Chemoprevention of rat hepatocarcinogenesis with histone deacetylase inhibitors: Efficacy of tributyrin, a butyric acid prodrug

Hepatocellular carcinoma (HCC) ranks in prevalence and mortality among top 10 cancers worldwide. Butyric acid (BA), a member of histone deacetylase inhibitors (HDACi) has been proposed as an anticareinogenic agent. However, its short half-life is a therapeutical limitation. This problem could be circumvented with tributyrin (TB), a proposed BA prodrug. To investigate TB effectiveness for chemoprevention, rats were treated with the compound during initial phases of ""resistant hepatocyte"" model of hepatocarcinogenesis, and cellular and molecular parameters were evaluated. TB inhibited (p < 0.05) development of hepatic preneoplastic lesions (PNL) including persistent ones considered HCC progression sites. TB increased (p < 0.05) PNL remodeling, a process whereby they tend to disappear. TB did not inhibit cell proliferation in PNL, but induced (p < 0.05) apoptosis in remodeling ones. Compared to controls, rats treated with TB presented increased (P < 0.05) hepatic levels of BA indicating its effectiveness as a prodrug. Molecular mechanisms of TB-induced hepatocarcinogenesis chemoprevention were investigated. TB increased (p < 0.05) hepatic nuclear histone H3K9 hyperacetylation specifically in PNL and p21 protein expression, which could be associated with inhibitory HDAC effects. Moreover, it reduced (p < 0.05) the frequency of persistent PNL with aberrant cytoplasmic p53 accumulation, an alteration associated with increased malignancy. Original data observed in our study support the effectiveness of TB as a prodrug of BA and as an HDACi in hepatocarcinogenesis chemoprevention. Besides histone acetylation and p21 restored expression, molecular mechanisms involved with TB anticarcinogenic actions could also be related to modulation of p53 pathways. (C) 2008 Wiley-Liss, Inc.

Efeitos de dietas com níveis crescentes de milho no metabolismo ruminal de energia e proteína em bubalinos

Avaliaram-se os efeitos de dietas com níveis crescentes de milho em grão moído (0, 22, 37 e 49% na MS) em substituição ao feno de coast-cross mantendo-se diferentes relações proteína:carboidratos não-fibrosos (PB:CNF = 1,01; 0,39; 0,33 e 0,27) sobre o metabolismo ruminal de búfalos. Utilizaram-se quatro búfalos fistulados no rúmen, mantidos em delineamento quadrado latino 4 × 4, para a coleta de amostras do líquido ruminal, colhidas em cada período experimental (de 28 dias) nos tempos 0, 2, 4 e 8 horas após a alimentação. Em geral, os bubalinos apresentaram boa capacidade tamponante no rúmen, com pH médio alto (6,70) e aumento da ingestão de milho em grão moído. O acréscimo nos níveis de milho na dieta promoveu aumento da produção de ácido butírico. Somente a dieta com 49% de milho promoveu melhor fermentação ruminal, com menor propoção de ácidos acético:propiônico. A relação PB:CNF de 1,01 indica deficiência de energia da dieta disponível para microrganismos no rúmen ao longo do dia, enquanto dietas com PB:CNF entre 0,39 e 0,27 promovem fermentações ruminais semelhantes, o que indica sincronismo na utilização de nitrogênio e energia pelos microrganismos no rúmen nessas condições.

Effects of dietary energy and nitrogen supplements on rumen fermentation and protozoa population in buffalo and zebu cattle

The effects were assessed of two energy sources in concentrate (ground grain corn vs. citrus pulp) and two nitrogen sources (soybean meal vs. urea) on rumen metabolism in four buffaloes and four zebu cattle (Nellore) with rumen cannula and fed in a 4 × 4 Latin square design with feeds containing 60% sugar cane. Energy supplements had no effect on the rumen ammonia concentration in cattle, but ground grain corn promoted higher ammonia level than citrus pulp in buffalo. Urea produced higher ammonia level than soybean meal in both animal species. On average, the buffaloes maintained a lower rumen ammonia concentration (11.7 mg/dL) than the cattle (14.5 mg/dL). Buffaloes had lower production of acetic acid than cattle (58.7 vs. 61.6 mol/100 mol) and higher of propionic acid (27.4 vs. 23.6 mol/100 mol). There was no difference in the butyric acid production between the buffaloes (13.6 mol/100 mol) and cattle (14.8 mol/100 mol) and neither in the total volatile fatty acids concentration (82.5 vs. 83.6 mM, respectively). The energy or nitrogen sources had no effect on rumen protozoa count in either animal species. The zebu cattle had higher rumen protozoa population (8.8 × 10(5)/mL) than the buffaloes (6.1 × 10(5)/mL). The rumen protozoa population differed between the animal species, except for Dasytricha and Charonina. The buffaloes had a lower Entodinium population than the cattle (61.0 vs 84.9%, respectively) and a greater percentage of species belonging to the Diplodiniinae subfamily than the cattle (28.6 vs. 1.4%, respectively). In cattle, ground corn is a better energy source than citrus pulp for use by Entodinium and Diplodiniinae. In the buffaloes, the Entodinium are favored by urea and Diplodiniinae species by soybean meal.

Características de fermentação da silagem de seis variedades de milho indicadas para a região semiárida Brasileira

Avaliaram-se as características fermentativas e a qualidade das silagens de seis variedades de milho, de ciclos precoce e superprecoce - BRS Caatingueiro, BRS Assum Preto, BR 5033 Asa Branca, BR 5028 São Francisco, Gurutuba e BRS 4103 - indicadas para a região semiárida brasileira. Foram utilizados silos experimentais, em delineamento inteiramente ao acaso, com seis tratamentos (variedades) e quatro repetições. Avaliaram-se: matéria seca (MS), matéria orgânica (MO), proteína bruta (PB), fibra em detergente neutro (FDN), fibra em detergente ácido (FDA), extrato etéreo (EE), carboidratos totais (CHO), carboidratos não fibrosos (CNF), pH, nitrogênio amoniacal como parte do nitrogênio total (N-NH3/NT), ácidos orgânicos e digestibilidade in vitro da matéria seca (DIVMS) das silagens. Os valores médios encontrados para a silagem foram: MS= 28,7%; MO= 94,9%; PB= 8,3%; FDN= 49,9%; FDA= 27,5%; EE= 3,8%; CHO= 82,7%; CNF= 32,8%; pH= 3,8; N-NH3/NT= 2,9%/NT; ácido láctico = 7,6%; ácido acético = 0,6%; ácido butírico = 0,3% e DIVMS= 57,9%. As variedades BR 5028 - São Francisco e Gurutuba destacaram-se das demais em relação ao teor de matéria seca. A variedade BRS Caatingueiro apresentou maior teor de carboidratos não fibrosos em relação às demais. As silagens de todas as variedades foram classificadas como de excelente qualidade, por apresentarem potencial para ensilagem no semiárido brasileiro

Evaluation of elephant grass silage with the addition of cassava scrapings

The objective of this experiment was to evaluate the effects of adding cassava scrapings on gas and effluent losses, dry matter recovery, pH, contents of N-NH3, organic acids and volatile fatty acids and the bromatological composition of elephant grass silages. It was used a randomized complete design, with four levels of cassava scrapings (0, 7, 15 or 30% natural matter) each one with four replications per level. The grass was cut at 50 days of regrowth and ensiled in 15-L silos, equipped with a Bunsen valve to allow gas outflow. The gas losses decreased quadratically with the addition of cassava scrapings, whereas effluent losses decreased linearly. Dry matter recovery increased quadratically with the addition of cassava scrapings. Dry matter (DM) concentration increased but crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF) and hemicellulose (HEM) decreased linearly with the addition of cassava scrapings. The pH value and lactic acid concentration increased quadratically with the addition of cassava scrapings. Contents of N-NH3 and butyric acid decreased quadratically with the addition of cassava scrapings, whereas acetic acid content decreased linearly. Addition of cassava scrapings reduced gas and effluent losses and improved the fermentation profile of elephant grass silages and the level of 7% already ensures this improvement.

Fermentative hydrogen production by microbial consortium

Heat pre-treatment of the inoculum associated to the pH control was applied to select hydrogen-producing bacteria and endospores-forming bacteria. The source of inoculum to the heat pre-treatment was from a UASB reactor used in the slaughterhouse waste treatment. The molecular biology analyses indicated that the microbial consortium presented microorganisms affiliated with Enterobacter cloacae (97% and 98%), Clostridium sp. (98%) and Clostridium acetobutyricum (96%), recognized as H, and volatile acids` producers. The following assays were carried out in batch reactors in order to verify the efficiencies of sucrose conversion to H-2 by the microbial consortium: (1) 630.0 mg sucrose/L, (2) 1184.0 mg sucrose/L, (3) 1816.0 mg sucrose/L and (4) 4128.0 mg sucrose/L. The subsequent yields were obtained as follows: 15% (1.2 mol H-2/mol sucrose), 20% (1.6 mol H-2/mol sucrose), 15% (1.2 mol H-2/mol sucrose) and 4% (0.3 mol H-2/mol sucrose), respectively. The intermediary products were acetic acid, butyric acid, methanol and ethanol in all of the anaerobic reactors. (C) 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.

Long-term stability of hydrogen and organic acids production in an anaerobic fluidized-bed reactor using heat treated anaerobic sludge inoculum

This study evaluates the stability of hydrogen and organic acids production in an anaerobic fluidized-bed reactor (AFBR) that contains expanded clay (2.8-3.35 mm in diameter) as a support medium and is operated on a long-term basis. The reactor was inoculated with thermally pre-treated anaerobic sludge and operated with decreasing hydraulic retention time (HRT), from 8 h to 1 h, at a controlled temperature of 30 degrees C and a pH of about 3.8. Glucose (2000 mg L(-1)) was used as the substrate, generating conversion rates of 92-98%. Decreasing the HRT from 8 h to 1 h led to an increase in average hydrogen-production rates, with a maximum value of 1.28 L h(-1) L(-1) for an HRT of 1 h. In general, hydrogen yield production increased as HRT decreased, reaching 2.29 mol of H(2)/mol glucose at an HRT of 2 h and yielding a maximum hydrogen content of 37% in the biogas. No methane was detected in the biogas throughout the period of operation. The main soluble metabolites (SMP) were acetic acid (46.94-53.84% of SMP) and butyric acid (34.51-42.16% of SMP), with less than 15.49% ethanol. The steady performance of the AFBR may be attributed to adequate thermal treatment of the inoculum, the selection of a suitable support medium for microbial adhesion, and the choice of satisfactory environmental conditions imposed on the system. The results show that stable hydrogen production and organic acids production were maintained in the AFBR over a period of 178 days. (C) 2009 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.

Anaerobic fluidized bed reactor with expanded clay as support for hydrogen production through dark fermentation of glucose

This study evaluated hydrogen production in an anaerobic fluidized bed reactor (AFBR) fed with glucose-based synthetic wastewater. Particles of expanded clay (2.8-3.35 mm) were used as a support material for biomass immobilization. The reactor was operated with hydraulic retention times (HRT) ranging from 8 to 1 h. The hydrogen yield production increased from 1.41 to 2.49 mol H(2) Mol(-1) glucose as HRT decreased from 8 to 2 h. However, when HRT was 1 h, there was a slight decrease to 2.41 mol H(2) Mol(-1) glucose. The biogas produced was composed of H(2) and CO(2), and the H(2) content increased from 8% to 35% as HRT decreased. The major soluble metabolites during H(2) fermentation were acetic acid (HAc) and butyric acid (HBu), accounting for 36.1-53.3% and 37.7-44.9% of total soluble metabolites, respectively. Overall, the results demonstrate the potential of using expanded clay as support material for hydrogen production in AFBRs. (c) 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.

`Swingle` citrumelo propagation by cuttings for citrus nursery tree production or inarching

`Swingle` citrumelo [Citrus paradisi MacFaden x Poncirus trifoliata (L.) Raf.] has been extensively used as a rootstock in several citrus growing regions of the World, including Southern Brazil where `Rangpur` lime (Citrus limonia Osbeck) is still the predominant variety despite being affected by several important pathogens. in this case, `Swingle` citrumelo is used to produce nursery trees to establish new orchards or to be inarched in adult and healthy groves in order to change the rootstock. We report herein a system to produce trees on `Swingle` citrumelo more rapidly by budding onto non-rooted cuttings, as well as assessing potential to rapidly multiply `Swingle` through rooting of non-budded cuttings. Therefore, two potential products are described: budded trees and rooted rootstock cuttings. `Valencia` sweet orange [Citrus sinensis (L.) Osbeck] was budded at different heights on cuttings derived from eight-month old rootstocks. Grafted and additional non-budded cuttings were then treated with indole-3-butyric acid (500 mg L(-1)) or left untreated before rooting. Three types of cuttings were evaluated: softwood, semi-hardwood and hardwood. The use of nursery trees derived from pre-budded hardwood cuttings of `Swingle` citrumelo is an alternative grafting method on this cultivar. Softwood cuttings with one leaf pair were considered the most adequate material for rapid multiplication of `Swingle` citrumelo by cutting. This could be particularly useful for inarching production or conventional budding after transplant of cutting-derived rootstocks. (C) 2008 Elsevier B.V. All rights reserved.

Absence of Melatonin Induces Night-Time Hepatic Insulin Resistance and Increased Gluconeogenesis Due to Stimulation of Nocturnal Unfolded Protein Response

It is known that the circadian rhythm in hepatic phosphoenolpyruvate carboxykinase expression (a limiting catalytic step of gluconeogenesis) and hepatic glucose production is maintained by both daily oscillation in autonomic inputs to the liver and night feeding behavior. However, increased glycemia and reduced melatonin (Mel) levels have been recently shown to coexist in diabetic patients at the end of the night period. In parallel, pinealectomy (PINX) is known to cause glucose intolerance with increased basal glycemia exclusively at the end of the night. The mechanisms that underlie this metabolic feature are not completely understood. Here, we demonstrate that PINX rats show night-time hepatic insulin resistance characterized by reduced insulin-stimulated RAC-alpha serine/threonine-protein kinase phosphorylation and increased phosphoenolpyruvate carboxykinase expression. In addition, PINX rats display increased conversion of pyruvate into glucose at the end of the night. The regulatory mechanism suggests the participation of unfolded protein response (UPR), because PINX induces night-time increase in activating transcription factor 6 expression and prompts a circadian fashion of immunoglobulin heavy chain-binding protein, activating transcription factor 4, and CCAAT/enhancer-binding protein-homologous protein expression with Zenith values at the dark period. PINX also caused a night-time increase in Tribble 3 and regulatory-associated protein of mammalian target of rapamycin; both were reduced in liver of PINX rats treated with Mel. Treatment of PINX rats with 4-phenyl butyric acid, an inhibitor of UPR, restored night-time hepatic insulin sensitivity and abrogated gluconeogenesis in PINX rats. Altogether, the present data show that a circadian oscillation of UPR occurs in the liver due to the absence of Mel. The nocturnal UPR activation is related with night-time hepatic insulin resistance and increased gluconeogenesis in PINX rats. (Endocrinology 152: 1253-1263, 2011)

Surface and subsurface erosion of primary enamel by acid beverages over time

This study evaluated the influence of a cola-type soft drink and a soy-based orange juice on the surface and subsurface erosion of primary enamel, as a function of the exposure time. Seventy-five primary incisors were divided for microhardness test (n=45) or scanning electron microscopy (SEM) analysis (n=30). The specimens were randomly assigned to 3 groups: 1 - artificial saliva (control); 2 - cola-type soft drink; and 3 - soy-based orange juice. Immersion cycles in the beverages were undertaken under agitation for 5 min, 3 times a day, during 60 days. Surface microhardness was measured at 7, 15, 30, 45 and 60 days. After 60 days, specimens were bisected and subsurface microhardness was measured at 30, 60, 90, 120, 150 and 200 µm from the surface exposed. Data were analyzed by ANOVA and Tukey’s test (a=0.05). Groups 2 and 3 presented similar decrease of surface microhardness. Regarding subsurface microhardness, group 2 presented the lowest values. SEM images revealed that after 60 days the surfaces clearly exhibited structural loss, unlike those immersed in artificial saliva. It may be concluded that erosion of the surfaces exposed to the cola-type soft drink was more accentuated and directly proportional to the exposure time.

Can previous acid etching increase the bond strength of a self-etching primer adhesive to enamel?

Because a greater research effort has been directed to analyzing the adhesive effectiveness of self etch primers to dentin, the aim of this study was to evaluate, by microtensile testing, the bond strength to enamel of a composite resin combined with a conventional adhesive system or with a self-etching primer adhesive, used according to its original prescription or used with previous acid etching. Thirty bovine teeth were divided into 3 groups with 10 teeth each (n= 10). In one of the groups, a self-etching primer (Clearfil SE Bond - Kuraray) was applied in accordance with the manufacturer's instructions and, in the other, it was applied after previous acid etching. In the third group, a conventional adhesive system (Scotchbond Multipurpose Plus - 3M-ESPE) was applied in accordance with the manufacturer's instructions. The results obtained by analysis of variance revealed significant differences between the adhesive systems (F = 22.31). The self-etching primer (Clearfil SE Bond) presented lower enamel bond strength values than the conventional adhesive system (Scotchbond Multipurpose Plus) (m = 39.70 ± 7.07 MPa) both when used according to the original prescription (m = 27.81 ± 2.64 MPa) and with previous acid etching (m = 25.08 ± 4.92 MPa).

The role of γ -aminobutyric acid (Gaba) in somatic embryogenesis of Acca sellowiana Berg. (Myrtaceae)

The γ-aminobutyric acid (Gaba) is a non-protein amino acid found in prokaryotes and eukaryotes. Its role in plant development has not been fully established. This study reports a quantification of the levels of endogenous Gaba, as well as investigation of its role in different stages of somatic embryogenesis in Acca sellowiana Berg. (Myrtaceae). Zygotic embryos were used as explants and they were inoculated into the culture medium contained different concentrations of Gaba (0,2, 4, 6, 8 and 10 µM). The highest concentrations of endogenous Gaba were detected between the third and nine days after inoculation, reaching the value of 12.77 µmol.g-1FW. High frequency of somatic embryogenesis was observed in response to 10 µM Gaba. This treatment also resulted in a large number of normal embryos, and the lowest percentage of formation of fused somatic embryos, phenotypic characteristic of most deformed embryos in all treatments. Also, all treatments promoted the formation of the somatic embryos with positive characteristics of development resumption, which however did not originate the seedlings.

Tensile bond strength and SEM analysis of enamel etched with Er:YAG laser and phosphoric acid: a comparative study In vitro

Er:YAG laser has been studied as a potential tool for restorative dentistry due to its ability to selectively remove oral hard tissue with minimal or no thermal damage to the surrounding tissues. The purpose of this study was to evaluate in vitro the tensile bond strength (TBS) of an adhesive/composite resin system to human enamel surfaces treated with 37% phosphoric acid, Er:YAG laser (lambda=2.94 mum) with a total energy of 16 J (80 mJ/pulse, 2Hz, 200 pulses, 250 ms pulse width), and Er:YAG laser followed by phosphoric acid etching. Analysis of the treated surfaces was performed by scanning electron microscopy (SEM) to assess morphological differences among the groups. TBS means (in MPa) were as follows: Er:YAG laser + acid (11.7 MPa) > acid (8.2 MPa) > Er:YAG laser (6.1 MPa), with the group treated with laser+acid being significantly from the other groups (p=0.0006 and p= 0.00019, respectively). The groups treated with acid alone and laser alone were significantly different from each other (p=0.0003). The SEM analysis revealed morphological changes that corroborate the TBS results, suggesting that the differences in TBS means among the groups are related to the different etching patterns produced by each type of surface treatment. The findings of this study indicate that the association between Er:YAG laser and phosphoric acid can be used as a valuable resource to increase bond strength to laser-prepared enamel.