Abstract  Background: Metabolomic studies have been applied to disease biomarkers selection. With the metabolomic technique, gas chromatography/mass spectrometry (GC/MS), human serum metabolites can be detected and identified. The purpose of this study was to investigate the serum metabolic profile of hepatitis B virus (HBV) infected cirrhosis patients and to detect disease biomarkers. Methods: HBV infected non-cirrhosis male subjects (n=20) and HBV infected cirrhosis male patients (n=20) participated in this experiment. Serum metabolome was detected through chemical derivatization followed by GC/MS. The high-flux metabolomic data were analyzed by stepwise discriminant analysis. Results: Out of the 41 metabolites detected in serum, we selected metabolites, including acetic acid, sorbitol, D-lactic acid, hexanoic acid, 1-naphthalenamine, butanoic acid, phosphoric acid, D-glucitol, and glucose, which in combination with each other could segregate the two groups. The error count was 0% for the non-cirrhosis group and 25% for the cirrhosis group. Conclusions: This technique can be used to select biomarkers for hepatic cirrhosis. Clin Chem Lab Med 2009;47.

Abstract  The gut microflora in some patients with Crohn's disease can be reduced in numbers of butyrate-producing bacteria and this could result in metabolic stress in the colonocytes. Thus, we hypothesized that the short-chain fatty acid, butyrate, is important in the maintenance and regulation of the barrier function of the colonic epithelium.

Confluent monolayers of the human colon-derived T84 or HT-29 epithelial cell lines were exposed to dinitrophenol (DNP (0.1 mM), uncouples oxidative phosphorylation) + Escherichia coli (strain HB101, 10(6) cfu) +/- butyrate (3-50 mM). Transepithelial resistance (TER), and bacterial internalization and translocation were assessed over a 24-hour period. Epithelial ultrastructure was assessed by transmission electron microscopy.

Epithelia under metabolic stress display decreased TER and increased numbers of pseudopodia that is consistent with increased internalization and translocation of the E. coli. Butyrate (but not acetate) significantly reduced the bacterial translocation across DNP-treated epithelia but did not ameliorate the drop in TER in the DNP+E. coli exposed monolayers. Inhibition of bacterial transcytosis across metabolically stressed epithelia was associated with reduced I-kappaB phosphorylation and hence NF-kappaB activation.

Reduced butyrate-producing bacteria could result in increased epithelial permeability particularly in the context of concomitant exposure to another stimulus that reduces mitochondria function. We speculate that prebiotics, the substrate for butyrate synthesis, is a valuable prophylaxis in the regulation of epithelial permeability and could be of benefit in preventing relapses in IBD.

Abstract  Metabolite accumulation has pleiotropic, toxic, or beneficial effects on cell physiology, but such effects are not well understood at the molecular level. Cells respond and adapt to metabolite stress by mechanisms largely unexplored, especially in the context of multiple and simultaneous stresses. Solventogenic and related clostridia have an inherent advantage for production of biofuels and chemicals directly from cellulosic material and other complex carbohydrates, but issues of product/metabolite tolerance and related culture productivities remain. Using DNA microarray-based gene expression analysis, the transcriptional-stress responses of Clostridium acetobutylicum to fermentation acids acetate and butyrate and the solvent product butanol were analyzed and compared in the context of cell physiology. Ontological analysis demonstrated that stress by all three metabolites resulted in upregulation of genes related to post-translational modifications and chaperone activity, and downregulation of the translation-machinery genes. Motility genes were downregulated by acetate-stress only. The general metabolite stress included upregulation of numerous stress genes (dnaK, groES, groEL, hsp90, hsp18, clpC, and htrA), the solventogenic operon aad-ctfA-ctfB, and other solventogenic genes. Acetate stress downregulated expression of the butyryl-CoA- and butyrate-formation genes, while butyrate stress downregulated expression of acetate-formation genes. Pyrimidine-biosynthesis genes were downregulated by most stresses, but purine-biosynthesis genes were upregulated by acetate and butyrate, possibly for thiamine and histidine biosynthesis. Methionine-biosynthesis genes were upregulated by acetate stress, indicating a possibly conserved stress response mechanism also observed in Escherichia coli. Nitrogen-fixation gene expression was upregulated by acetate stress. Butyrate stress upregulated many iron-metabolism genes, riboflavin-biosynthesis genes, and several genes related to cellular repair from oxidative stress, such as perR and superoxide dismutases. Butanol stress upregulated the glycerol metabolism genes glpA and glpF. Surprisingly, metabolite stress had no apparent effect on the expression of the sporulation-cascade genes. It is argued that the list of upregulated genes in response to the three metabolite stresses includes several genes whose overexpression would likely impart tolerance, thus making the information generated in this study, a valuable source for the development of tolerant recombinant strains.

Abstract  Anaerobic bacteria such as the solventogenic clostridia can ferment a wide range of carbon sources (e.g., glucose, galactose, cellobiose, mannose, xylose, and arabinose) to produce carboxylic acids (acetic and butyric) and solvents such as acetone, butanol, and ethanol (ABE). The fermentation process typically proceeds in two phases (acidogenic and solventogenic) in a batch mode. Poor solvent resistance by the solventogenic clostridia and other fermenting microorganisms is a major limiting factor in the profitability of ABE production by fermentation. The toxic effect of solvents, especially butanol, limits the concentration of these solvents in the fermentation broth, limiting solvent yields and adding to the cost of solvent recovery from dilute solutions. The accepted dogma is that toxicity in the ABE fermentation is due to chaotropic effects of butanol on the cell membranes of the fermenting microorganisms, which poses a challenge for the biotechnological whole-cell bio-production of butanol. This mini-review is focused on (1) the effects of solvents on inhibition of cell metabolism (nutrient transport, ion transport, and energy metabolism); (2) cell membrane fluidity, death, and solvent tolerance associated with the ability of cells to tolerate high concentrations of solvents without significant loss of cell function; and (3) strategies for overcoming poor solvent resistance in acetone and butanol-producing microorganisms.

Abstract  The solventogenic bacterium Clostridium acetobutylicum is the most important species of Clostridium used in the fermentation industry. However, the intolerance to butanol hampers the efficient production of solvents. Butanol toxicity has been attributed to the chaotropic effect on the cell membrane, but the knowledge on the effect of butanol on membrane associated proteins is quite limited. Using 2-DE combined with MALDI-TOF MS/MS and 1-DE integrated with LC-MS/MS, 341 proteins in the membrane fractions of cell lysate were identified, thus establishing the first comprehensive membrane proteome of C. acetobutylicum. The identified proteins are mainly involved in transport, cellular membrane/wall machinery, formation of surface coat and flagella, and energy metabolism. Comparative analysis on the membrane proteomes of the wild type strain DSM 1731 and its butanol-tolerant mutant Rh8 revealed 73 differentially expressed proteins. Hierarchical clustering analysis suggested that mutant Rh8 may have evolved a more stabilized membrane structure, and have developed a cost-efficient energy metabolism strategy, to cope with the butanol challenge. This comparative membrane proteomics study, together with our previous published work on comparative cytoplasmic proteomics, allows us to obtain a systemic understanding of the effect of butanol on cellular physiology of C. acetobutylicum.

Abstract  Rhodococcus erythropolis is a promising Gram-positive bacterium capable of numerous bioconversions including those involving alcohol dehydrogenases (ADHs). In this work, we compared and optimized the redox biocatalytic performances of 1- butanol-grown R. erythropolis NCIMB 13064 cells in aqueous and in non-conventional gas phase using the 1-butanol-hexanal oxidation-reduction as model reaction. Oxidation of 1-butanol to butanal is tightly coupled to the reduction of hexanal to 1-hexanol at the level of a nicotinoprotein-ADH-like enzyme. Cell viability is dispensable for reaction. In aqueous batch conditions, fresh and lyophilized cells are efficient redox catalysts (oxidation-reduction rate = 765 mu mol min(-1) g cell dry mass(-1)) being also reactive towards benzyl alcohol, (S)-2- pentanol, and geraniol as reductants. However, butanol-hexanal oxidation-reduction is strongly limited by product accumulation and by hexanal toxicity that is a major factor influencing cell behavior and performance. Reaction rate is maximal at 40 degrees C-pH 7.0 in aqueous phase and at 60 degrees C-pH 7.0-9.0 in gas phase. Importantly, lyophilized cells also showed to be promising redox catalysts in the gas phase (at least 65 mu mol min(-1) g cell dry mass(-1)). The system is notably stable for several days at moderate thermodynamic activities of hexanal (0.06-0.12), 1- butanol (0.12) and water (0.7). (C) 2008 Elsevier Inc. All rights reserved.

Abstract  Using fermentation to replace chemical processes in the production of acetone and butanol depends largely on the availability of inexpensive and abundant raw materials and efficient conversion of these materials to solvents. In this study solvent production of Clostridium acetobutylicum ATCC824 from nano-membrane concentrated hemicellulosic hydrolysate was investigated. Alkali pretreatment methods were applied to improve fermentability of nano-membrane concentrated hemicellulosic hydrolysate and solvent production by ATCC824. Results demonstrated that though nanofiltration could remove nearly all small molecular organic acids (acetic acid, formic acid), furfural and HMF, the resulting hydrolysate found to be still inhibiting solvent production of C. acetobutylicum. Solid particles separated from filtering hydrolysate were found not toxic to cells when xylose or glucose was used as carbon resource. Overliming treatment can significantly improve the ultimate butanol concentration to 7g l(-1) from 0.8 g l(-1). Providing cells with more carbon source at the final stage of fermentation was found to have no impact on butanol production, but acetic acid and butyric acid production were found to increase significantly. The reasons leading to low solvent yield at later fermentation stages is not cell degeneration, but the toxicity of butanol and inhibitors remaining in the hydrolysate. (C) 2010 Elsevier Ltd. All rights reserved.

Abstract  Formation of compounds that are toxic to fermenting organisms during pretreatment and hydrolysis of lignocellulosic biomass has long been identified as one of the key factors affecting bioconversion of biomass to biofuels. In this study, fermentation was carried out to convert electrolyzed water pretreated dried distillers' grains and solubles (DDGS) hydrolysates to acetone butanol ethanol (ABE) using the hyper-butanol producing Clostridium beijerinckii BA 101. The presence of inhibitors in the DDGS hydrolysates resulted in a 40 h adjustment phase before the initiation of fermentation while the mixed sugar control fermentation was completed in less than 16 hours. While the ABE yield (ABE produced per unit of sugar utilized) obtained from the fermentation of DDGS hydrolysates was comparable to the yield obtained from the mixed sugar control fermentation, ABE productivity (ABE produced per unit of fermentation time) from the DDGS hydrolysates was lower than that of the mixed sugar control due to the long adjustment time experienced by C. beijerinckii BA101 in DGGS hydrolysates fermentation. Consequently, C. beijerinckii BA101 spores which had sporulated in DDGS hydrolysates were collected and used to ferment inhibitors-containing DDGS hydrolysates. Results demonstrated that inhibitor-adapted C. beijerinckii cells were able to adjust to the inhibitory environment in less than 20 hours and produce approximately the same amount of ABE as in the case for the control fermentation. Challenging the microorganism using the inhibitors present in the hydrolysates is believed to have enhanced the tolerance of C. beijerinckii to the inhibitors and resulted in significantly shorter lag phase. The results from this study demonstrated that the use of inhibitor-adapted Clostridium may be a promising option for ABE production.

Abstract  Interest in biobutanol, a sustainable vehicle fuel, is increasing due to rising oil prices and concerns of surrounding climate change and the energy crisis. However, the costs of biobutanol with conventional ABE fermentation by Clostridium are higher than the cost of butanol from today's petrochemical processes. Two major problems in the economic production of biobutanol are difficulty controlling the induction of a metabolic shift from acidogenesis to solventogenesis and limitations imposed by severe product inhibition. With developments in biotechnology, and the completion of genome sequencing of Clostridium, genetic modification is a viable method to improve the solvent yield and the butanol production ratio. The present article aims to highlight the latest research progress on overexpressing, inserting, knocking out, and knocking down genes of the key enzymes in the ABE fermentation pathway and other relative genes (such as genes coding for heat-shock proteins, operon, transcription, etc). Recombinant manipulations of these genes in Escherichiacoli and yeast have also been reported recently, although their butanol yields are lower than in Clostridium. Butanol production with solventogenic clostridia from various feedstocks is also evaluated in this review.

Abstract  Recent advances in systems biology, omics, and computational studies allow us to carry out data mining for improving biofuel production bioprocesses. Of particular interest are bioprocesses that center on microbial capabilities to biotransform both the hexose and pentose fractions present in crop residues. This called for a systematic exploration of the components of the media to obtain higher-density cultures and more-productive fermentation operations than are currently found. By using a meta-analysis approach of the transcriptional responses to butanol stress, we identified the nutritional requirements of solvent-tolerant strain Clostridium beijerinckii SA-1 (ATCC 35702). The nutritional requirements identified were later validated using the chemostat pulse-and-shift technique. C. beijerinckii SA-1 was cultivated in a two-stage single-feed-stream continuous production system to test the proposed validated medium formulation, and the coutilization of D-glucose and D-xylose was evaluated by taking advantage of the well-known ability of solventogenic clostridia to utilize a large variety of carbon sources such as mono-, oligo-, and polysaccharides containing pentose and hexose sugars. Our results indicated that C. beijerinckii SA-1 was able to coferment hexose/pentose sugar mixtures in the absence of a glucose repression effect. In addition, our analysis suggests that the solvent and acid resistance mechanisms found in this strain are differentially regulated compared to strain NRRL B-527 and are outlined as the basis of the analysis toward optimizing butanol production.

Abstract  BACKGROUND: Colon cancer is one of the leading causes of cancer related deaths. Its impact on African Americans (AAs) is higher than in the general population both in the incidence and mortality from the disease. Colon cancer aggressiveness in AAs as well as non-frequent check-ups and follow up in this population have been proposed as ways to explain the observed discrepancies. These facts made the detection of early carcinogenesis markers in this population a priority.

MATERIALS AND METHODS: Here, we analyzed 50 colon adenomas from AA patients for both microsatellite instability (MSI) and the methylation status of SLC5A8 gene. This gene's product is involved in the transport of butyrate that has anti-proliferative properties through its effects on histone acetylation and gene expression. A proteomic analysis to check the expressed histones in adenoma and normal tissues was also performed.

RESULTS: The analyzed samples displayed 82% (n = 41) methylation level of SLC5A8 gene in adenomas. The MSI-H (high) adenoma were about 18% (n = 9) while the rest were mostly MSS (microsatellite stable) with few MSI-L (Low). No association was found between SLC5A8 methylation and the MSI status. Also, there was no association between SLC5A8 methylation and the sex and age of the patients. However, there were more right sided adenomas with SLC5A8 methylation than the left sided ones. The proteomic analysis revealed distinct histone expression profiles between normal and adenoma tissues.

CONCLUSION: SLC5A8 is highly methylated in AA colon adenomas which points to its potential use as a marker for early detection. The MSI rate is similar to that found in colon cancer tumors in AAs. These findings suggest that both processes stem from the same epigenetic and genetic events occurring at an early stage in colon carcinogenesis in AAs.

Abstract  The combination of sodium butyrate (NaB) and ganciclovir (GCV) was considered to be a noteworthy therapeutic strategy in Epstein-Barr virus (EBV)-associated cancers. However, clinical studies have indicated that an extremely high dose of NaB is required to obtain the expected curative efficacy. This obviously limits the practical clinical application of the two drugs combined. In this study, we investigated the possibility of sensitizing tumor cells to NaB and GCV mediated cytotoxicity by modulating intracellular signal pathways. The results showed that the disruption of Ras/Raf activity by expressing dominant negative forms of both Ras and Raf-1 did not alter the potency of the NaB and GCV combination in the EBV-positive cell line, B95-8. However, blocking Akt activity by expressing its dominant negative form remarkably promoted NaB and GCV-mediated cytotoxicity via a thymidine kinase (TK)-independent mechanism. Interestingly, it was found that the constitutive activation of mitogen-activated protein kinase kinase kinase 1 (MEKK1) dramatically enhanced the sensitization of the cells to the combination of NaB and GCV, accompanied with an increase in TK expression in B95-8 cells. These results suggest that interfering with either the Akt or MEKK1 signaling pathway may be a useful therapeutic strategy to increase the sensitivity of EBV-positive tumor cells to the combination of NaB and GCV.

Abstract  The antiangiogenic and antineoplastic activities of the butyric acid prodrugs AN-7 and AN-9 were demonstrated in vitro with HUVEC by inhibition of proliferation and vascular tubes formation, enhanced apoptosis, and inhibition of 22Rv-1 cells migration. In the sc implanted human prostate tumors (22Rv-1) in nude mice, AN-7 significantly inhibited Ki-67, HIF-1alpha, HER-2/neu, bFGF and increased PTEN level. AN-7 and AN-9 reduced hemoglobin accumulation in matrigel plugs implanted sc in Balb-c mice. Herein, we show that the anticancer activity of AN-7 and AN-9 can be attributed in part to their antiangiogenic activities suggesting potential therapeutic benefits for prostate cancer patients.

Abstract  OBJECTIVE: There is a need for otoprotective agents that can be administered systemically without compromising cancer treatment. Histone deacetylase inhibitors are anticancer agents that act by upregulating the expression of cell-cycle control genes. They are also neuroprotective, leading us to hypothesize that they might be otoprotective. The goal of this study was to determine if the antitumor agent sodium butyrate (a histone deacetylase inhibitor) protects against cisplatin ototoxicity when administered systemically.

STUDY DESIGN: This was an animal study.

METHODS: : Cisplatin was administered to guinea pigs who received either 12 days of sodium butyrate (7 d before and 5 d after cisplatin) or equivolume saline injections. Hearing was tested with distortion product otoacoustic emission (DPOAE) analysis before the start of the study and 2 weeks after cisplatin treatment.

RESULTS: Guinea pigs given a single intraperitoneal injection of 14 mg/kg cisplatin experience a mean hearing loss of 8 dB across the frequencies of 3.5, 5, 7, 10, 14, and 20 kHz. Intraperitoneal injection of 1.2 mg/kg sodium butyrate per day for 7 days before and 5 days after cisplatin almost completely eliminates this threshold shift (P=.0011).

CONCLUSIONS: The histone deacetylase inhibitor sodium butyrate gives almost complete protection in a single-dose model of cisplatin ototoxicity in guinea pigs. Because histone deacetylase inhibitors are anticancer agents with very few side effects, they may be candidates for clinical use during cisplatin chemotherapy.

Abstract  While conservation of ATP is often a desirable trait for microbial production of chemicals, we demonstrate that additional consumption of ATP may be beneficial to drive product formation in a nonnatural pathway. Although production of 1-butanol by the fermentative coenzyme A (CoA)-dependent pathway using the reversal of β-oxidation exists in nature and has been demonstrated in various organisms, the first step of the pathway, condensation of two molecules of acetyl-CoA to acetoacetyl-CoA, is thermodynamically unfavorable. Here, we show that artificially engineered ATP consumption through a pathway modification can drive this reaction forward and enables for the first time the direct photosynthetic production of 1-butanol from cyanobacteria Synechococcus elongatus PCC 7942. We further demonstrated that substitution of bifunctional aldehyde/alcohol dehydrogenase (AdhE2) with separate butyraldehyde dehydrogenase (Bldh) and NADPH-dependent alcohol dehydrogenase (YqhD) increased 1-butanol production by 4-fold. These results demonstrated the importance of ATP and cofactor driving forces as a design principle to alter metabolic flux.

Abstract  An Escherichia coli strain was engineered to synthesize 1-hexanol from glucose by extending the coenzyme A (CoA)-dependent 1-butanol synthesis reaction sequence catalyzed by exogenous enzymes. The C4-acyl-CoA intermediates were first synthesized via acetyl-CoA acetyltransferase (AtoB), 3-hydroxybutyryl-CoA dehydrogenase (Hbd), crotonase (Crt), and trans-enoyl-CoA reductase (Ter) from various organisms. The butyryl-CoA synthesized was further extended to hexanoyl-CoA via β-ketothiolase (BktB), Hbd, Crt, and Ter. Finally, hexanoyl-CoA was reduced to yield 1-hexanol by aldehyde/alcohol dehydrogenase (AdhE2). Enzyme activities for the C6 intermediates were confirmed by assays using HPLC and GC. 1-Hexanol was secreted to the fermentation medium under anaerobic conditions. Furthermore, co-expressing formate dehydrogenase (Fdh) from Candida boidinii increased the 1-hexanol titer. This demonstration of 1-hexanol production by extending the 1-butanol pathway provides the possibility to produce other medium chain length alcohols using the same strategy.

Abstract  2,4,5-trimethyl-3-thiazoline (TMT), a component of fox feces, is a widely used odorant to induce innate fear behavior in rats and mice. However, based on the slight acrid smell it was argued that the observed behavioral effects are a result of the aversive and not of the fear-inducing properties of TMT. In the present study, we tried to directly compare the aversive and fear-inducing properties of TMT with those of the aversive control odor butyric acid. We first identified concentrations of butyric acid and TMT that induce similar amounts of avoidance behavior in rats, indicating that these concentrations have similar aversive properties. In a second experiment, these two concentrations were then tested for their ability to induce freezing, a species-specific defensive response. Only TMT but not butyric acid induced freezing in the rats. This supports the hypothesis that TMT indeed has specific fear-inducing properties and that the observed behavioral effects could not simply be reduced to the aversive properties of TMT.

Abstract  Our previous study has revealed that providing dry feeds increased the plasma concentration of antidiuretic hormone (ADH) in suckling calves, leading to altered water balance. To examine whether ketone bodies formed from ruminal fermentation-derived butyrate induced ADH secretion in suckling calves, the effects of intraruminal administration of butyrate on plasma concentration of ADH and ketone bodies, plasma and urine osmolality, and urine volume were examined. Six male Holstein calves aged 4 wk were used. Three levels of butyrate (0 g, 22 g and 44 g) were intraruminally administrated in a 3 x 3 Latin square design, and blood plasma and urine were analyzed. Plasma concentration of ketone bodies was increased by intraruminal administration of butyrate within 15 min in a dose-dependent manner, and the elevation of plasma levels continued until 4 h. Plasma concentration of ADH was also increased by the butyrate treatment, and it was higher in the 44 g butyrate group than in the 22 g butyrate group from 15 min to 2 h. The duration of the elevated plasma concentration of ADH was shorter than that of plasma concentration of ketone bodies. The relationship between plasma concentrations of ADH and those of ketone bodies was statistically significant, although the relationship was weaker. In accordance with the elevation of plasma ADH levels, the butyrate treatment resulted in the decreases in urine volume and increases in urine osmolality. Plasma osmolality was not different among the groups. The present results suggest that ruminal butyrate-derived ketone bodies are at least partly responsible for ADH secretion in suckling calves fed with dry feeds, and that the secreted ADH decreases urine volume through the increase in urine osmolality. (c) 2006 Elsevier B.V. All rights reserved.

Abstract  In the healthy adult brain, neurogenesis normally occurs in the subventricular zone (SVZ) and hippocampal dentate gyrus (DG). Cerebral ischemia enhances neurogenesis in neurogenic and non-neurogenic regions of the ischemic brain of adult rodents. This study demonstrated that post-insult treatment with a histone deacetylase inhibitor, sodium butyrate (SB), stimulated the incorporation of bromo-2'-deoxyuridine (BrdU) in the SVZ, DG, striatum, and frontal cortex in the ischemic brain of rats subjected to permanent cerebral ischemia. SB treatment also increased the number of cells expressing polysialic acid-neural cell adhesion molecule, nestin, glial fibrillary acidic protein, phospho-cAMP response element-binding protein (CREB), and brain-derived neurotrophic factor (BDNF) in various brain regions after cerebral ischemia. Furthermore, extensive co-localization of BrdU and polysialic acid-neural cell adhesion molecule was observed in multiple regions after ischemia, and SB treatment up-regulated protein levels of BDNF, phospho-CREB, and glial fibrillary acidic protein. Intraventricular injection of K252a, a tyrosine kinase B receptor antagonist, markedly reduced SB-induced cell proliferation detected by BrdU and Ki67 in the ipsilateral SVZ, DG, and other brain regions, blocked SB-induced nestin expression and CREB activation, and attenuated the long-lasting behavioral benefits of SB. Together, these results suggest that histone deacetylase inhibitor-induced cell proliferation, migration and differentiation require BDNF-tyrosine kinase B signaling and may contribute to long-term beneficial effects of SB after ischemic injury.