In vitro culture and characterization of alveolar bone osteoblasts isolated from type 2 diabetics

In order to understand the mechanisms of poor osseointegration following dental implants in type 2 diabetics, it is important to study the biological properties of alveolar bone osteoblasts isolated from these patients. We collected alveolar bone chips under aseptic conditions and cultured them in vitro using the tissue explants adherent method. The biological properties of these cells were characterized using the following methods: alkaline phosphatase (ALP) chemical staining for cell viability, Alizarin red staining for osteogenic characteristics, MTT test for cell proliferation, enzyme dynamics for ALP contents, radio-immunoassay for bone gla protein (BGP) concentration, and ELISA for the concentration of type I collagen (COL-I) in the supernatant. Furthermore, we detected the adhesion ability of two types of cells from titanium slices using non-specific immunofluorescence staining and cell count. The two cell forms showed no significant difference in morphology under the same culture conditions. However, the alveolar bone osteoblasts received from type 2 diabetic patients had slower growth, lower cell activity and calcium nodule formation than the normal ones. The concentration of ALP, BGP and COL-I was lower in the supernatant of alveolar bone osteoblasts received from type 2 diabetic patients than in that received from normal subjects (P < 0.05). The alveolar bone osteoblasts obtained from type 2 diabetic patients can be successfully cultured in vitro with the same morphology and biological characteristics as those from normal patients, but with slower growth and lower concentration of specific secretion and lower combining ability with titanium than normal ones.

Challenges in the research and development of new human vaccines

The field of vaccinology was born from the observations by the fathers of vaccination, Edward Jenner and Louis Pasteur, that a permanent, positive change in the way our bodies respond to life-threatening infectious diseases can be obtained by specific challenge with the inactivated infectious agent performed in a controlled manner, avoiding the development of clinical disease upon exposure to the virulent pathogen. Many of the vaccines still in use today were developed on an empirical basis, essentially following the paradigm established by Pasteur, “isolate, inactivate, and inject” the disease-causing microorganism, and are capable of eliciting uniform, long-term immune memory responses that constitute the key to their proven efficacy. However, vaccines for pathogens considered as priority targets of public health concern are still lacking. The literature tends to focus more often on vaccine research problems associated with specific pathogens, but it is increasingly clear that there are common bottlenecks in vaccine research, which need to be solved in order to advance the development of the field as a whole. As part of a group of articles, the objective of the present report is to pinpoint these bottlenecks, exploring the literature for common problems and solutions in vaccine research applied to different situations. Our goal is to stimulate brainstorming among specialists of different fields related to vaccine research and development. Here, we briefly summarize the topics we intend to deal with in this discussion.

In vitro proliferation and differentiation of hepatic oval cells and their potential capacity for intrahepatic transplantation

Hepatic oval cells (HOCs) are recognized as facultative liver progenitor cells that play a role in liver regeneration after acute liver injury. Here, we investigated the in vitro proliferation and differentiation characteristics of HOCs in order to explore their potential capacity for intrahepatic transplantation. Clusters or scattered HOCs were detected in the portal area and interlobular bile duct in the liver of rats subjected to the modified 2-acetylaminofluorene and partial hepatectomy method. Isolated HOCs were positive for c-kit and CD90 staining (99.8% and 88.8%, respectively), and negative for CD34 staining (3.6%) as shown by immunostaining and flow cytometric analysis. In addition, HOCs could be differentiated into hepatocytes and bile duct epithelial cells after leukemia inhibitory factor deprivation. A two-cuff technique was used for orthotopic liver transplantation, and HOCs were subsequently transplanted into recipients. Biochemical indicators of liver function were assessed 4 weeks after transplantation. HOC transplantation significantly prolonged the median survival time and improved the liver function of rats receiving HOCs compared to controls (P=0.003, Studentt-test). Administration of HOCs to rats also receiving liver transplantation significantly reduced acute allograft rejection compared to control liver transplant rats 3 weeks following transplantation (rejection activity index score: control=6.3±0.9; HOC=3.5±1.5; P=0.005). These results indicate that HOCs may be useful in therapeutic liver regeneration after orthotopic liver transplantation.

Serum uric acid and disorders of glucose metabolism: the role of glycosuria

Hyperuricemia has been associated with hypertension, diabetes mellitus, and metabolic syndrome. We studied the association between hyperuricemia and glycemic status in a nonrandomized sample of primary care patients. This was a cross-sectional study of adults ≥20 years old who were members of a community-based health care program. Hyperuricemia was defined as a value >7.0 mg/dL for men and >6.0 mg/dL for women. The sample comprised 720 participants including controls (n=257) and patients who were hypertensive and euglycemic (n=118), prediabetic (n=222), or diabetic (n=123). The mean age was 42.4±12.5 years, 45% were male, and 30% were white. The prevalence of hyperuricemia increased from controls (3.9%) to euglycemic hypertension (7.6%) and prediabetic state (14.0%), with values in prediabetic patients being statistically different from controls. Overall, diabetic patients had an 11.4% prevalence of hyperuricemia, which was also statistically different from controls. Of note, diabetic subjects with glycosuria, who represented 24% of the diabetic participants, had a null prevalence of hyperuricemia, and statistically higher values for fractional excretion of uric acid, Na excretion index, and prevalence of microalbuminuria than those without glycosuria. Participants who were prediabetic or diabetic but without glycosuria had a similarly elevated prevalence of hyperuricemia. In contrast, diabetic patients with glycosuria had a null prevalence of hyperuricemia and excreted more uric acid and Na than diabetic subjects without glycosuria. The findings can be explained by enhanced proximal tubule reabsorption early in the course of dysglycemia that decreases with the ensuing glycosuria at the late stage of the disorder.

Electric paralyzation and reduction of weight loss in the processing of round-cooked spiny lobsters

This study proposes alternatives to the current methods of processing round-cooked lobster. The paralyzation of lobsters with direct electric shock consumes 10.526 x 10-3 kWh, which is significantly less than the 11 kWh required by the traditional thermal-shock method (based on 60 kg of lobsters). A better weight gain was obtained by immersion of paralyzed lobsters in brine before cooking. Systematic trials combining 3, 6, or 9% brine concentrations with immersion periods of 15, 30, or 45 minutes were performed in order to determine the best combinations. A mathematical model was designed to predict the weight gain of lobsters of different sizes in any combination of treatments. For small lobsters, a 45 minutes immersion in 6% brine gave the best response in terms of weight gain (4.7%) and cooking produced a weight loss of only 1.34% in relation to fresh lobster weight. For medium-sized lobsters, a 45 minutes immersion in 9% brine produced a weight gain of 2.64%, and cooking a weight gain of 1.08%. For large lobsters, a 45 minutes immersion in 6% brine produced a weight gain of 3.87%, and cooking a weight gain of 1.62%.

Safety and outcome of coronary interventions with special reference to anticoagulation and stent type

University of Turku, Faculty of Medicine, Department of Cardiology and Cardiovascular Medicine, Doctoral Programme of Clinical Investigation, Heart Center, Turku University Hospital, Turku, Finland Division of Internal Medicine, Department of Cardiology, Seinäjoki Central Hospital, Seinäjoki, Finland Heart Center, Satakunta Central Hospital, Pori, Finland Annales Universitatis Turkuensis Painosalama Oy, Turku, Finland 2015 Antithrombotic therapy during and after coronary procedures always entails the challenging establishment of a balance between bleeding and thrombotic complications. It has been generally recommended to patients on long-term warfarin therapy to discontinue warfarin a few days prior to elective coronary angiography or intervention to prevent bleeding complications. Bridging therapy with heparin is recommended for patients at an increased risk of thromboembolism who require the interruption of anticoagulation for elective surgery or an invasive procedure. In study I, consecutive patients on warfarin therapy referred for diagnostic coronary angiography were compared to control patients with a similar disease presentation without warfarin. The strategy of performing coronary angiography during uninterrupted therapeutic warfarin anticoagulation appeared to be a relatively safe alternative to bridging therapy, if the international normalized ratio level was not on a supratherapeutic level. In-stent restenosis remains an important reason for failure of long-term success after a percutaneous coronary intervention (PCI). Drug-eluting stents (DES) reduce the problem of restenosis inherent to bare metal stents (BMS). However, a longer delay in arterial healing may extend the risk of stent thrombosis (ST) far beyond 30 days after the DES implantation. Early discontinuation of antiplatelet therapy has been the most important predisposing factor for ST. In study II, patients on long-term oral anticoagulant (OAC) underwent DES or BMS stenting with a median of 3.5 years’follow-up. The selective use of DESs with a short triple therapy seemed to be safe in OAC patients, since late STs were rare even without long clopidogrel treatment. Major bleeding and cardiac events were common in this patient group irrespective of stent type. In order to help to predict the bleeding risk in patients on OAC, several different bleeding risk scorings have been developed. Risk scoring systems have also been used also in the setting of patients undergoing a PCI. In study III, the predictive value of an outpatient bleeding risk index (OBRI) to identify patients at high risk of bleeding was analysed. The bleeding risk seemed not to modify periprocedural or long-term treatment choices in patients on OAC after a percutaneous coronary intervention. Patients with a high OBRI often had major bleeding episodes, and the OBRI may be suitable for risk evaluation in this patient group. Optical coherence tomography (OCT) is a novel technology for imaging intravascular coronary arteries. OCT is a light-based imaging modality that enables a 12–18 µm tissue axial resolution to visualize plaques in the vessel, possible dissections and thrombi as well as, stent strut appositions and coverage, and to measure the vessel lumen and lesions. In study IV, 30 days after titanium-nitride-oxide (TITANOX)-coated stent implantation, the binary stent strut coverage was satisfactory and the prevalence of malapposed struts was low as evaluated by OCT. Long-term clinical events in patients treated with (TITANOX)-coated bio-active stents (BAS) and paclitaxel-eluting stents (PES) in routine clinical practice were examined in study V. At the 3-year follow-up, BAS resulted in better long-term outcome when compared with PES with an infrequent need for target vessel revascularization. Keywords: anticoagulation, restenosis, thrombosis, bleeding, optical coherence tomography, titanium

The Brazilian consumer's understanding and perceptions of organic vegetables: a Focus Group approach

Focus Group is a tool which generates, through interview sessions with a small number of participants, preliminary data to be used in subsequent quantitative stages. Many consumer studies use qualitative research with the aim of obtaining information and opinions on a specific product or situation. The objective of the present study was to obtain knowledge on the opinion, understanding and perception of the Brazilian consumer with respect to vegetables, focusing on organic products, using Focus Group Sessions. Four Focus Group Sessions were held with men and women in different environments, following a previously elaborated interview guide. In this study, it was observed that the consumers demonstrated being interested in having a healthy diet, based on fruit, vegetables and natural products. However, only a few declared consuming organic foods. Some participants did not know what the term organic meant, and most of them think that organic products are still very expensive, are not easily available in the supermarkets, do not have a good appearance, mainly in terms of size and packaging, and their certification is not always trustworthy. Almost all participants stated that they read package labels and among the items most observed were best-before date, nutritional information, production system and price. This study has identified important vegetable attributes perceived by the consumer, favouring the planning of a subsequent quantitative research. The results suggest that more information on the benefits of organic agriculture has to be passed on to consumers in order to contribute to a higher consumption of such products.

Partial characterization and inactivation of peroxidases and polyphenol-oxidases of umbu-cajá (Spondias spp.)

A crude extract of Spondias spp. was evaluated for the influence of pH and temperature on the activity and stability of its peroxidases and polyphenol-oxidases. In order to evaluate the conditions for the inactivation of the enzymes by heat treatment and by addition of a reducing agent, a factorial experimental design (n = 3) was employed using the Statistica (6.0) software package for data analysis. The optimal conditions found for peroxidases were: pH = 5.0 and temperature = 40 ºC, and for polyphenol-oxidases they were pH = 7.0 and temperature = 40 ºC. The peroxidases and polyphenol-oxidases were stable at all pH values tested (3.0 - 10.0) and maintained more than 60% of their activity at temperatures above 30 and 40 ºC, respectively. To achieve the total inactivation of these enzymes, two alternatives can be suggested: incubation at 92 ºC for 3.15 minutes with 200 mg.L-1 of ascorbic acid or incubation at 96 ºC for 2.80 minutes with 100 mg.L-1 of ascorbic acid.

Determination of thermobacteriological parameters and size of Bacillus stearothermophilus ATCC 7953 spores

In order to determine thermobacteriological parameters for B. stearothermophilus spores, they were diluted in a saline solution medium and in ground corn-soybean mix, distributed in TDT tube, and submitted to heat for a specific period of time. The D value (time to reduce 1 log cycle of microbial count under a certain temperature) and z value (variation of temperature to cause 10-fold change in D value) were estimated. To estimate their dimensions, the spores were visualized by using a scanning electron microscope. D121.1 ºC and z values for these spores, as determined in the saline solution, were 8.8 minutes and 12.8 ºC, respectively. D121,1 ºC and z values determined in the corn-soy mix were 14.2 minutes and 23.7 ºC, respectively. The micrographs indicated that the spores have homogeneous shape and size, with length and diameter of 2 and 1 µm, respectively. These results confirm that the spore is highly thermal-resistant, and it is a good biological indicator to evaluate the extrusion process as a feed sterilizer.

Hygienic-sanitary quality of vegetables and evaluation of treatments for the elimination of indigenous E. coli and E. coli O157:H7 from the surface of leaves of lettuce (Lactuca sativa L.)

The purpose of this study is to evaluate the hygienic-sanitary quality of vegetables and irrigation water and assess the effectiveness of lemon juice and vinegar in reducing E. coli strains inoculated on lettuce. One hundred and forty samples of vegetables and 45 samples of irrigation water were investigated for thermotolerant coliforms and Salmonella spp. In order to verify the effectiveness of natural household sanitizers in reducing E. coli in inoculated lettuce, four treatment solutions were tested: fresh lemon juice, alcohol vinegar, lemon juice-vinegar mixture, and lemon juice-vinegar-water mixture. The microbiological analysis revealed high rates of contamination by thermotolerant coliforms and identified the presence of E. coli in 32% of the tested vegetable samples and 56% of the water samples. While no significant statistical difference (p < 0, 05) was identified in the tested solutions, the treatment with a combination of lemon juice and vinegar resulted in the highest Decimal Reductions (DR) of E. coli O157: H7 while the treatment with vinegar alone was the most effective against the indigenous E. coli strain

Determination of pectin methylesterase activity in commercial pectinases and study of the inactivation kinetics through two potentiometric procedures

Pectinases are enzymes that degrade pectic substances and are widely used in juice and fruit beverages to improve the quality of the process. The objective of this study was to determine the optimum pH and temperature of two samples of commercial pectinases and propose an alternative procedure to determine the residual activity comparing the data with those of the traditional procedure. The pectin methylesterase (PME) activity in Pectinex 100 L Plus and Panzyn Clears was determined by potentiometry. The reaction consisted of 5.00 mg.mL-1 apple pectin, 0.100 mol.L-1 NaCl, and 50 µL enzyme to a total volume of 30 mL. The pectin reaction in the presence of PME in all experiments revealed a first order kinetics. The PME in the two enzyme preparations showed higher activity at pH 4.0 to 4.5 and temperature of 45 ºC. From the results of both procedures ΔV NaOH/Δt and ΔpH/Δt, it was concluded that the inactivation of PME occurred at 75 ºC. The results obtained from the ratio ΔpH/Δt showed good correlation with those obtained from the ratio ΔV NaOH/Δt. In the reaction accompanied by the ratio ΔpH/Δt, the release of H3O+ occurred in the real time reaction.

Antioxidant activity, fatty acid profile and tocopherols of Tamarindus indica L. seeds

This study aimed to characterize Tamarindus indica L. seeds regarding its composition and to evaluate its antioxidant potential, fatty acid profile and content of tocopherols. In order to obtain the extract, the dried and crushed seeds were extracted with ethanol for 30 minutes in a 1:3 seeds: ethanol ratio under continuous stirring at room temperature. After that, the mixtures were filtered and subjected to roto-evaporation at 40 ºC in order to determine, through direct weighing, the dry matter yields of the extracts. According to the results, Tamarindus indica L. seeds showed high content of total carbohydrates (71.91%) and offered relevant content and antioxidant activity of phenolic compounds. Tamarindus indica L. seeds oil presents high oxidative stability (15.83 hours) and significant total tocopherol content (57.77 mg.kg-1), besides presenting a higher percentage of unsaturated fatty acids - the main component being linolenic (59.61%), which is considered an essential fatty acid.

Isolation and recovery of glycomacropeptide from milk whey by means of thermal treatment

During enzymatic process of cheese manufacturing, rennin cleaves κ-casein releasing two fractions: para-κ-casein and glycomacropeptide (GMP), which remains soluble in milk whey. GMP is a peptide with structural particularities such as chain carbohydrates linked to specific threonine residues, to which a great variety of biological activities is attributed. Worldwide cheese production has increased generating high volumes of milk whey that could be efficiently used as an alternative source of high quality peptide or protein in foodstuff formulations. In order to evaluate isolation and recovery on whey GMP by means of thermal treatment (90 °C), 18 samples (2 L each) of sweet whey, resuspended commercial whey (positive control) and acid whey (negative control) were processed. Indirect presence of GMP was verified using chemical tests and PAGE-SDS 15%. At 90 °C treated sweet whey, 14, 20 and 41 kDa bands were observed. These bands may correspond to olygomers of GMP. Peptide recovery showed an average of 1.5 g/L (34.08%). The results indicate that industrial scale GMP production is feasible; however, further research must be carried out for the biological and nutritional evaluation of GMP's incorporation to foodstuff as a supplement.

Cytotoxicity and mutagenicity of cola and grape flavored soft drinks in bone marrow cells of rodents

Due to the large consumption of soft drinks in Brazil and worldwide in recent years and considering that some of the components present in their composition pose potential risks to human health, the aim of this study was to evaluate the cytotoxic and mutagenic potential of specific cola and grape-flavored soft drink brands. Bone marrow cells of Wistar rats were initially treated by gavage with one single dose of Cola or Grape soft drink, which was next offered ad libitum (instead of water) for 24 hours. A negative control treatment was performed by administering one single dose of water and a positive control administering cyclophosphamide intraperitoneally. Statistical analysis showed that the Cola and Grape soft drinks studied were not cytotoxic. However, the Cola soft drink proved mutagenic in this experiment treatment time. Therefore, this study serves as a warning about the consumption of Cola-flavored soft drink and for the need for further subchronic and chronic studies on soft drinks in order to evaluate the long term mutagenic and cytotoxic effects of these substances.

Characterization and classification of pequi trees (Caryocar brasiliense Camb.) based on the profile of volatile constituents using headspace solid-phase microextraction - gas chromatography - mass spectrometry and multivariate analysis

In order to determine the variability of pequi tree (Caryocar brasiliense Camb.) populations, volatile compounds from fruits of eighteen trees representing five populations were extracted by headspace solid-phase microextraction and analyzed by gas chromatography-mass spectrometry. Seventy-seven compounds were identified, including esters, hydrocarbons, terpenoids, ketones, lactones, and alcohols. Several compounds had not been previously reported in the pequi fruit. The amount of total volatile compounds and the individual compound contents varied between plants. The volatile profile enabled the differentiation of all of the eighteen plants, indicating that there is a characteristic profile in terms of their origin. The use of Principal Component Analysis and Cluster Analysis enabled the establishment of markers (dendrolasin, ethyl octanoate, ethyl 2-octenoate and β-cis-ocimene) that discriminated among the pequi trees. According to the Cluster Analysis, the plants were classified into three main clusters, and four other plants showed a tendency to isolation. The results from multivariate analysis did not always group plants from the same population together, indicating that there is greater variability within the populations than between pequi tree populations.