STAT, Toll and Nod like receptors, Angiotensin II, Arachidonic acid metabolism, Wnt and coagulation cascade. Also, new insights were gained about the underlying protection mechanisms against renal IRI promoted by IPC and Hemin treatment. Venn diagram analysis allowed us to uncover common and exclusively differentially expressed genes between these two protective maneuvers, underscoring potential common and exclusive biological functions regulated in each case. In summary, IPC exclusively regulated the expression of genes belonging to stress, protein modification and apoptosis, highlighting the role of IPC in controlling exacerbated stress response. Treatment with the Hmox1 inducer Hemin, in turn, exclusively regulated the expression of genes associated with cell differentiation, metabolic pathways, cell cycle, mitosis, development, regulation of actin cytoskeleton and arachidonic acid metabolism, suggesting a pleiotropic effect for Hemin. These findings improve the biological understanding of how the kidney behaves after IRI. They also illustrate some possible underlying molecular mechanisms involved in kidney protection observed with IPC or Hemin treatment maneuvers.

Peat palynomorphs from Eugenio de Melo, middle valley of Paraiba do Sul River, Sao Paulo, Brazil.

Peat palynomorphs from Eugenio de Melo, middle valley of Paraiba do Sul River, Sao Paulo, Brazil. This paper presents palynomorphs identified in the peats of the Eugenio de Melo region, the middle valley of the Paraiba do Sul River, Sao Paulo State. The samples were collected at intervals of 10 cm from a core with 3.90 m deep, aged between 11400-11220 and 490-290 cal. years B.P. Three types of algae were identified and presented, and ten spores, two gymnosperm pollen grains and thirty-nine angiosperms were described, with their own ecological information.

Light emission and current rectification in a molecular device: Experiment and theory

We have investigated optical and transport properties of the molecular structure 2,3,4,5-tetraphenyl-1-phenylethynyl-cyclopenta-2,4-dienol experimentally and theoretically. The optical spectrum was calculated using Hartree-Fock-intermediate neglect of differential overlap-configuration interaction model. The experimental photoluminescence spectrum showed a peak around 470nm which was very well described by the modeling. Electronic transport measurements showed a diode-like effect with a strong current rectification. A phenomenological microscopic model based on non-equilibrium Green's function technique was proposed and a very good description electronic transport was obtained. (C) 2012 American Institute of Physics. [http://dx.doi.org/10.1063/1.4767457]

Human Endometrial-Derived Mesenchymal Stem Cells Suppress Inflammation in the Central Nervous System of EAE Mice

FAPESP [2009/13109-5]

Exploring the Role of Soluble Factors Associated with Immune Regulatory Properties of Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are characterized as multipotent stromal cells with the capacity for both self-renewal and differentiation into mesodermal cell lineages. MSCs also have a fibroblast-like phenotype and can be isolated from several tissues. In recent years, researchers have found that MSCs secrete several soluble factors that exert immunosuppressive effects by modulating both innate (macrophages, dendritic and NK cells) and adaptive (B cells and CD4+ and CD8+ T cells) immune responses. This review summarizes the principal trophic factors that are related to immune regulation and secreted by MSCs under both autoimmune and inflammatory conditions. The understanding of mechanisms that regulate immunity in MSCs field is important for their future use as a novel cellular-based immunotherapy with clinical applications in several diseases.

Low-Level Laser Therapy Decreases Renal Interstitial Fibrosis

Objective: the purpose of this study was to investigate the effect of low-level laser therapy (LLLT) on chronic kidney disease (CKD) in a model of unilateral ureteral obstruction (UUO). Background data: Regardless of the etiology, CKD involves progressive widespread tissue fibrosis, tubular atrophy, and loss of kidney function. This process also occurs in kidney allograft. At present, effective therapies for this condition are lacking. We investigated the effects of LLLT on the interstitial fibrosis that occurs after experimental UUO in rats. Methods: The occluded kidney of half of the 32 Wistar rats that underwent UUO received a single intraoperative dose of LLLT (AlGaAs laser, 780 nm, 22.5 J/cm(2), 30mW, 0.75W/cm(2), 30 sec on each of nine points). After 14 days, renal fibrosis was assessed by Sirius red staining under polarized light. Immunohistochemical analyses quantitated the renal tissue cells that expressed fibroblast (FSP-1) and myofibroblast (alpha-SMA) markers. Reverse transcriptase polymerase chain reaction (RT-PCR) was performed to determine the mRNA expression of interleukin (IL)-6, monocyte chemotactic protein-1 (MCP-1), transforming growth factor (TGF)-beta 1 and Smad3. Results: The UUO and LLLT animals had less fibrosis than the UUO animals, as well having decreased expression inflammatory and pro-fibrotic markers. Conclusions: For the first time, we showed that LLLT had a protective effect regarding renal interstitial fibrosis. It is conceivable that by attenuating inflammation, LLLT can prevent tubular activation and transdifferentiation, which are the two processes that mainly drive the renal fibrosis of the UUO model.

Evidence of contamination by oil and oil products in the Santos-São Vicente estuary, São Paulo, Brazil

Different components of the mixed function oxidase (MFO) system and the levels of fluorescent aromatic compounds in bile (FACs) were measured in Cathorops spixii in order to assess the impact of polycyclic aromatic hydrocarbons (PAHs). Fish were sampled in an estuary (Santos/Sao Vicente) with a history of contamination by PAHs, mainly due to the presence of the industrial complex of Cubatao city and of another of low anthropogenic influence (Cananeia) on the Brazilian coast. FACs were higher in fish from the polluted site, and the PAH 5 and 6-ring metabolites were the most frequent - with 14% and 15%, respectively. Levels of the different components of the MFO system showed the same variation profile as the FACs for both estuaries. Therefore, the values found for somatic indexes and biomarkers with data of bile PAH metabolites indicate the presence of organic contaminants, especially in the area subject to the influence of the industrial complex on the Santos/Sao Vicente estuary.