Functional characterization of the Pneumocystis jirovecii potential drug targets dhfs and abz2 involved in folate biosynthesis.

Pneumocystis species are fungal parasites colonizing mammal lungs with strict host specificity. Pneumocystis jirovecii is the human-specific species and can turn into an opportunistic pathogen causing severe pneumonia in immunocompromised individuals. This disease is currently the second most frequent life-threatening invasive fungal infection worldwide. The most efficient drug, cotrimoxazole, presents serious side effects, and resistance to this drug is emerging. The search for new targets for the development of new drugs is thus of utmost importance. The recent release of the P. jirovecii genome sequence opens a new era for this task. It can now be carried out on the actual targets to be inhibited instead of on those of the relatively distant model Pneumocystis carinii, the species infecting rats. We focused on the folic acid biosynthesis pathway because (i) it is widely used for efficient therapeutic intervention, and (ii) it involves several enzymes that are essential for the pathogen and have no human counterparts. In this study, we report the identification of two such potential targets within the genome of P. jirovecii, the dihydrofolate synthase (dhfs) and the aminodeoxychorismate lyase (abz2). The function of these enzymes was demonstrated by the rescue of the null allele of the orthologous gene of Saccharomyces cerevisiae.

HBV genotypes and response to tenofovir disoproxil fumarate in HIV/HBV-coinfected persons.

BACKGROUND: Hepatitis B virus (HBV) genotypes can influence treatment outcome in HBV-monoinfected and human immunodeficiency virus (HIV)/HBV-coinfected patients. Tenofovir disoproxil fumarate (TDF) plays a pivotal role in antiretroviral therapy (ART) of HIV/HBV-coinfected patients. The influence of HBV genotypes on the response to antiviral drugs, particularly TDF, is poorly understood. METHODS: HIV/HBV-co-infected participants with detectable HBV DNA prior to TDF therapy were selected from the Swiss HIV Cohort Study. HBV genotypes were identified and resistance testing was performed prior to antiviral therapy, and in patients with delayed treatment response (>6 months). The efficacy of TDF to suppress HBV (HBV DNA <20 IU/mL) and the influence of HBV genotypes were determined. RESULTS: 143 HIV/HBV-coinfected participants with detectable HBV DNA were identified. The predominant HBV genotypes were A (82 patients, 57 %); and D (35 patients, 24 %); 20 patients (14 %) were infected with multiple genotypes (3 % A + D and 11 % A + G); and genotypes B, C and E were each present in two patients (1 %). TDF completely suppressed HBV DNA in 131 patients (92 %) within 6 months; and in 12 patients (8 %), HBV DNA suppression was delayed. No HBV resistance mutations to TDF were found in patients with delayed response, but all were infected with HBV genotype A (among these, 5 patients with genotype A + G), and all had previously been exposed to lamivudine. CONCLUSION: In HIV/HBV-coinfected patients, infection with multiple HBV genotypes was more frequent than previously reported. The large majority of patients had an undetectable HBV viral load at six months of TDF-containing ART. In patients without viral suppression, no TDF-related resistance mutations were found. The role of specific genotypes and prior lamivudine treatment in the delayed response to TDF warrant further investigation.

Integrin-Specific Mechanoresponses to Compression and Extension Probed by Cylindrical Flat-Ended AFM Tips in Lung Cells

Cells from lung and other tissues are subjected to forces of opposing directions that are largely transmitted through integrin-mediated adhesions. How cells respond to force bidirectionality remains ill defined. To address this question, we nanofabricated flat-ended cylindrical Atomic Force Microscopy (AFM) tips with ~1 µm2 cross-section area. Tips were uncoated or coated with either integrin-specific (RGD) or non-specific (RGE/BSA) molecules, brought into contact with lung epithelial cells or fibroblasts for 30 s to form focal adhesion precursors, and used to probe cell resistance to deformation in compression and extension. We found that cell resistance to compression was globally higher than to extension regardless of the tip coating. In contrast, both tip-cell adhesion strength and resistance to compression and extension were the highest when probed at integrin-specific adhesions. These integrin-specific mechanoresponses required an intact actin cytoskeleton, and were dependent on tyrosine phosphatases and Ca2+ signaling. Cell asymmetric mechanoresponse to compression and extension remained after 5 minutes of tip-cell adhesion, revealing that asymmetric resistance to force directionality is an intrinsic property of lung cells, as in most soft tissues. Our findings provide new insights on how lung cells probe the mechanochemical properties of the microenvironment, an important process for migration, repair and tissue homeostasis.

Vulnerability of stream biota to climate change in mediterranean climate regions: a synthesis of ecological responses and conservation challenges

Freshwater species worldwide are experiencing dramatic declines partly attributable to ongoing climate change. It is expected that the future effects of climate change could be particularly severe in mediterranean climate (med-) regions, which host many endemic species already under great stress from the high level of human development. In this article, we review the climate and climate-induced changes in streams of med-regions and the responses of stream biota, focusing on both observed and anticipated ecological responses. We also discuss current knowledge gaps and conservation challenges. Expected climate alterations have already been observed in the last decades, and include: increased annual average air temperatures; decreased annual average precipitation; hydrologic alterations; and an increase in frequency, intensity and duration of extreme events, such as floods, droughts and fires. Recent observations, which are concordant with forecasts built, show stream biota of med-regions when facing climate changes tend to be displaced towards higher elevations and upper latitudes, communities tend to change their composition and homogenize, while some life-history traits seem to provide biota with resilience and resistance to adapt to the new conditions (as being short-lived, small, and resistant to low streamflow and desiccation). Nevertheless, such responses may be insufficient to cope with current and future environmental changes. Accurate forecasts of biotic changes and possible adaptations are difficult to obtain in med-regions mainly because of the difficulty of distinguishing disturbances due to natural variability from the effects of climate change, particularly regarding hydrology. Long-term studies are needed to disentangle such variability and improve knowledge regarding the ecological responses and the detection of early warning signals to climate change. Investments should focus on taxa beyond fish and macroinvertebrates, and in covering the less studied regions of Chile and South Africa. Scientists, policy makers and water managers must be involved in the climate change dialogue because the freshwater conservation concerns are huge.

Characterization of nontypable haemophilus influenzae isolates recovered from adult patients with underlying chronic lung disease reveals genotypic and phenotypic traits associated with persistent infection

Nontypable Haemophilus influenzae (NTHi) has emerged as an important opportunistic pathogen causing infection in adults suffering obstructive lung diseases. Existing evidence associates chronic infection by NTHi to the progression of the chronic respiratory disease, but specific features of NTHi associated with persistence have not been comprehensively addressed. To provide clues about adaptive strategies adopted by NTHi during persistent infection, we compared sequential persistent isolates with newly acquired isolates in sputa from six patients with chronic obstructive lung disease. Pulse field gel electrophoresis (PFGE) identified three patients with consecutive persistent strains and three with new strains. Phenotypic characterisation included infection of respiratory epithelial cells, bacterial self-aggregation, biofilm formation and resistance to antimicrobial peptides (AMP). Persistent isolates differed from new strains in showing low epithelial adhesion and inability to form biofilms when grown under continuous-flow culture conditions in microfermenters. Self-aggregation clustered the strains by patient, not by persistence. Increasing resistance to AMPs was observed for each series of persistent isolates; this was not associated with lipooligosaccharide decoration with phosphorylcholine or with lipid A acylation. Variation was further analyzed for the series of three persistent isolates recovered from patient 1. These isolates displayed comparable growth rate, natural transformation frequency and murine pulmonary infection. Genome sequencing of these three isolates revealed sequential acquisition of single-nucleotide variants in the AMP permease sapC, the heme acquisition systems hgpB, hgpC, hup and hxuC, the 3-deoxy-D-manno-octulosonic acid kinase kdkA, the long-chain fatty acid transporter ompP1, and the phosphoribosylamine glycine ligase purD. Collectively, we frame a range of pathogenic traits and a repertoire of genetic variants in the context of persistent infection by NTHi.

Variability of the wheat powdery mildew pathogen Blumeria graminis f. sp. tritici in the 2003 crop season

Wheat (Triticum aestivum) powdery mildew, caused by the biotrophic fungus Blumeria graminis f. sp. tritici, is one of the most severe foliar diseases attacking this crop, reducing grain yields by 10% to 62% in Brazil. The disease can be controlled by genetic resistance of the host, but the pathogen has physiological specialization, which enables it to infect wheat cultivars that have remained resistant for years. The objective of this work was to evaluate the variability of pathogenic strains of B. graminis f. sp. tritici collected in Brazil and the effectiveness of wheat resistance genes to powdery mildew in the 2003 crop season. Plants of a differential series were inoculated with each monopustular isolate. Thirty-one combinations of effective and ineffective resistance genes were identified. Only the gene Pm4a+... remained totally effective to all isolates, and gene Pm6 was highly effective (below 10% of susceptibility), whereas genes Pm3a and Pm8 were totally ineffective (susceptible to all isolates). Genes Pm3c, D1, and D2 showed low effectiveness (above 50% of susceptibility), and genes Pm1, 2, 4a, 1+?, and 2+Mld had mean effective results to most strains (susceptibility between 10% and 49%). The virulence formula Pm1, 3c, 4a, 6, 1+?, 2+Mld, 4a+..., D2 (effective genes) / 2, 3a, 8, D1 (ineffective genes) was most frequently found, accounting for 15% of the occurrences. The most frequent number of ineffective genes was seven, ranging from three to ten.

NATURAL DURABILITY OF Eucalyptus dunnii Maiden, Eucalyptus robusta Sm., Eucalyptus tereticornis Sm. AND Hovenia dulcis Thunb. WOOD IN FIELD AND FOREST ENVIRONMENT

AB STRACT This study aimed at evaluating the natural durability of Eucalyptus dunnii, Eucalyptus robusta, Eucalyptus tereticornis and Hovenia dulcis woods submitted to a deterioration test in two environments, field and forest. The test samples were buried until half of their length (150 mm). Evaluations were carried out each 45 days, totalizing a 405-day period, with three-repetition withdrawal of each species for environment, totalizing nine samples from each environment, making up 24 test samples for evaluation. After percentage calculations of mass loss and resistance degree classification, the deterioration index was adopted for decomposition evaluation and fungal decay potential determination of test samples. The study has been carried out in completely randomized design (CRD), evaluated through analysis of variance (ANOVA) with subsequent comparison of means by Turkey' s test, in a 5%-level of probability of error, along with regression analysis. Eucalyptus tereticornis wood presented lesser mass loss in both environments. Hovenia dulcis presented lesser deterioration probability in both environments. Forest environment test samples presented greater mass loss percentages and lesser deterioration index.

Corrosion behaviour of boiler tube materials during combustion of fuels containing Zn and Pb

Många förbränningsanläggningar som bränner utmanande bränslen såsom restfraktioner och avfall råkar ut för problem med ökad korrosion på överhettare och/eller vattenväggar pga. komponenter i bränslena som är korrosiva. För att minimera problemen i avfallseldade pannor hålls ångparametrarna på en relativt låg nivå, vilket drastiskt minskar energiproduktionen. Beläggningarna i avfallseldade pannor består till största delen av element som är förknippade med högtemperaturkorrosion: Cl, S, alkalimetaller, främst K och Na, och tungmetaller som Pb och Zn, och det finns också indikationer av Br-förekomst. Det låga ångtrycket i avfallseldade pannor påverkar också stålrörens temperatur i pannväggarna i eldstaden. I dagens läge hålls temperaturen normalt vid 300-400 °C. Alkalikloridorsakad (KCl, NaCl) högtemperaturkorrosion har inte rapporterats vara relevant vid såpass låga temperaturer, men närvaro av Zn- och Pb-komponenter i beläggningarna har påvisats förorsaka ökad korrosion redan vid 300-400 °C. Vid förbränning kan Zn och Pb reagera med S och Cl och bilda klorider och sulfater i rökgaserna. Dessa tungmetallföreningar är speciellt problematiska pga. de bildar lågsmältande saltblandningar. Dessa lågsmältande gasformiga eller fasta föreningar följer rökgasen och kan sedan fastna eller kondensera på kallare ytor på pannväggar eller överhettare för att sedan bilda aggressiva beläggningar. Tungmetallrika (Pb, Zn) klorider och sulfater ökar risken för korrosion, och effekten förstärks ytterligare vid närvaro av smälta. Motivet med den här studien var att få en bättre insikt i högtemperaturkorrosion förorsakad av Zn och Pb, samt att undersöka och prediktera beteendet och motståndskraften hos några stålkvaliteter som används i överhettare och pannväggar i tungmetallrika förhållanden och höga materialtemperaturer. Omfattande laboratorie-, småskale- och fullskaletest utfördes. Resultaten kan direkt utnyttjas i praktiska applikationer, t.ex. vid materialval, eller vid utveckling av korrosionsmotverkande verktyg för att hitta initierande faktorer och förstå deras effekt på högtemperaturkorrosion.

Uterine Doppler velocimetry of the uterine arteries in the second and third trimesters for the prediction of gestational outcome

PURPOSE: The aim of this longitudinal study was to investigate the value of uterine artery Doppler sonography during the second and third trimesters in the prediction of adverse pregnancy outcome in low-risk women. METHODS: From July 2011 to August 2012, a total of 205 singleton pregnant women presenting at our antenatal clinic were enrolled in this prospective study and were assessed for baseline demographic and obstetric data. They underwent ultrasound evaluation at the time of second and third trimesters, both included Doppler assessment of bilateral uterine arteries to determine the values of the pulsatility index (PI) and resistance index (RI) and presence of early diastolic notch. The endpoint of this study was assessing the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of Doppler ultrasonography of the uterine artery, for the prediction of adverse pregnancy outcomes including preeclampsia, stillbirth, placental abruption and preterm labor. RESULTS: The mean age of cases was 26.4±5.11. The uterine artery PI and RI values for both second (PI: 1.1±0.42 versus 1.53±0.59, p=0.002; RI: 0.55±0.09 versus 0.72±0.13, p=0.000 respectively) and third-trimester (PI: 0.77±0.31 versus 1.09±0.46, p=0.000; RI: 0.46±0.10 versus 0.60±0.14, p=0.010 respectively) evaluations were significantly higher in patients with adverse pregnancy outcome than in normal women. Combination of PI and RI >95th percentile and presence of bilateral notch in second trimester get sensitivity and specificity of 36.1 and 97% respectively, while these measures were 57.5 and 98.2% in third trimester. CONCLUSIONS: According to our study, it seems that uterine artery Doppler may be a valuable tool for the prediction of a variety of adverse outcomes in second and third trimesters.

Absence of intestinal colonization by vancomycin-resistant enterococci in nonhuman primates

The animal reservoirs of vancomycin-resistant enterococci (VRE) have important role in the epidemiology of the bacteria and resistant genes. The present work searched fecal samples taken off nonhuman primates for the presence of VRE. Resistance profiles, virulence traits, and genetic variability among enterococci isolates were also analyzed. The samples included Capuchin monkeys (Cebus apella, n=28) and Common marmoset (Callithrix penicillata, n=37) housed in the Primate Center of the University of Brasília, Brazil. Most individuals were captive monkeys from the Central-West and South-East regions of Brazil (n=48). We collected rectal swabs and carried out selective isolation followed by multiplex Polymerase Chain Reaction (PCR) to identify species and resistance genes. No vanA or vanB-containing enterococci were found. The carriage rates ranged from 1.5% for the VanC-type E. casseliflavus and E. gallinarum until 12.3% (n=8) for Enterococcus faecalis. All E. faecalis isolates showed susceptibility to vancomycin, teicoplanin, ampicillin, gentamicin, and streptomycin. The virulence genes ace and esp were prevalent (100.0%, 87.5%). Multilocus variable number of tandem repeats (MLVA) revealed diversity in the number of repeats among E. faecalis isolates and targets, which was higher for espC, efa5, and efa6. We identified six different MLVA genotypes that were divergent from those described in human beings. Also, they were clustered into two genogroups that showed host-specificity for the species Cebus apella or Callithrix penicillata. In conclusion, no vanA- or vanB-containing enterococci were found colonizing those primate individuals. This finding suggested that the primate individuals investigated in our study are not directly involved in the epidemiological chain of high-level vancomycin-resistant genes vanA or vanB in Brazil. Our study also showed that E. faecalis isolated from nonhuman primates carry virulence traits and have ability to spread their lineages among different individuals.

Irinotecan and oxaliplatin: an overview of the novel chemotherapeutic options for the treatment of advanced colorectal cancer

Colorectal cancer is one of the most frequent malignancies in humans and an important cause of cancer death. Metastatic colorectal cancer remains incurable with available systemic therapeutic options. The most active cytotoxic drug against this malignancy, the antimetabolite 5-fluorouracil, was developed more than forty years ago, and as a single agent produces responses in only 10 to 15% of patients which in general last less than one year. Efforts to ameliorate these poor results resulted in the 5-fluorouracil/leucovorin combination, which enhances response rates about two-fold, without, however, significantly improving survival rates. The recent emergence of a handful of new 5-fluorouracil analogues and folate antagonists, as well as the topoisomerase I inhibitor irinotecan, and the third-generation platinum compound oxaliplatin, is likely to alter this gloomy scenario. These agents are at least as effective as 5-fluorouracil in patients with advanced colorectal carcinoma, both untreated and previously treated with 5-fluorouracil-based regimens. This has led to the approval of irinotecan as second-line treatment for 5-fluorouracil-refractory disease, while the use of oxaliplatin has been suggested for patients having a defective 5-fluorouracil catabolism. Recently, FDA approved the combination of irinotecan with 5-fluorouracil and leucovorin for first-line treatment of advanced colon cancer. Based on the synergistic preclinical antitumor effects of some of these agents, their meaningful single-agent activity, distinct mechanisms of cytotoxicity and resistance, and only partially overlapping toxicity profiles, effective combination regimens are now being developed, which are likely to lead to a new, more hopeful era for patients suffering from advanced colorectal carcinoma.

Rapid detection of multidrug-resistant Mycobacterium tuberculosis using the mycobacteria growth indicator tube (MGIT) system

The emergence of multidrug-resistant strains of Mycobacterium tuberculosis has increased the need for rapid drug susceptibility tests, which are needed for adequate patient treatment. The objective of the present study was to evaluate the mycobacteria growth indicator tube (MGIT) system to detect multidrug-resistant M. tuberculosis strains. The MGIT system was compared with two standard methods (proportion and resistance ratio methods). One hundred clinical M. tuberculosis isolates [25 susceptible to isoniazid (INH) and rifampicin (RIF), 20 resistant to INH, 30 resistant to INH-RIF, and 25 resistant to INH-RIF and other drugs] obtained in the State of São Paulo were tested for INH and RIF susceptibility. Full agreement among the tests was found for all sensitive and all INH-resistant strains. For RIF-resistant strains results among the tests agreed for 53 (96.4%) of 55 isolates. Results were obtained within 6 days (range, 5 to 8 days), 28 days and 12 days when using MGIT, the proportion method and the resistance ratio methods, respectively. The MGIT system presented an overall agreement of 96% when compared with two standard methods. These data show that the MGIT system is rapid, sensitive and efficient for the early detection of multidrug-resistant M. tuberculosis.

Effect of cyhalothrin on Ehrlich tumor growth and macrophage activity in mice

Cyhalothrin, a pyrethroid insecticide, induces stress-like symptoms, increases c-fos immunoreactivity in the paraventricular nucleus of the hypothalamus, and decreases innate immune responses in laboratory animals. Macrophages are key elements in cellular immune responses and operate at the tumor-host interface. This study investigated the relationship among cyhalothrin effects on Ehrlich tumor growth, serum corticosterone levels and peritoneal macrophage activity in mice. Three experiments were done with 10 experimental (single gavage administration of 3.0 mg/kg cyhalothrin daily for 7 days) and 10 control (single gavage administration of 1.0 mL/kg vehicle of cyhalothrin preparation daily for 7 days) isogenic BALB/c mice in each experiment. Cyhalothrin i) increased Ehrlich ascitic tumor growth after ip administration of 5.0 x 106 tumor cells, i.e., ascitic fluid volume (control = 1.97 ± 0.39 mL and experimental = 2.71 ± 0.92 mL; P < 0.05), concentration of tumor cells/mL in the ascitic fluid (control = 111.95 ± 16.73 x 106 and experimental = 144.60 ± 33.18 x 106; P < 0.05), and total number of tumor cells in the ascitic fluid (control = 226.91 ± 43.22 x 106 and experimental = 349.40 ± 106.38 x 106; P < 0.05); ii) increased serum corticosterone levels (control = 200.0 ± 48.3 ng/mL and experimental = 420.0 ± 75.5 ng/mL; P < 0.05), and iii) decreased the intensity of macrophage phagocytosis (control = 132.3 ± 19.7 and experimental = 116.2 ± 4.6; P < 0.05) and oxidative burst (control = 173.7 ± 40.8 and experimental= 99.58 ± 41.7; P < 0.05) in vitro in the presence of Staphylococcus aureus. These data provide evidence that cyhalothrin simultaneously alters host resistance to Ehrlich tumor growth, hypothalamic-pituitary-adrenocortical (HPA) axis function, and peritoneal macrophage activity. The results are discussed in terms of data suggesting a link between stress, HPA axis activation and resistance to tumor growth.

Endothelial dysfunction in cardiovascular and endocrine-metabolic diseases: an update

The endothelium plays a vital role in maintaining circulatory homeostasis by the release of relaxing and contracting factors. Any change in this balance may result in a process known as endothelial dysfunction that leads to impaired control of vascular tone and contributes to the pathogenesis of some cardiovascular and endocrine/metabolic diseases. Reduced endothelium-derived nitric oxide (NO) bioavailability and increased production of thromboxane A2, prostaglandin H2 and superoxide anion in conductance and resistance arteries are commonly associated with endothelial dysfunction in hypertensive, diabetic and obese animals, resulting in reduced endothelium-dependent vasodilatation and in increased vasoconstrictor responses. In addition, recent studies have demonstrated the role of enhanced overactivation ofβ-adrenergic receptors inducing vascular cytokine production and endothelial NO synthase (eNOS) uncoupling that seem to be the mechanisms underlying endothelial dysfunction in hypertension, heart failure and in endocrine-metabolic disorders. However, some adaptive mechanisms can occur in the initial stages of hypertension, such as increased NO production by eNOS. The present review focuses on the role of NO bioavailability, eNOS uncoupling, cyclooxygenase-derived products and pro-inflammatory factors on the endothelial dysfunction that occurs in hypertension, sympathetic hyperactivity, diabetes mellitus, and obesity. These are cardiovascular and endocrine-metabolic diseases of high incidence and mortality around the world, especially in developing countries and endothelial dysfunction contributes to triggering, maintenance and worsening of these pathological situations.

Nitric oxide synthesis and biological functions of nitric oxide released from ruthenium compounds

During three decades, an enormous number of studies have demonstrated the critical role of nitric oxide (NO) as a second messenger engaged in the activation of many systems including vascular smooth muscle relaxation. The underlying cellular mechanisms involved in vasodilatation are essentially due to soluble guanylyl-cyclase (sGC) modulation in the cytoplasm of vascular smooth cells. sGC activation culminates in cyclic GMP (cGMP) production, which in turn leads to protein kinase G (PKG) activation. NO binds to the sGC heme moiety, thereby activating this enzyme. Activation of the NO-sGC-cGMP-PKG pathway entails Ca2+ signaling reduction and vasodilatation. Endothelium dysfunction leads to decreased production or bioavailability of endogenous NO that could contribute to vascular diseases. Nitrosyl ruthenium complexes have been studied as a new class of NO donors with potential therapeutic use in order to supply the NO deficiency. In this context, this article shall provide a brief review of the effects exerted by the NO that is enzymatically produced via endothelial NO-synthase (eNOS) activation and by the NO released from NO donor compounds in the vascular smooth muscle cells on both conduit and resistance arteries, as well as veins. In addition, the involvement of the nitrite molecule as an endogenous NO reservoir engaged in vasodilatation will be described.