Comparison of vitrification and slow cooling for umbilical tissues

The tissue cryopreservation maintains the cellular metabolism in a quiescence state and makes the conservation possible for an indefinite period of time. The choice of an appropriate cryopreservation protocol is essential for maintenance of cryopreserved tissue banks. This study evaluated 10 samples of umbilical cord, from which small fragments of tissue (Wharton’s jelly and cord lining membrane) were subjected to two protocols of cryopreservation: slow cooling and vitrification. The samples were frozen for a period of time ranging from 5 to 78 days. The efficiency of cryopreservation was evaluated by testing cell viability, histological analysis, cell culture, cytogenetic analysis and comparison with the results of the fresh samples. The results showed that the slow cooling protocol was more efficient than the vitrification for cryopreservation of umbilical cord tissue, because it has caused fewer changes in the structure of tissue (edema and degeneration of the epithelium) and, despite the significant decrease cell viability compared to fresh samples, the ability of cell proliferation in vitro was preserved in most samples. In conclusion, this study showed that it is possible to cryopreserve small fragments of tissue from the umbilical cord and, to obtain viable cells capable of proliferation in vitro after thawing, contributing to the creation of a frozen tissue bank.     

Predictors of Attrition and Immunological Failure in HIV-1 Patients on Highly Active Antiretroviral Therapy from Different Healthcare Settings in Mozambique

In Mozambique, the evaluation of retention in HIV care and ART programmes is limited. To assess rate and predictors of attrition (no retention in care) and HAART effectiveness in HIV-1 infected patients who pay for medication and laboratory testing in Mozambique, we conducted a multicenter survey of HIV-1-infected patients who started HAART during 2002–2006. Cox proportional hazard models were used to assess risk of attrition and of therapy failure. Overall, 142 patients from 16 healthcare centers located in the capital city Maputo were followed-up for 22.2 months (12.1–46.7). The retention rate was 75%, 48% and 37% after one, two and three years, respectively. Risk of attrition was lower in patients with higher baseline CD4 count (P = 0.022) and attending healthcare center 1 (HCC1) (P = 0.013). The proportion of individuals with CD4 count ≤200 cells/µL was 55% (78/142) at baseline and decreased to 6% (3/52) at 36 months. Among the patients with available VL, 86% (64/74) achieved undetectable VL levels. The rate of immunologic failure was 17.2% (95% CI: 12.6–22.9) per 100 person-years. Risk of failure was associated to higher baseline CD4 count (P = 0.002), likely reflecting low adherence levels, and decreased with baseline VL ≥10,000 copies/mL (P = 0.033). These results suggest that HAART can be effective in HIV-1 infected patients from Mozambique that pay for their medication and laboratory testing. Further studies are required to identify the causes for low retention rates in patients with low CD4 counts and to better understand the association between healthcare setting and attrition rate.     

Insulin/IGF-I Signaling Pathways Enhances Tumor Cell Invasion through Bisecting GlcNAc N-glycans Modulation. An Interplay with E-Cadherin

Changes in glycosylation are considered a hallmark of cancer, and one of the key targets of glycosylation modifications is E-cadherin. We and others have previously demonstrated that E-cadherin has a role in the regulation of bisecting GlcNAc N-glycans expression, remaining to be determined the E-cadherin-dependent signaling pathway involved in this N-glycans expression regulation. In this study, we analysed the impact of E-cadherin expression in the activation profile of receptor tyrosine kinases such as insulin receptor (IR) and IGF-I receptor (IGF-IR). We demonstrated that exogenous E-cadherin expression inhibits IR, IGF-IR and ERK 1/2 phosphorylation. Stimulation with insulin and IGF-I in MDA-MD-435 cancer cells overexpressing E-cadherin induces a decrease of bisecting GlcNAc N-glycans that was accompanied with alterations on E-cadherin cellular localization. Concomitantly, IR/IGF-IR signaling activation induced a mesenchymal-like phenotype of cancer cells together with an increased tumor cell invasion capability. Altogether, these results demonstrate an interplay between E-cadherin and IR/IGF-IR signaling as major networking players in the regulation of bisecting N-glycans expression, with important effects in the modulation of epithelial characteristics and tumor cell invasion. Here we provide new insights into the role that Insulin/IGF-I signaling play during cancer progression through glycosylation modifications.     

Leishmania amazonensis Promastigotes Present Two Distinct Modes of Nucleus and Kinetoplast Segregation during Cell Cycle

Here, we show the morphological events associated with organelle segregation and their timing in the cell cycle of a reference strain of Leishmania (L.) amazonensis promastigotes, the main causative agent of Tegumentary leishmaniasis in the Americas. We show evidences that during the cell cycle, L. amazonensis promastigotes present two distinct modes of nucleus and kinetoplast segregation, which occur in different temporal order in different proportions of cells. We used DAPI-staining and EdU-labeling to monitor the segregation of DNA-containing organelles and DNA replication in wild-type parasites. The emergence of a new flagellum was observed using a specific monoclonal antibody. The results show that L. amazonensis cell cycle division is peculiar, with 65% of the dividing cells duplicating the kinetoplast before the nucleus, and the remaining 35% doing the opposite or duplicating both organelles concomitantly. In both cases, the new flagellum appeared during S to G2 phase in 1N1K cells and thus before the segregation of both DNA-containing organelles; however, we could not determine the exact timing of flagellar synthesis. Most of these results were confirmed by the synchronization of parasites using hydroxyurea. Altogether, our data show that during the cell cycle of L. amazonensis promastigotes, similarly to L. donovani, the segregation of nucleus and kinetoplast do not follow a specific order, especially when compared to other trypanosomatids, reinforcing the idea that this characteristic seems to be species-specific and may represent differences in cellular biology among members of the Leishmania genus.     

Development of a New Approach to Aid in Visual Identification of Murine iPS Colonies Using a Fuzzy Logic Decision Support System

The a priori identification of induced pluripotent stem cells remains a challenge. Being able to quickly identify the most embryonic stem cell-similar induced pluripotent stem cells when validating results could help to reduce costs and save time. In this context, tools based on non-classic logic can be useful in creating aid-systems based on visual criteria. True colonies when viewed at 100x magnification have been found to have the following 3 characteristics: a high degree of border delineation, a more uniform texture, and the absence of a cracked texture. These visual criteria were used for fuzzy logic modeling. We investigated the possibility of predicting the presence of alkaline phosphatase activity, typical of true induced pluripotent stem cell colonies, after 25 individuals, with varying degrees of experience in working with murine iPS cells, categorized the images of 136 colonies based on visual criteria. Intriguingly, the performance evaluation by area under the ROC curve (16 individuals with satisfactory performance), Spearman correlation (all statistically significant), and Cohen''s Kappa agreement analysis (all statistically significant) demonstrates that the discriminatory capacity of different evaluators are similar, even those who have never cultivated cells. Thus, we report on a new system to facilitate visual identification of murine- induced pluripotent stem cell colonies that can be useful for staff training and opens the possibility of exploring visual characteristics of induced pluripotent stem cell colonies with their functional peculiarities. The fuzzy model has been integrated as a web-based tool named “2see-iPS” which is freely accessed at http://genetica.incor.usp.br/2seeips/.     

Enzymatic synthesis of biodiesel from palm oil assisted by microwave irradiation

Optimal conditions for enzymatic synthesis of biodiesel from palm oil and ethanol were determined with lipase from Pseudomonas fluorescens immobilized on epoxy polysiloxane–polyvinyl alcohol hybrid composite under a microwave heating system. The main goal was to reduce the reaction time preliminarily established by a process of conventional heating. A full factorial design assessed the influence of ethanol-to-palm oil (8:1–16:1) molar ratio and temperature (43–57 °C) on the transesterification yield. Microwave irradiations varying from 8 to 15 W were set up according to reaction temperature. Under optimal conditions (8:1 ethanol-to-oil molar ratio at 43 °C), 97.56 % of the fatty acids present in the palm oil were converted into ethyl esters in a 12-h reaction, corresponding to a productivity of 64.2 mg ethyl esters g^?1 h^?1. This represents a sixfold increase from the process carried out under conventional heating, thus proving to be a potential tool for enhancing biochemical modification of oils and fats. In general, advantages of the new process include: (1) microwaves speed up the enzyme-catalyzed reactions; (2) there are no destructive effects on the enzyme properties, such as stability and substrate specificity, and (3) the microwave assistance allows the entire reaction volume to be heated uniformly. These bring benefits of a low energy demand and a faster conversion of palm oil into biodiesel.     

Characterization and Antilisterial Effect of Phosphatidylcholine Nanovesicles Containing the Antimicrobial Peptide Pediocin

Encapsulation may provide increased stability and antimicrobial efficiency to bacteriocins. In this work, the antilisterial peptide pediocin was encapsulated in nanovesicles prepared from partially purified soybean phosphatidylcholine. The maintenance of antimicrobial activity and properties of free and encapsulated pediocin was observed during 13 days at 4 °C, and after this period, the encapsulated pediocin retained 50 % its initial activity. The maintenance of the bioactive properties of free and encapsulated pediocin was observed against different species of Listeria, inhibiting Listeria monocytogenes, Listeria innocua and Listeria ivanovii. The size of vesicles containing pediocin was determined by dynamic light scattering as an average of 190 nm, with little change throughout the observation period. Polydispersity index values were around 0.201 and are considered satisfactory, indicating an adequate size distribution of liposomes. The efficiency of encapsulation was 80 %. Considering these results, the protocol used was appropriate for the encapsulation of this bacteriocin. Results demonstrate the production of stable nanoparticulate material. The maintenance of the properties of pediocin encapsulated in liposomes is fundamental to prospect the stability in different conditions of the food matrix.     

FAS/FASL Expression Profile as a Prognostic Marker in Squamous Cell Carcinoma of the Oral Cavity

FAS/FASL altered expression may cause tumor protecting immunomodulation, with a direct impact on patient prognosis. FAS expression was studied in 60 squamous cell carcinomas of the oral cavity. FAS expression did not show a significant association with tumor histopathological characteristics, but was significantly associated with lymph node positivity. FAS expression was significantly associated with disease specific death and negative FAS expression was an independent risk factor, increasing risk 4 times when compared to positive expression. When FAS and FASL expression results were combined, we were able to define high, intermediate and low risk profiles. Disease-free and disease-specific survival were significantly correlated with FAS/FASL expression profiles. The high risk category was an independent marker for earlier disease relapse and disease-specific death, with approximately 4- and 6-fold increased risk, respectively, when compared to the low risk profile. Risk profiles based on FAS/FASL expression showed that high risk was significantly associated with increased disease relapse and death, as well as shorter disease-free or disease-specific survival. This categorization, added to patient clinical data, may facilitate the choice of therapy, minimizing treatment failure and increasing disease control.     

The Use of Bayesian Latent Class Cluster Models to Classify Patterns of Cognitive Performance in Healthy Ageing

The main focus of this study is to illustrate the applicability of latent class analysis in the assessment of cognitive performance profiles during ageing. Principal component analysis (PCA) was used to detect main cognitive dimensions (based on the neurocognitive test variables) and Bayesian latent class analysis (LCA) models (without constraints) were used to explore patterns of cognitive performance among community-dwelling older individuals. Gender, age and number of school years were explored as variables. Three cognitive dimensions were identified: general cognition (MMSE), memory (MEM) and executive (EXEC) function. Based on these, three latent classes of cognitive performance profiles (LC1 to LC3) were identified among the older adults. These classes corresponded to stronger to weaker performance patterns (LC1>LC2>LC3) across all dimensions; each latent class denoted the same hierarchy in the proportion of males, age and number of school years. Bayesian LCA provided a powerful tool to explore cognitive typologies among healthy cognitive agers.     

Small tropical forest trees have a greater capacity to adjust carbon metabolism to long-term drought than large canopy trees

The response of small understory trees to long-term drought is vital in determining the future composition, carbon stocks and dynamics of tropical forests. Long-term drought is, however, also likely to expose understory trees to increased light availability driven by drought-induced mortality. Relatively little is known about the potential for understory trees to adjust their physiology to both decreasing water and increasing light availability. We analysed data on maximum photosynthetic capacity (J_max, V_cmax), leaf respiration (R_leaf), leaf mass per area (LMA), leaf thickness and leaf nitrogen and phosphorus concentrations from 66 small trees across 12 common genera at the world's longest running tropical rainfall exclusion experiment and compared responses to those from 61 surviving canopy trees. Small trees increased J_max, V_cmax, R_leaf and LMA (71, 29, 32, 15% respectively) in response to the drought treatment, but leaf thickness and leaf nutrient concentrations did not change. Small trees were significantly more responsive than large canopy trees to the drought treatment, suggesting greater phenotypic plasticity and resilience to prolonged drought, although differences among taxa were observed. Our results highlight that small tropical trees have greater capacity to respond to ecosystem level changes and have the potential to regenerate resilient forests following future droughts.