Human Stem Cell Cultures from Cleft Lip/Palate Patients Show Enrichment of Transcripts Involved in Extracellular Matrix Modeling By Comparison to Controls

Nonsyndromic cleft lip and palate (NSCL/P) is a complex disease resulting from failure of fusion of facial primordia, a complex developmental process that includes the epithelial-mesenchymal transition (EMT). Detection of differential gene transcription between NSCL/P patients and control individuals offers an interesting alternative for investigating pathways involved in disease manifestation. Here we compared the transcriptome of 6 dental pulp stem cell (DPSC) cultures from NSCL/P patients and 6 controls. Eighty-seven differentially expressed genes (DEGs) were identified. The most significant putative gene network comprised 13 out of 87 DEGs of which 8 encode extracellular proteins: ACAN, COL4A1, COL4A2, GDF15, IGF2, MMP1, MMP3 and PDGFa. Through clustering analyses we also observed that MMP3, ACAN, COL4A1 and COL4A2 exhibit co-regulated expression. Interestingly, it is known that MMP3 cleavages a wide range of extracellular proteins, including the collagens IV, V, IX, X, proteoglycans, fibronectin and laminin. It is also capable of activating other MMPs. Moreover, MMP3 had previously been associated with NSCL/P. The same general pattern was observed in a further sample, confirming involvement of synchronized gene expression patterns which differed between NSCL/P patients and controls. These results show the robustness of our methodology for the detection of differentially expressed genes using the RankProd method. In conclusion, DPSCs from NSCL/P patients exhibit gene expression signatures involving genes associated with mechanisms of extracellular matrix modeling and palate EMT processes which differ from those observed in controls. This comparative approach should lead to a more rapid identification of gene networks predisposing to this complex malformation syndrome than conventional gene mapping technologies.

Thermal and spectrophotometric studies of new crosslinking method for collagen matrix with glutaraldehyde acetals

Despite the many existing crosslinking procedures, glutaraldehyde (GA) is still the method of choice used in the manufacture of bioprosthesis. The major problems with GA are: (a) uncontrolled reactivity due to the chemical complexity or GA solutions; (b) toxicity due to the release of GA from polymeric crosslinks; and (c) tissue impermeabilization due to polymeric and heterogeneous crosslinks formation, partially responsible for the undesirable calcification of the bioprosthesis. A new method of crosslinking glutaraldehyde acetals has been developed with GA in acid ethanolic solution, and after the distribution inside de matrix, GA is released to crosslinking. Concentrations of hydrochloride acid in ethanolic solutions between 0.1 and 0.001 mol/L with GA concentration between 0.1 and 1.0% were measured in an ultraviolet spectrophotometer to verify the presence of free aldehyde groups and polymeric compounds of GA. After these measurements, the solutions were used to crosslink bovine pericardium. The spectrophotometric results showed that GA was better protected in acetal forms for acid ethanolic solution with HCl at 0.003 mol/L and GA 1.0%(v/v). The shrinkage temperature results of bovine pericardium crosslinked with acetal solutions showed values near 85 C after the exposure to triethylamine vapors.

Determination of polyamines in organic extracts from roots of Canavalia ensiformis by capillary electrophoresis

A rapid, selective and specific capillary zone electrophoresis method to determine polyamines in organic extracts from roots of Canavalia ensiformis (Jack Beans) was developed using ultra violet (UV) detection. Canavalia ensiformis is relatively free from diseases and it is used as reference in allelopathy studies. Polyamines are widely distributed in plant and it could be involved in plant pathogen interactions. Optimal separation was achieved using 15 mmol.L-1formic acid (pH 3.0) + 4 mmol.L-1 imidazole as a background electrolyte. It was possible to identify and quantify the polyamines on herbal samples in the presence of other phytochemical substances and analyze them quickly (up to 6 min). The applicability of this method was evaluated in crude organic extracts from roots of Canavalia ensiformis.

Increased CLA content in organic milk fermented by bifidobacteria or yoghurt cultures

This study investigates the kinetics of acidification, fatty acid (FA) profile and conjugated linoleic acid (CLA, C18:2 c9, t11) content in fermented milks prepared from organic and conventional milk. Fermented milks were manufactured with five mixed cultures: four different strains of Bifidobacterium animalis subsp. lactis (BL04, B94, BB12 and HN019) and Lactobacillus delbrueckii subsp. bulgaricus LB340, in co-culture with Streptococcus thermophilus TA040. The composition of milk was evaluated, and the kinetics of acidification was followed by continuous pH measurement using the Cinac system. The profile of FA, including CLA, was analyzed by gas chromatography. The chemical composition of conventional and organic milk was similar, with the exception of protein and Fe, the concentrations of which were higher in the organic milk. The rate of acidification was significantly influenced by the type of milk and the bacterial strain used. Co-cultures St-HN019 and St-BB12 showed higher maximal acidification rates in both milks. Final counts of S. thermophilus (9.0-10.1 log(10) colony forming units (CFU) . mL(-1), L)actobacillus bulgaricus (8.2-8.5 log(10) CFU . mL(-1)) and B. animalis subsp. lactis strains (8.3-9.3 log(10) CFU . mL(-1)) did not differ significantly in either milk. Unexpectedly, all fermented organic milks contained significantly higher amounts of CLA than the same milk before fermentation, whereas CLA amounts did not change during fermentation of conventional milk. Regardless of the type of milk, CLA was found to be significantly positively correlated with trans-vaccenic acid and negatively correlated with linoleic acid. Moreover, the CLA contents were significantly higher in fermented milks showing shorter fermentation times.

Application of the essential work of fracture method in ranking the performance in service of high-density polyethylene resins employed in pressure pipes

High-density polyethylene resins have increasingly been used in the production of pipes for water- and gas-pressurized distribution systems and are expected to remain in service for several years, but they eventually fail prematurely by creep fracture. Usual standard methods used to rank resins in terms of their resistance to fracture are expensive and non-practical for quality control purposes, justifying the search for alternative methods. Essential work of fracture (EWF) method provides a relatively simple procedure to characterize the fracture behavior of ductile polymers, such as polyethylene resins. In the present work, six resins were analyzed using the EWF methodology. The results show that the plastic work dissipation factor, beta w(p), is the most reliable parameter to evaluate the performance. Attention must be given to specimen preparation that might result in excessive dispersion in the results, especially for the essential work of fracture w(e).

MULTIVARIATE STATISTICAL METHOD IN OPTIMIZATION OF PROTEIN EXTRACTION FROM SOYBEAN FLOUR WITH DAIRY WHEY

The aim objective of this project was to evaluate the protein extraction of soybean flour in dairy whey, by the multivariate statistical method with 2(3) experiments. Influence of three variables were considered: temperature, pH and percentage of sodium chloride against the process specific variable ( percentage of protein extraction). It was observed that, during the protein extraction against time and temperature, the treatments at 80 degrees C for 2h presented great values of total protein (5.99%). The increasing for the percentage of protein extraction was major according to the heating time. Therefore, the maximum point from the function that represents the protein extraction was analysed by factorial experiment 2(3). By the results, it was noted that all the variables were important to extraction. After the statistical analyses, was observed that the parameters as pH, temperature, and percentage of sodium chloride, did not sufficient for the extraction process, since did not possible to obtain the inflection point from mathematical function, however, by the other hand, the mathematical model was significant, as well as, predictive.

MICROORGANISMS IN ORGANIC AND NON ORGANIC HONEY SAMPLES OF AFRICANIZED HONEYBEES

This research was carried out to evaluate and compare 11 organic honey samples and six non organic honey samples, respectively, harvested from islands of the triple frontier (Sao Paulo, Parana and Mato Grosso do Sul states) and from the state of Parana, Brazil. The samples were studied for the presence of coliforms from 35 degrees C, to 45 degrees C and the enumeration of moulds and yeast, a minimum of 1.9 x 10(2) and a maximum of 1.1 x 10(3) CFU/g were observed in organic honey and a minimum of 1.8 x 10(1) and a maximum of 2.5 x 10(2) CFU/g were in non organic honey. In this studied region, the organic honey presented a microbiological quality inferior to the non organic honey.

Topliss method in the optimization of salicylic acid derivatives as potential antimycobacterial agents

The Topliss method was used to guide a synthetic path in support of drug discovery efforts toward the identification of potent antimycobacterial agents. Salicylic acid and its derivatives, p-chloro, p-methoxy, and m-chlorosalicylic acid, exemplify a series of synthetic compounds whose minimum inhibitory concentrations for a strain of Mycobacterium were determined and compared to those of the reference drug, p-aminosalicylic acid. Several physicochemical descriptors (including Hammett`s sigma constant, ionization constant, dipole moment, Hansch constant, calculated partition coefficient, Sterimol-L and -B-4 and molecular volume) were considered to elucidate structure-activity relationships. Molecular electrostatic potential and molecular dipole moment maps were also calculated using the AM1 semi-empirical method. Among the new derivatives, m-chlorosalicylic acid showed the lowest minimum inhibitory concentration. The overall results suggest that both physicochemical properties and electronic features may influence the biological activity of this series of antimycobacterial agents and thus should be considered in designing new p-aminosalicylic acid analogs.

Differences in both matrix metalloproteinase 9 concentration and zymographic profile between plasma and serum with clot activators are due to the presence of amorphous silica or silicate salts in blood collection devices

Matrix metalloproteinases (MMPs) are promising diagnostic tools, and blood sampling/handling alters MMP concentrations between plasma and serum and between serum with and without clot activators. To explain the higher MMP-9 expression in serum collected with clot accelerators relative to serum with no additives and to plasma, we analyzed the effects of increasing amounts of silica and silicates (components of clot activators) in,citrate plasma, serum, and huffy coats collected in both plastic and glass tubes from 50 healthy donors, and we analyzed the effects of silica and silicate on cultured leukemia cells. The levels of MMP-2 did not show significant changes between glass and plastic tubes, between serum and plasma, between serum with and without clot accelerators, or between silica and silicate treatments. No modification of MMP-9 expression was obtained by the addition of silica or silicate to previously separated plasma and serum. Increasing the amounts of nonsoluble silica and soluble silicate added to citrate and empty tubes prior to blood collection resulted in increasing levels of MMP-9 relative to citrate plasma and serum. Silica and silicate added to buffy coats and leukemia cells significantly induced MMP-9 release/secretion, demonstrating that both silica and silicate induce the release of pro- and complexed MMP-9 forms. We recommend limiting the misuse of serum and avoiding the interfering effects of clot activators. (c) 2007 Elsevier Inc. All rights reserved.

Digital image analysis to standardize a photometric method in colorimetric quantification

Techniques applying digital images increasingly have been used in biology, medicine, physics, and other research areas. The image coordinates can represent light intensities values to be detected by a CCD. Based on this concept, a photometric system composed of a LED source and a digital camera as a detector was used for optical density measurements. Standards for permanganate, glucose, and protein solutions were detemined by colorimetric methods using our device. Samples of protein of Pasteurella mutocida bacteria membrane and, also, fractions of rabbit kidney membrane, rich in Na, K-ATPase, with unknown concentrations were dosed through the Hartree method using our photometric system.

Jacobsen catalyst immobilized on chitosan membrane as interface catalyst in organic/aqueous system for alkene oxidation

The Jacobsen catalyst, Mn(salen), was immobilized in chitosan membrane. The obtained Mn(salen)-Chit was characterized by thermogravimetric analysis (TC), differential thermal analysis (DTA), differential scanning calorimetry (DSC), infrared spectroscopy (FT-IR), degree of N-acetylation by (1)H NMR, and UV-vis spectroscopy. The UV-vis absorption spectrum of the encapsulated catalyst displayed the typical bands of the Jacobsen catalyst, and the FT-IR presented an absorption band characteristic of the imines present in the Jacobsen catalyst. The chitosan membranes were available, in a biphasic system, as a catalytic barrier between two different phases: an organic substrate phase (cyclooctene or styrene) and an aqueous solution of either m-CPBA, t-BuOOH or H(2)O(2), and dismissing the need for phase transfer agents and leading to better product yields compared with the catalyst in homogeneous medium. This new catalyst did not leach from the support and was reused many times, leading to high turnover frequencies. (C) 2009 Elsevier B.V. All rights reserved.

Scanning electron microscopy as an auxiliary method in the study of exhumed bones

Scanning electronmicroscopy (SEM) has been used in forensic science in many ways. The reports of cases in which SEM has been used as an auxiliary method in the investigation of exhumed bones are rare. In this article, we report an exhumation that was made to determine if a seized weapon could have been used in a homicide. We used SEM to analyze a fracture in the interior of the skull of the victim. The findings described in this article showed us that it is possible to develop new researches in this field. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Increases in circulating matrix metalloproteinase-9 levels following fibrinolysis for acute pulmonary embolism

Introduction: Fibrinolyis is one of the first line therapies in high risk pulmonary embolism (PE) according to current guidelines. Previous studies showed that brinolytic therapy with tPA (tissue plasminogen activator, or alteplase) upregulates the concentrations of matrix metalloproteinases (MMPs) and contributes to hemorrhagic transformation after cardioembolic stroke. However, no previous study has described the circulating MMPs levels following fibrinolysis for acute PE. Materials and Methods: We serially measured the circulating levels of MMPs (MMP-9 and MMP-2) and their endogenous inhibitors, the tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 in alteplase and in streptokinase-treated patients with acute PE by gelatin zymography and by enzyme-linked immunosorbent assays, respectively. Results: We found that therapy of PE streptokinase or with alteplase is associated increased pro-MMP-9, but not MMP-2, concentrations for up to 24 hours, whereas no significant changes were found in TIMP-1 or TIMP-2 concentrations. This alteration returned to normal 3 to 5 days after thrombolysis. This is the first study reporting on MMPs alterations following fibrinolysis for acute PE. Conclusions: We found transient increases in circulating pro-MMP-9 levels following fibrinolysis for acute PE. Our findings support the hypothesis that increased MMP-9 levels may underlie the risk of intracerebral hemorrhage or other bleeding complication of thrombolysis for acute PE, and the use of MMP inhibitors may decrease such risk. (C) 2010 Elsevier Ltd. All rights reserved.

Nitrite or sildenafil, but not BAY 41-2272, blunt acute pulmonary embolism-induced increases in circulating matrix metalloproteinase-9 and oxidative stress

Introduction: Inhibition of matrix metalloproteinases (MMPs) improves the hemodynamics during acute pulmonary embolism (APE) and oxidative stress upregulates MMPs. We compared the effects of different NO-cGMP pathway activators on APE-induced increases in MMPs. Materials and Methods: Hemodynamic and biochemical evaluations were performed in non-embolized dogs treated with saline (N = 5), and in microspheres embolized dogs receiving saline (n = 9), or nitrite (6.75 mu mol/kg i.v. over 15 min followed by 0.28 mu mol/kg/min; n = 5), or sildenafil (0.25 mg/kg; n = 5), or BAY 41-2272 (0.03, 0.1, 0.3, and 1 mg/kg/h; n = 5). Plasma thiobarbituric acid reactive substances (TBARS) concentrations were determined. Zymograms of plasma samples were performed, and in vitro antioxidant effects or inhibition of MMPs by these drugs were examined. Results: APE increased mean pulmonary artery pressure by similar to 25 mmHg. Nitrite, BAY 41-2272, or sildenafil reversed this increase by similar to 40% (P < 0.05). Similar effects were seen on the pulmonary vascular resistance. While both nitrite and sildenafil produced no systemic effects, the highest dose of BAY 41-2272 produced systemic hypotension (P<0.05). While nitrite and sildenafil blunted the increases in plasma pro-MMP-9 levels and TBARS (all P < 0.05), BAY 41-2272 produced no such effects. Nitrite and sildenafll produced in vitro antioxidant effects and inhibited MMPs only at high concentrations. BAY 41-2272 produced no such effects. Conclusions: Activation of the NO-cGMP pathway with nitrite or sildenafil, but not with BAY 41-2272, attenuates APE-induced oxidative stress and increased MMP-9 levels. These findings are consistent with the idea that NO-cGMP pathway activators with antioxidant effects prevent the release of MMP-9 during APE. (c) 2008 Elsevier Ltd. All rights reserved.

Effect of the C-factor and Dentin Preparation Method in the Bond Strength of a Mild Self-etch Adhesive

This study evaluated the effect of the C-factor and dentin preparation method (DPM) in the bond strength (BS) of a mild self-etch adhesive; the study also observed the SEM superficial aspects of the corresponding smear layer. For purposes of this study, 25 molars (n=5) were used in a bond strength test. The molars were divided into two parts (buccal and lingual): one part received a Class V cavity (C-factor=3) and the other received a flat surface (C-factor=0) with the same bur type (coarse diamond or carbide bur and fine diamond or carbide bur), both within the same dentin depth. Five teeth were prepared with wet 60-grit and 600-grit SiC papers. After restoration with Clearfil SE Bond, microtensile beans (0.8 mm(2)) were prepared and tested after 24 hours in a universal testing machine (0.5 mm/minute). An additional two teeth for each DPM were prepared for SEM evaluation of the smear layer superficial aspects. The BS values were submitted to one-way ANOVA, considering only the DPM (flat surfaces) and two-way ANOVA (C-Factor x DPM, considering only burs) with p=0.05. Although the DPM in the flat surfaces was not significant, the standard deviations of carbide bur-prepared specimens were markedly lower. The BS was significantly lower in cavities. The fine carbide bur presented the most favorable smear layer aspect. It was concluded that different dentin preparation methods could not prevent the adverse effect in bond strength of a high C-factor. A coarse cut carbide bur should be avoided prior to a mild self-etch adhesive, because it adversely affected bond strength. In contrast, a fine cut carbide bur provided the best combination: high bond strength with low variability, which suggests a more reliable bond strength performance.