Expression of genes related to apoptosis,cell cycle and signaling pathways are independent of <Emphasis Type="Italic">TP53</Emphasis> status in urinary bladder cancer cells

Urinary bladder cancer is the fourth most common malignancy in the Western world. Transitional cell carcinoma (TCC) is the most common subtype, accounting for about 90% of all bladder cancers. The TP53 gene plays an essential role in the regulation of the cell cycle and apoptosis and therefore contributes to cellular transformation and malignancy; however, little is known about the differential gene expression patterns in human tumors that present with the wild-type or mutated TP53 gene. Therefore, because gene profiling can provide new insights into the molecular biology of bladder cancer, the present study aimed to compare the molecular profiles of bladder cancer cell lines with different TP53 alleles, including the wild type (RT4) and two mutants (5637, with mutations in codons 280 and 72; and T24, a TP53 allele encoding an in-frame deletion of tyrosine 126). Unsupervised hierarchical clustering and gene networks were constructed based on data generated by cDNA microarrays using mRNA from the three cell lines. Differentially expressed genes related to the cell cycle, cell division, cell death, and cell proliferation were observed in the three cell lines. However, the cDNA microarray data did not cluster cell lines based on their TP53 allele. The gene profiles of the RT4 cells were more similar to those of T24 than to those of the 5637 cells. While the deregulation of both the cell cycle and the apoptotic pathways was particularly related to TCC, these alterations were not associated with the TP53 status.     

Insights into HLA-restricted T cell responses in a novel mouse model of dengue virus infection point toward new implications for vaccine design

The frequency of dengue virus (DENV) infection has increased dramatically in the last few decades, and the lack of a vaccine has led to significant morbidity and mortality worldwide. To date, a convenient murine system to study human T cell responses to DENV has not been available. Mice transgenic for HLA are widely used to model human immune responses, and it has been shown that mouse-passaged DENV is able to replicate to significant levels in IFN-α/βR(-/-) mice. To cover a wide range of HLA phenotypes, we backcrossed IFN-α/βR(-/-) mice with HLA A*0201, A*0101, A*1101, B*0702, and DRB1*0101-transgenic mice. A DENV proteome-wide screen identified a total of 42 epitopes across all HLA-transgenic IFN-α/βR(-/-) strains tested. In contrast, only eight of these elicited responses in the corresponding IFN-α/βR(+/+) mice. We were able to identify T cell epitopes from 9 out of the 10 DENV proteins. However, the majority of responses were derived from the highly conserved nonstructural proteins NS3 and NS5. The relevance of this model is further demonstrated by the fact that most of the epitopes identified in our murine system are also recognized by PBMC from DENV-exposed human donors, and a dominance of HLA B*0702-restricted responses has been detected in both systems. Our results provide new insights into HLA-restricted T cell responses against DENV, and we describe in this study a novel murine model that allows the investigation of T cell-mediated immune mechanisms relevant to vaccine design.     

DNA damage in patients who underwent minimally invasive surgery under inhalation or intravenous anesthesia

Recent studies have demonstrated the genotoxicity of anesthetics in patients who have undergone surgery and in personnel who are occupationally exposed to anesthetics. However, these findings are controversial. Herein, we used the comet assay (single-cell gel electrophoresis) to investigate the genotoxic effects of two volatile compounds [isoflurane (ISF) and sevoflurane (SVF)] that are used in inhalation anesthesia, and of one intravenous (iv) anesthetic compound [propofol (PF)]. The groups consisted of 45 patients who underwent minimally invasive surgery that lasted at least 2h. Patients were classified as physical status I using the criteria of the American Society of Anesthesiologists (ASA) and were randomly allocated to receive ISF, SVF or PF anesthesia. Venous blood samples were collected at three time points as follows: before the premedication and the induction of anesthesia (T(0)); 2h after the beginning of anesthesia (T(1)); and on the day following surgery (T(2)). DNA damage (strand breaks and alkali-labile sites) was evaluated in peripheral blood lymphocytes. For each patient, one hundred nucleoids were analyzed per time point using a semi-automated image system. Patients did not differ with respect to their demographic characteristics, the duration of surgery, or the total doses of intraoperative drugs. The amount of DNA damage was not different among the three groups before anesthesia (T(0)). No statistically significant (p>0.05) increase in DNA damage was detected during (T(1)) or after anesthesia (T(2)) using three different protocols (ISF, SVF or PF). In conclusion, general anesthesia with inhaled ISF and SVF or iv PF did not induce DNA strand breaks or alkali-labile sites in peripheral lymphocytes. Therefore, our results show that the genotoxic risk of these anesthetics, for healthy patients undergoing minimally invasive otorhinological surgery, is low or even absent.     

Itk functions to control actin polymerization at the immune synapse through localized activation of Cdc42 and WASP

Actin polymerization at the immune synapse is required for T cell activation and effector function; however, the relevant regulatory pathways remain poorly understood. We showed previously that binding to antigen presenting cells (APCs) induces localized activation of Cdc42 and Wiskott-Aldrich Syndrome protein (WASP) at the immune synapse. Several lines of evidence suggest that Tec kinases could interact with WASP-dependent actin regulatory processes. Since T cells from Rlk-/-, Itk-/-, and Rlk-/- x Itk-/- mice have defects in signaling and development, we asked whether Itk or Rlk function in actin polymerization at the immune synapse. We find that Itk-/- and Rlk-/- x Itk-/- T cells are defective in actin polymerization and conjugate formation in response to antigen-pulsed APCs. Itk functions downstream of the TCR, since similar defects were observed upon TCR engagement alone. Using conformation-specific probes, we show that although the recruitment of WASP and Arp2/3 complex to the immune synapse proceeds normally, the localized activation of Cdc42 and WASP is defective. Finally, we find that the defect in Cdc42 activation likely stems from a requirement for Itk in the recruitment of Vav to the immune synapse. Our results identify Itk as a key element of the pathway leading to localized actin polymerization at the immune synapse.     

ABCA1-dependent lipid efflux to apolipoprotein A-I mediates HDL particle formation and decreases VLDL secretion from murine hepatocytes

High levels of expression of the ATP binding cassette transporter A1 (ABCA1) in the liver and the need to over- or underexpress hepatic ABCA1 to impact plasma HDL levels in mice suggest a major role of the liver in HDL formation and in determining circulating HDL levels. Cultured murine hepatocytes were used to examine the role of hepatic ABCA1 in mediating the lipidation of apolipoprotein A-I (apoA-I) for HDL particle formation. Exogenous apoA-I stimulated cholesterol efflux to the medium from wild-type hepatocytes, but not from ABCA1-deficient (abca1(-/-)) hepatocytes. ApoA-I induced the formation of new HDL particles and enhanced the lipidation of endogenously secreted murine apoA-I in ABCA1-expressing but not abca1(-/-) hepatocytes. ABCA1-dependent cholesterol mobilization to apoA-I increased new cholesterol synthesis, indicating depletion of the regulatory pool of hepatocyte cholesterol during HDL formation. Secretion of triacylglycerol and apoB was decreased following apoA-I incubation with ABCA1-expressing but not abca1(-/-) hepatocytes. These results support a major role for hepatocyte ABCA1 in generating a critical pool of HDL precursor particles that enhance further HDL generation and passive cholesterol mobilization in the periphery. The results also suggest that diversion of hepatocyte cholesterol into the "reverse" cholesterol transport pathway diminishes cholesterol availability for apoB-containing lipoprotein secretion by the liver.     

In Vitro Characterization of the Impact of Different Substrates on Metabolite Production,Energy Extraction and Composition of Gut Microbiota from Lean and Obese Subjects

The aim of this study was to investigate the effect of galacto-oligosaccharides, lactulose, apple fiber and sugar beet pectin on the composition and activity of human colonic microbiota of lean and obese healthy subjects using an in vitro model of the proximal colon: TIM-2. Substrate fermentation was assessed by measuring the production of short-chain and branched-chain fatty acids, lactate and ammonia and by studying the composition of the bacterial communities over time. The results suggest that energy harvest (in terms of metabolites) of lean and obese microbiotas is different and may depend on the fermentable substrate. For galacto-oligosaccharides and lactulose, the cumulative amount of short-chain fatty acids plus lactate produced in TIM-2 was lower in the fermentation experiments with the lean microbiota (123 and 155 mmol, respectively) compared to the obese (162 and 173 mmol, respectively). This was reversed for the pectin and the fiber. The absolute amount produced of short-chain fatty acids including lactate was higher after 72 h in the fermentation experiments with apple fiber-L (108 mmol) than with apple fiber-O (92 mmol). Sugar beet-L was also higher (130 mmol) compared to sugar beet-O (103 mmol). Galacto-oligosaccharides and lactulose boosted the balance of health-promoting over toxic metabolites produced by the microbiota from obese subjects. Firmicutes were more predominant in the inoculum prepared from feces of obese subjects compared to lean subjects. The average abundance at time zero was 92% and 74%, respectively. On the other hand, Bacteroidetes were more dominant in the microbiota prepared with homogenates from lean subjects with an average abundance of 22% compared with the microbiota prepared with homogenates from obese subjects (3.6%). This study brings evidence that different fermentable carbohydrates are fermented differently by lean and obese microbiotas, which contributes to the understanding of the role of diet and the microbiota in tackling obesity.     

Isolation and Identification of Environmental Mycobacteria in the Waters of a Hemodialysis Center

The use of poorly treated water during hemodialysis may lead to contamination with nontuberculous mycobacteria (NTM). This study aimed to isolate and identify NTM species in the water of a Brazilian hemodialysis center. We collected 210 samples of water from the hydric system of the unit (post-osmosis system, hemodialysis rooms, reuse system, and hemodialysis equipment) and from the municipal supply network; we isolated the NTM by a classic microbiological technique and identified them by the PCR restriction enzyme pattern of the hsp65 gene (PRA). Fifty-one (24.3 %) of the collected samples tested positive for NTM; both the municipal supply network (2 samples, 3.2 %) and the hydric system of the hemodialysis center (49 samples, 96.1 %) contained NTM. We isolated and identified potentially pathogenic bacteria such as Mycobacterium lentiflavum (59.0 %) and M. kansasii (5.0 %), as well as rarely pathogenic bacteria like M. gordonae (24.0 %), M. gastri (8.0 %), and M. szulgai (4.0 %). The ability of NTM to cause diseases is well documented in the literature. Therefore, the identification of NTM in the water of a Brazilian hemodialysis center calls for more effective water disinfection procedures in this unit.     

Effects of El Niño Southern Oscillation on avian breeding phenology

Aim

Climate oscillations are known to influence the reproductive phenology of birds. Here, we quantify the effects of cyclic climatic variation, specifically El Niño Southern Oscillation (ENSO), on birds that breed opportunistically. We aim to show how inter‐decadal climate fluctuations influence opportunistic breeding. This knowledge is essential for tracking the phenological responses of birds to climate change.

Location

Temperate and arid Australia.

Methods

We assessed variation in egg‐laying (start, peak, conclusion, length) during the three phases of ENSO (El Niño, La Niña and Neutral) for 64 temperate and 15 arid region species using ~80,000 observations. Linear mixed‐effect models and analysis of variance were used to (1) determine if, on average within each region, egg‐laying dates differed significantly among species between Neutral‐El Niño and Neutral‐La Niña phases, and (2) assess how La Niña and El Niño episodes influence egg‐laying in birds which breed early in the year.

Results

During La Niña phases, which are characterized by mild/wet conditions, most bird species in the temperate and arid regions exhibited longer egg‐laying periods relative to Neutral phases. However, there was substantial variation across species. This effect was strongly seasonal; species breeding in spring experienced the greatest increases in egg‐laying periods during La Niña. Further, we found only small differences in peak egg‐laying dates during Neutral and La Niña in the arid region; suggesting that hot temperatures may constrain breeding regardless of rainfall. The effects of El Niño on breeding phenology were not consistent in the temperate and arid regions and may be confounded by highly mobile species opportunistically moving and breeding with localized rainfall during dry periods.

Main conclusions

In both arid and temperate regions, increased rainfall associated with La Niña phases positively influences avian breeding, and likely recruitment. However, dry El Niño phases may not have the dramatic impacts on breeding phenology that are commonly assumed.

     

Contrasting species responses to continued nitrogen and phosphorus addition in tropical montane forest tree seedlings

Global changes in nutrient deposition rates are likely to have profound effects on plant communities, particularly in the nutrient‐limited systems of the tropics. We studied the effects of increased nutrient availability on the seedlings of six tree species in montane forests of southern Ecuador in situ. After five years of continued N, P, or N+P addition, naturally grown seedlings of each of the two most common species at each elevation (1000, 2000, and 3000 m asl) were harvested for analyses of leaf morphology, nutrient content, herbivory, and tissue biomass allocation. Most species showed increased foliar N and P concentrations after addition of each respective element. Leaf tissue N:P ratios of >20 in the control plants of all species suggest that P is more growth‐limiting in these forests than N. Leaf morphological responses to nutrient addition were species and nutrient specific, with some species (Hedyosmum purparescens, Graffenrieda emarginata) exhibiting increased specific leaf area (SLA), and others (Graffenrieda harlingii) increased leaf area ratios (LAR). Pouteria torta (1000 m) had lower SLA and LAR after P addition. Increased herbivory was only evident in G. emarginata (after N and N+P addition). Only the species from 3000 m asl modified biomass allocation after nutrient addition. In general, N and N+P addition more strongly affected the species studied at the upper elevations, whereas P addition had a similar range of effects on the species at all elevations. We conclude that the responses of the studied tropical montane forest tree seedlings to chronic N and P addition are highly species‐specific and that successful adaptation to increased nutrient availability will depend on species‐specific morphological and physiological plasticity.