NMR spectroscopy and chemometrics as a tool for anti-TNFα activity screening in crude extracts of grapes and other berries

The identification of active ingredients in crude plant extracts offers great advantages. In this study, nuclear magnetic resonance and chemometrics were used for the screening of in vitro anti-TNF?? activity in different berry types. Solid phase extraction was applied and the resulting water, methanol?Cwater (1:1), and methanol fractions were tested for the activity. The methanol?Cwater fraction contained most of the phenolics and showed significantly higher activity than the other two fractions. In the second phase of this study, grapes from ??Trincadeira??, ??Touriga Nacional??, and ??Aragonês??, at four developmental stages were metabolically classified and tested for the TNF?? inhibition. The initial stages of grape development, green and veraison, were found more active against TNF?? production as compared to the later ripe and harvest stages. Among the cultivars, ??Touriga Nacional?? was found to be the most potent inhibitor. Different multivariate data analyses algorithms based on projections to latent structures were applied to correlate the NMR and TNF?? inhibition data. The variable importance in the projections plot showed that phenolics like quercetin, myricetin, (+)-catechin, (?)-epicatechin, caftarate, and coutarate, were positively correlated with high activity. This work demonstrates the great potential of NMR spectroscopy in combination with chemometrics for the screening of large set of crude extracts, to study the effects of different variables on the activity, and identifying active compounds in complex mixtures like plant extracts.     

Differences in worst-case scenarios calculated by fixed length and rolling average methods in professional soccer match-play

The aims of this study were to describe the worst-case scenarios (WCS) in professional soccer players calculated by fixed length and rolling average methods with regards to each playing position. This was done, firstly, by comparing total distance (TD covered in the WCS; secondly, by comparing high-speed running distance (HSRD); and thirdly, by comparing sprint distance (SPD). The study was conducted over a three-mesocycle competitive period. The WCS of three distance-related variables (TD, HSRD, SPD) in four time windows (1, 3, 5, 10 minutes) were calculated according to playing position (central defender; full-back; midfielder, wide midfielder, and forward) using fixed length and rolling average methods. A significant effect of the type of method used to calculate the WCS in TD (F_{(1, 142)} = 151.49, p < 0.001, ηp² = 0.52), HSRD (F_{(1, 138)} = 336.95, p < 0.001, ηp² = 0.71) and SPD (F_{(1, 138)} = 76.74, p < 0.001, ηp² = 0.36) was observed. In addition, there was a significant interaction between type of method and WCS duration in TD (F_{(1.36, 193.53)} = 41.95, p < 0.001, ηp² = 0.23), HSRD (F_{(2.28, 315.11)} = 21.77, p < 0.001, ηp² = 0.14) and SPD (F_{(2.59, 358.41)} = 6.93, p < 0.001, ηp² = 0.05). In conclusion, the use of fixed length methods of different durations significantly underestimated the WCS of TD, HSRD and SPD across the most common playing positions in professional soccer players. Therefore, the application of rolling averages is recommended for an appropriate WCS analysis in professional soccer match-play.     

Enalapril treatment corrects the reduced response to bradykinin in diabetes increasing the B2 protein expression

Considering the growing importance of the interaction between components of kallikrein-kinin and renin-angiotensin systems in physiological and pathological processes, particularly in diabetes mellitus, the aim of the present study was to investigate the effect of enalapril on the reduced response of bradykinin and on the interaction between angiotensin-(1-7) (Ang-(1-7)) and bradykinin (BK), important components of these systems, in an insulin-resistance model of diabetes. For the above purpose, the response of mesenteric arterioles of anesthetized neonatal streptozotocin-induced (n-STZ) diabetic and control rats was evaluated using intravital microscopy. In n-STZ diabetic rats, enalapril treatment restored the reduced response to BK but not the potentiation of BK by Ang-(1-7) present in non-diabetic rats. The restorative effect of enalapril was observed at a dose that did not correct the altered parameters induced by diabetes such as hyperglycemia, glicosuria, insulin resistance but did reduce the high blood pressure levels of n-SZT diabetic rats. There was no difference in mRNA and protein expressions of B1 and B2 kinin receptor subtypes between n-STZ diabetic and control rats. Enalapril treatment increased the B2 kinin receptor expression. From our data, we conclude that in diabetes enalapril corrects the impaired BK response probably by increasing the expression of B2 receptors. The lack of potentiation of BK by Ang-(1-7) is not corrected by this agent.     

Hepatitis B virus screening in contacts of blood donors with antibodies against core protein (anti-HBC), but without surface antigen (HBsAg)

To increase blood safety Brazil introduced screening for anti-HBc among blood donors in 1993. There was a decrease in the hepatitis B virus (HB V) transmission, but this measure identified a great number ofHBsAg-negative, anti-HBc-positive donors. Surveillance policy determines that contacts of HBV carriers should be screened to HBV markers, but there is no recommendation about how to guide contacts of HBsAg-negative, anti-HBc-positive donors. Aiming to evaluate whether the contacts of this group are at greater risk for HB V infection, a cross-sectional study was performed to compare prevalence of HBV infection between contacts of HBsAg-positive blood donors (group I) and contacts of HBsAg-negative, anti-HBc-positive donors (group II). Contacts were submitted to a questionnaire and blood tests for HBV markers. In group I (n = 143), 53 (37.1%) were anti-HBc-positive and 11 (7.7%) were HBsAg-positive. In group II (n = 111), there were 9 and 0.9%, respectively. HB V exposure was associated with group I, sexual activity, blood transfusion, being one of the donor's parents, and living for more than ten years with the donor. Regarding the families as sample units, it was more common to find at least one member with HBV markers (p < 0.05) among the families of group I compared to group II. Contacts of HBsAg-negative, anti-HBc-positive individuals presented a much lower risk of having already been exposed to HBV and there is no need to screen them for HBV in low to moderate prevalence populations.     

Modulation of kinin B1 receptor expression by endogenous angiotensin II in hypertensive rats

We investigated the expression and localization of B1 receptor in tissues of rats submitted to a renin-dependent model of hypertension (2K-1C), and analyzed the influence of endogenous Ang II in modulating the in vivo expression of these receptors. B1 mRNA levels in the heart, kidney and thoracic aorta were quantified by real time PCR, B1 receptor protein expression was assessed by immunohistochemistry, plasma Ang II levels were analyzed by radioimmunoassay and the effects of AT1 receptor blockade were determined after losartan treatment. 2K-1C rats presented a marked increase in Ang II levels when compared to sham-operated rats. In parallel, cardiac- (but not renal and aortic) B1 mRNA levels were 15-fold higher in 2K-1C than in sham rats. In 2K-1C, B1 expression was detected in the endothelium of small cardiac arteries and in cardiomyocytes. Losartan completely reverted the increased B1 mRNA levels and significantly decreased the protein expression observed in 2K-1C rats, despite reducing, but not normalizing blood pressure. We conclude that in the 2K-1C rat, induction of cardiac B1 receptor might be tightly linked to AT1 receptor activation. These data suggest the existence of a new site of interaction between kinins and angiotensins, and might provide important contributions for a better understanding of the pathophysiology of hypertension.     

Natural Atypical Listeria innocua Strains with Listeria monocytogenes Pathogenicity Island 1 Genes

Identification of bona fide Listeria isolates into the six species of the genus normally requires only a few tests. Aberrant isolates do occur, but even then only one or two extra confirmatory tests are generally needed for identification to species level. We have discovered a hemolytic-positive, rhamnose and xylose fermentation-negative Listeria strain with surprising recalcitrance to identification to the species level due to contradictory results in standard confirmatory tests. The issue had to be resolved by using total DNA-DNA hybridization testing and then confirmed by further specific PCR-based tests including a Listeria microarray assay. The results show that this isolate is indeed a novel one. Its discovery provides the first fully documented instance of a hemolytic Listeria innocua strain. This species, by definition, is typically nonhemolytic. The L. innocua isolate contains all the members of the PrfA-regulated virulence gene cluster (Listeria pathogenicity island 1) of L. monocytogenes. It is avirulent in the mouse pathogenicity test. Avirulence is likely at least partly due to the absence of the L. monocytogenes-specific allele of iap, as well as the absence of inlA, inlB, inlC, and daaA. At least two of the virulence cluster genes, hly and plcA, which encode the L. monocytogenes hemolysin (listeriolysin O) and inositol-specific phospholipase C, respectively, are phenotypically expressed in this L. innocua strain. The detection by PCR assays of specific L. innocua genes (lin0198, lin0372, lin0419, lin0558, lin1068, lin1073, lin1074, lin2454, and lin2693) and noncoding intergenic regions (lin0454-lin0455 and nadA-lin2134) in the strain is consistent with its L. innocua DNA-DNA hybridization identity. Additional distinctly different hemolytic L. innocua strains were also studied.     

Effect of Campsurus notatus on NH+4, DOC Fluxes,O2 Uptake and Bacterioplankton Production in Experimental Microcosms with Sediment‐Water Interface of an Amazonian Lake Impacted by Bauxite Tailings

The aim of this study was to evaluate the influence of Campusurus notatus Eaton 1868 (Ephemeroptera: Polimitarciydae) and the impact of bauxite tailings on ammonium (NH₄⁺) and dissolved organic carbon (DOC) fluxes, oxygen uptake and bacterioplankton production in the sediment‐water interface of Lake Batata, a shallow Amazonian floodplain lake. Mesocosms were constructed from natural and impacted areas of Lake Batata, to reproduce the sediment‐water interface. The cores were incubated with 0 to 2,388 ind m^–2 of Campsurus notatus nymphs, and the changes in NH₄⁺, DOC, O₂ concentration and bacterioplankton production in the overlying water column were measured. Ammonium efflux (F = 9.8, p < 0.05, multiple regression) and oxygen uptake (F = 11.8, p < 0.05) showed a significant correlation with the density of C. notatus in the cores with natural sediment. No differences on DOC release were observed in cores with natural or impacted sediment. In the cores incubated with natural sediment and nymphs of C. notatus, a significant increase (Two‐way ANOVA, p < 0.05) in bacterial production (0.44 μg C l^–1 h^–1) was observed after 3 hours of incubation. In cores incubated with sediment impacted by bauxite tailings, there was no difference in bacterial production with and without C. notatus. We conclude that C. notatus is an important bioturbator in Lake Batata, increasing the turnover rate of nitrogen (NH₄⁺) at the sediment‐water interface and bacterial production in cores incubated with natural sediment. It is also clear that bauxite tailings reduce the nutrients turnover rates in impacted regions of Lake Batata and influence bacterial production.     

Metformin Induces Apoptosis and Cell Cycle Arrest Mediated by Oxidative Stress,AMPK and FOXO3a in MCF-7 Breast Cancer Cells

Recent studies have demonstrated that the anti-diabetic drug, metformin, can exhibit direct antitumoral effects, or can indirectly decrease tumor proliferation by improving insulin sensitivity. Despite these recent advances, the underlying molecular mechanisms involved in decreasing tumor formation are not well understood. In this study, we examined the antiproliferative role and mechanism of action of metformin in MCF-7 cancer cells treated with 10 mM of metformin for 24, 48, and 72 hours. Using BrdU and the MTT assay, it was found that metformin demonstrated an antiproliferative effect in MCF-7 cells that occurred in a time- and concentration- dependent manner. Flow cytometry was used to analyze markers of cell cycle, apoptosis, necrosis and oxidative stress. Exposure to metformin induced cell cycle arrest in G₀-G₁ phase and increased cell apoptosis and necrosis, which were associated with increased oxidative stress. Gene and protein expression were determined in MCF-7 cells by real time RT-PCR and western blotting, respectively. In MCF-7 cells metformin decreased the activation of IRβ, Akt and ERK1/2, increased p-AMPK, FOXO3a, p27, Bax and cleaved caspase-3, and decreased phosphorylation of p70S6K and Bcl-2 protein expression. Co-treatment with metformin and H₂O₂ increased oxidative stress which was associated with reduced cell number. In the presence of metformin, treating with SOD and catalase improved cell viability. Treatment with metformin resulted in an increase in p-p38 MAPK, catalase, MnSOD and Cu/Zn SOD protein expression. These results show that metformin has an antiproliferative effect associated with cell cycle arrest and apoptosis, which is mediated by oxidative stress, as well as AMPK and FOXO3a activation. Our study further reinforces the potential benefit of metformin in cancer treatment and provides novel mechanistic insight into its antiproliferative role.     

Multiple facets of biodiversity are threatened by mining-induced land-use change in the Brazilian Amazon

Aim

Mining is increasingly pressuring areas of critical importance for biodiversity conservation, such as the Brazilian Amazon. Biodiversity data are limited in the tropics, restricting the scope for risks to be appropriately estimated before mineral licensing decisions are made. As the distributions and range sizes of other taxa differ markedly from those of vertebrates—the common proxy for analysis of risk to biodiversity from mining—whether mining threatens lesser-studied taxonomic groups differentially at a regional scale is unclear.

Location

Brazilian Amazon.

Methods

We assess risks to several facets of biodiversity from industrial mining by comparing mining areas (within 70 km of an active mining lease) and areas unaffected by mining, employing species richness, species endemism, phylogenetic diversity and phylogenetic endemism metrics calculated for angiosperms, arthropods and vertebrates.

Results

Mining areas contained higher densities of species occurrence records than the unaffected landscape, and we accounted for this sampling bias in our analyses. None of the four biodiversity metrics differed between mining and nonmining areas for vertebrates. For arthropods, species endemism was greater in mined areas. Mined areas also had greater angiosperm species richness, phylogenetic diversity and phylogenetic endemism, although less species endemism than unmined areas.

Main Conclusions

Unlike for vertebrates, facets of angiosperm and arthropod diversity are relatively higher in areas of mining activity, underscoring the need to consider multiple taxonomic groups and biodiversity facets when assessing risk and evaluating management options for mining threats. Particularly concerning is the proximity of mining to areas supporting deep evolutionary history, which may be impossible to recover or replace. As pressures to expand mining in the Amazon grow, impact assessments with broader taxonomic reach and metric focus will be vital to conserving biodiversity in mining regions.