AroC, a chorismate synthase, is required for the formation of Edwardsiella tarda biofilms.

Biofilms contribute to the resistance of Edwardsiella tarda to antibiotics and host immunity. AroC in the shikimate pathway produces chorismate to synthesize crucial intermediates such as indole. In this study, the differences between biofilms produced by aroC mutants (△aroC), wild-type (WT) strains, and △aroC complementary strains (C△aroC) were detected both in vitro with 96-well plates, tubes, or coverslips and in vivo using a mouse model of subcutaneous implants. When examining potential mechanisms, we found that the diameters of the movement rings in soft agar plates and the flagellar sizes and numbers determined by silver staining were all lower for △aroC than for WT and C△aroC. Moreover, qRT-PCR showed that the transcription levels of flagellar synthesis genes, fliA and fliC, were reduced in △aroC. AroC, FliC, or FliA may accompany the motility of △aroC strains. In addition, compared with the WT and C△aroC, the amounts of indole in △aroC were significantly decreased. Notably, the formation of biofilms by these strains could be promoted by exogenous indole. Therefore, the aroC gene could affect the biofilm formation of E. tarda concerning its impact on flagella and indole.

Biomimetic smart nanoplatform for dual imaging-guided synergistic cancer therapy.

Developing an integrated multimodal diagnosis and therapeutics nanoplatform is of great importance to enhance the outcome of cancer therapy. Herein, we report a highly efficient and biocompatible nanoplatform based on the assembly of a graphene oxide (GO) and metal-organic framework (MOF) Fe-porphyrin, which was coated with a folate-functionalized erythrocyte membrane (FA-EM@GO-MOF). The nanoplatform could be targeted to cancer cells precisely, and could avoid immune elimination and had prolonged blood circulation due to the presence of FA-EM on its surface. The presence of GO and paramagnetic Fe ions endowed the nanoplatform with robust fluorescence imaging and T2-weighted magnetic resonance imaging capacities. The porous structure and large surface area of GO-MOF make it desirable for drug delivery in chemotherapy. More importantly, with one operation, under the same laser (808 nm) irradiation, both photothermal therapy and photodynamic therapy could be triggered for efficient synergistic treatment by using MOF as a photosensitizer. This synergistic anticancer therapy promoted the generation of tumor-associated antigens and evoked an antitumor immune response. In vitro and in vivo therapy studies highlighted that the as-fabricated biomimetic nanoplatform for dual imaging-guided synergistic cancer therapy was highly effective yet straightforward, paving a new avenue for cancer diagnosis and therapy.

Target genes directly regulated by Eha are required for Edwardsiella tarda survival within macrophages.

Eha is a virulence regulator in Edwardsiella tarda (E. tarda). The present study examined how Eha regulated its target genes to affect the bacterial survival within the cells. We constructed the reporter a pGEX-4T-ehaflag plasmid expressing Eha tagged at its C terminus with the flag epitope, and introduced the plasmid into an eha mutant ET13 strain, and obtained a Cehaflag strain. The expression and activity of an EhaFlag fusion protein restored the survival of the Cehaflag as the wild type in macrophages by Western blotting and intracellular survival experiments. We used a monoclonal anti-Flag antibody to precipitate EhaFlag-DNA complexes using chromatic immunoprecipitation (ChIP). We then designed primers based on the differentially-expressed genes identified from RNA-sequencing, and identified ten Eha-interacting genes by qPCR. We amplified the promoter regions of the ten genes and the eha gene from ET13 strain by PCR, constructed pBD-PtargetlacZ and pBD-PehalacZ plasmids. The eha gene directly and positively regulated these target genes, and be negatively auto-regulated by Eha in E. tarda, as determined by comparing their ß-Galactosidase activities. These target genes were distributed in the categories involved in the bacterial growth, movement and resistance to H2O2 or acid. We further constructed a ETATCC_RS15225 mutant (△dcuA1), a ETATCC_ RS14855 mutant (△flgK) anda ETATCC_RS07650 mutant (ΔtnaA), and a partial complementary strains of △eha-tnaA and △eha-flgK and the complementary strains of CΔflgK and CΔtnaA. The ETATCC_RS15225 gene probably encoded a transporter protein DcuA1 at outer membrane with SDS-PAGE and RT-PCR. The ETATCC _RS14855 gene probably encoded FlgK protein and affected the bacterial motility. The ETATCC_RS07650 gene encoded Tryptophanase, which affected the bacterial survival within macrophages. With the assistance of these above strains, our results showed that the eha gene was able to regulate the ETATCC_RS15225 gene to express its outer membrane protein DcuA1, the ETATCC _RS14855 gene to control the flagellar motility and the ETATCC_RS07650 to affect the bacterial survival within macrophages. With the combination of other functions of above three genes, our results suggested that Eha directly regulates the target genes to affect E. tarda to survive within the cells.

Hyaluronic acid-modified mesoporous silica-coated superparamagnetic Fe3O4 nanoparticles for targeted drug delivery.

Introduction: The targeted delivery of anti-cancer drugs to tumor tissue has been recognized as a promising strategy to increase their therapeutic efficacy and reduce side effects. Mesoporous silica-coated superparamagnetic Fe3O4 nanoparticles (NH2-MSNs), a kind of nanocarrier, can passively enter tumor tissues to enhance the permeability and retention of drugs. However, NH2-MSNs do not specifically bind to cancer cells. This drawback encouraged us to develop a more efficient nanocarrier for cancer therapy. Methods: Herein, we describe the development of an effective nanocarrier based on NH2-MSNs, which were modified with hyaluronic acid on their surface (HA-MSNs) and loaded with doxorubicin (DOX). We have successfully fabricated uniform spherical HA-MSNs nanocarriers. The targeting ability of this delivery system was evaluated through specific uptake by cells and IVIS imaging. Results: DOX-HA-MSNs nanocarriers displayed more dramatic cytotoxic activity against 4T1 breast cancer cells compared to GES-1 gastric mucosa cells. In vivo results revealed that once DOX-HA-MSNs nanocarriers are exposed to an external magnetic field, they could be rapidly attracted to the magnet and effectively cross the cytoplasmic membrane via CD44 receptor-mediated transcytosis. This allows them to access the cancer cell cytoplasm and release DOX based on changes in the physiological environment. Both in vitro and in vivo results demonstrated that the HA-MSNs nanocarriers provided better therapeutic efficacy. Conclusion: The HA-MSNs nanocarriers represent an effective new paradigm to treat cancers due to active targeting to the tumor cells. Moreover, the specific uptake by the tumor effectively protects normal tissues to reduce off-target side effects. The reported findings support further investigation of HA-MSNs for cancer therapy.

EsR240, a non-coding sRNA, is required for the resistance of Edwardsiella tarda to stresses in macrophages and for virulence.

Bacterial small non-coding RNAs (sRNAs) are gene expression modulators that respond to environmental changes and pathogenic conditions. In this study, 13 novel sRNAs were identified in the intracellular pathogen, Edwardsiella tarda (E. tarda) ET13 strain, based on RNA sequencing and bioinformatic analyses. Eight of the 13 putative sRNAs from the ET13 strain were transcribed (as indicated by RT-PCR) following exposure to different stresses. The transcription levels of three sRNAs (EsR128, EsR139 and EsR240) were all highly induced under these stress conditions. Northern blot hybridization was employed to verify that EsR240 was expressed in the ET13 strain under both logarithmic and stationary growth phases, and that it formed a single copy transcript in the chromosomes of the ET13 strain. The precise start and end points of EsR240 were determined using 5'and 3' RACE. The conservation of EsR240 was in agreement with the characteristics of sRNA, as indicated by a BLAST analysis. Furthermore, the survival rates of EsR240 mutant were lower than the rates of the wild type ET13 under stress conditions. When the infection time was extended 4 or 6 h, the CFUs of the wild type bacteria increased more significantly within macrophages compared to the mutant. When the intra-peritoneal (i.p.) route of infection was used in mice, the bacterial loads of the tissues in the mice infected with the wild type bacteria were significantly higher than in the mice infected with the mutants. The virulence of the EsR240 mutant was 6.79-fold lower than the wild type bacterium based on the LD50. In addition, the IntaRNA program was used to predict the target genes of EsR240. Out of the top 10 predicted target genes, 9 genes were regulated by EsR240. These target genes may encode FtsH protease modulator YccA, Na+ and H+ antiporters, FtsX-like permease family protein, glycoside hydrolases or various other proteins. Therefore, EsR240 may positively regulate its target genes in E. tarda to maintain intracellular survival within host macrophages and to increase its virulence.

Eha, a regulator of Edwardsiella tarda, required for resistance to oxidative stress in macrophages.

Edwardsiella tarda is distributed widely in a variety of hosts. Eha has recently been found to be its virulence regulator. In order to explore the mechanism of its regulation, we investigated the survival rates of wild type strain ET13, and its eha mutant and complemented strains in RAW264.7 macrophages under light microscopic observation as well as by counting bacterial CFUs on the plates. All of the different strains could live within the macrophages; however, the intracellular numbers of the wild type were significantly higher than the mutant when the incubation time extended 4 h or 6 h (P < 0.05). Furthermore, more ROS were produced by the mutant-infected cells, indicating that Eha may enhance ET13's capacity to detoxify ROS. In agreement with this, we found that the mutant exhibited more sensitivity by H2O2 disk inhibitory assay and less survival ability with H2O2 treatment. We further demonstrated that the bacterial antioxidant enzymes SodC and KatG were regulated by Eha with qRT-PCR and ß-galactosidase assay. Collectively, our data show Eha is required for E. tarda to resist the oxidative stress from the macrophages.

The role of regulator Eha in Edwardsiella tarda pathogenesis and virulence gene transcription.

Edwardsiella tarda is a pathogen with a broad host range that infects both animals and humans. Eha is a new transcriptional regulator identified in ET13, which is involved in the bacterial hemolytic activity. This study explored the effect of the Eha in the pathogenesis of E. tarda and the transcriptional regulation of the bacterial virulence genes (eseC, fliC, pagC and fimA). Our results found that the virulence of the eha mutant was 2.5-fold less than the one of its wild ET13 by LD50 in a murine model of i.p. infection, and the bacterial loads of the mutant displayed a different profile from the one of the wild strain. Most significantly, the mice infected with the mutant have greatly reduced acute inflammation in the liver, spleen and kidney compared to the ones infected with the wild. We further demonstrated that eseC, fliC and pagC were regulated directly by the Eha with qRT-PCR and ß-Galactosidase assay, but fimA wasn't done. The promoter regions of the genes modulated and the cly gene reported before had been found to contain a common conserved motif by using software. In addition, we found that the wild strain was more toxic to RAW264.7 macrophages, and induced less the host cell apoptotic responses than the eha mutant did. Altogether, these data suggested that the Eha was required for the bacterial infection and the transcriptive regulation of the important virulence genes of E. tarda.

Eha, a transcriptional regulator of hemolytic activity of Edwardsiella tarda.

Hemolysis causes major symptoms such as the reddening skin and systemic hemorrhagic septicemia of diseased fish infected by Edwardsiella tarda. Cytolysin A (ClyA) is a pore-forming cytotoxic protein encoded by the clyA gene in Escherichia coli K-12. In this study, we observed that the heterologous expression of the eha gene from E. tarda could confer hemolytic activity upon a hemolytic-silent E. coli strain. The transcription of clyA is positively controlled by the eha gene in E. tarda by RT-PCR. We cloned and purified Eha protein which had shown preferential binding ability to the clyA sequences in its promoter region, as evidenced by gel shift assay. The eha controls the transcriptional start predominantly at 72 bp upstream in the clyA promoter region, as determined by primer extension assays. We suggest that Eha protein is a new positive regulator found in E. tarda. In addition, we constructed the eha mutant and complementary strains of E. tarda. The hemolytic activity of the eha mutant was found to be attenuated compared with the wild-type strain. The complementary strains restored the hemolytic activity to levels between those of the wild type and the eha mutation. Our results indicate that the Eha protein is an important positive regulator in the hemolytic properties of E. tarda.

Effect of hOGG1 over-expression on cisplatin resistance in esophageal squamous carcinoma cells.

PURPOSE: Human 8-oxoguanine DNA glycosylase (hOGG1) is an ubiquitous protein. It initiates the DNA base excision repair (BER) pathway to repair the 8-oxoguanine lesion. This may be associated with chemotherapeutics. In this article, the effect of hOGG1 over-expression on cisplatin resistance in esophageal squamous cell carcinoma (ESCC) EC9706 and ET13 cells was investigated. METHODS: Recombinant adenovirus pAd/CMV/V5-DEST-hogg1 and control adenovirus pAd/CMV/5-GW/lacZ were constructed and transferred into EC9706 and ET13 cells, respectively. The protein expression and localization were determined by Western blot and by immunofluorescence assay. The cell growth viability was determined by 3-(4,5-dimethylthiazol-2yl)-2,5 diphe-nyltetrazolium bromide (MTT) assay and clonogenic survival assay. The apoptotic cells were detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) staining and flow cytometry. The oxidative DNA damage (8-Hydroxyguanine [8-oxoG] DNA level) was semi-quantified by immunohistochemistry assay. RESULTS: The over-expression of hOGG1 protein was mainly in the nucleus in hOGG1 cells. After exposure to a common chemotherapeutic agent cisplatin, hOGG1 over-expression cells exhibited longer survival ability, lower cell apoptosis, and less 8-oxoG oxidative damage, compared with vector-treated cells and no-treated cells (p<0.05). CONCLUSION: BER pathway to repair 8-oxoG lesion may be associated with ESCC sensitivity to cisplatin, and over-expression of hOGG1 in the nucleus can repair more 8-oxoG oxidative damage. The findings implied that over-expression of hOGG1 can protect ESCC cells from cisplatin-induced apoptosis and prolong cancer cell survival time. Modulation of DNA damage repair activity in the nucleus or in the mitochondria may lead to a different approach regarding cisplatin-induced resistance to chemotherapy.

Selective decrease in the DNA base excision repair pathway in squamous cell cancer of the esophagus.

OBJECTIVES: Oxidative damage can lead to a highly mutagenic 8-oxoguanine lesion, which mispairs with adenosine residues, leading to G:C-->T:A transversions. In mammalian cells 8-oxoguanine glycosylase initiates the DNA base excision repair pathway to repair the 8-oxoguanine lesion. To date, there is no information regarding oxidative DNA damage and repair pathways in esophageal cancer. Therefore we designed the current study to demonstrate the DNA damage and repair pathways in esophageal cancer by expression of 8-oxoguanine glycosylase in reflux-induced and mutagen (methyl-n-amyl nitrosamine)-induced DNA damage and apoptosis in esophageal tumors. METHODS: Gastroduodenal reflux was surgically created in male Sprague Dawley rats (n = 120). Half of the animals received methyl-n-amyl nitrosamine. Animals not undergoing operations served as control animals (n = 10). The experiment concluded 30 weeks postoperatively. Immunohistochemistry for 8-oxoguanine and 8-oxoguanine glycosylase was assessed by 2 independent observers. Protein expression was assessed by using the Western blot method. RESULTS: There was significantly more DNA damage in both adenocarcinoma (n = 15) and squamous cell carcinoma (n = 19), as exemplified by positive 8-oxoguanine expression compared with that seen in control animals (P < .05). 8-Oxoguanine glycosylase was several folds upregulated in adenocarcinoma (P < .05), but there was significantly decreased expression in squamous cell carcinoma (P < .01). The apoptosis was assessed as caspase-dependent and caspase-independent pathways, and both were active and correlated well with 8-oxoguanine expression. CONCLUSION: These results demonstrate the selective decrease in the DNA base excision repair pathway in combined reflux and methyl-n-amyl nitrosamine-induced squamous cell cancer of the esophagus.

Duodenal reflux leads to down regulation of DNA mismatch repair pathway in an animal model of esophageal cancer.

BACKGROUND: Gastroduodenal reflux is implicated in esophageal carcinogenesis. This effect is mediated by reactive oxygen species. We hypothesized that this is mediated by DNA mismatch lesion 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxoG), which is repaired by the Mut Y homologue (MYH). We tested the effect of reflux, either alone or in combination with the human dietary mutagen methyl-n-amyl nitrosamine (MNAN), on DNA damage in adenocarcinoma and squamous cell cancer of the esophagus in a rat model. METHODS: Reflux was promoted in male Sprague-Dawley rats by duodenoesophageal anastomosis (8 weeks) without gastric bypass. MNAN treatment (25 mg/kg per week intraperitoneally for four doses) commenced at 10 weeks age. Ten animals served as controls. Quantification of 8-oxoG was performed by using immunohistochemistry, and MYH was analyzed by Western blot. Apoptosis was assessed by terminal deoxynucleotide transferase-mediated deoxy uridine triphosphate nick-end labeling (TUNEL), cytochrome C, and caspase. RESULTS: Tumors (adenocarcinoma) developed in 15 (50%) of 30 animals with reflux alone; this increased to 26 (86.6%) of 30 when reflux was combined with MNAN treatment, with tumor histology consistent with adenosquamous and squamous cell cancer. DNA damage, as reflected by positive 8-oxoG staining in reflux groups, was significantly increased compared with control (p < 0.01), and this was maximal in tissues with malignant transformation. Protein levels of the DNA repair enzyme MYH were significantly less in tissues subjected to reflux compared with controls (p < 0.05). TUNEL, cytochrome C, and caspase positivity confirmed increased apoptosis in cancer lesions. CONCLUSIONS: Gastroduodenal reflux leads to increased DNA damage and downregulation of the DNA mismatch repair pathway. This pathway has an important role in esophageal carcinogenesis in rats.

The mitochondrial K-ATP channel opener, diazoxide, prevents ischemia-reperfusion injury in the rabbit spinal cord.

Paraplegia resulting from ischemia is a catastrophic complication of thoracoabdominal aortic surgery. The current study was designed to investigate the effects of diazoxide (DZ) on mitochondrial structure, neurological function, DNA damage-repair, and apoptosis in spinal cord ischemia-reperfusion injury. Rabbits were subjected to 30 minutes of spinal cord ischemia and reperfusion (1 hour) with or without diazoxide (n = 6 in each group) by clamping and releasing the infrarenal aorta. The neurological functional score was significantly improved in the DZ-treated ischemia-reperfusion injury group. Electron microscopic studies demonstrated that mitochondrial damage in the spinal cord after injury was significantly reduced by DZ. Mitochondrial superoxide and hydrogen peroxide levels were also markedly decreased in the DZ-treated injury group compared with the untreated group. DZ decreased levels of the oxidative DNA damage product 8-oxoG and increased levels of the DNA repair enzyme OGG-1. Furthermore, DZ inhibited apoptosis via caspase-dependent and -independent pathways. These studies indicate for the first time that the mitochondrial K-ATP channel opener diazoxide improves neurological function after spinal cord ischemia and reperfusion by diminishing levels of reactive oxygen species, decreasing DNA oxidative damage, and inhibiting caspase-dependent and -independent apoptotic pathways while preserving mitochondrial structure.

Increased mitochondrial reactive oxygen species production in newborn brain during hypoglycemia.

Hypoglycemia is associated with gray and white matter injury in immature brain, but the specific mechanisms responsible for hypoglycemic brain injury remain poorly defined. We postulated that mitochondrial electron transport chain function is altered during hypoglycemia due to the decreased availability of reducing equivalents, and that altered activity of the electron transport chain would increase mitochondrial production of free radicals and lead to mitochondrial oxidant injury. The present study tests the hypothesis that production of reactive oxygen species (ROS) by cerebral mitochondria is increased during acute hypoglycemia. Studies were performed in an awake, chronically catheterized newborn piglet model. Hypoglycemia (blood glucose 1 mmol/L for 2 h) was induced using a bolus of intravenous lispro insulin, 25 U/kg. Superoxide and hydrogen peroxide production by mitochondria isolated from cerebral cortex of normoglycemic and hypoglycemic newborn piglets was measured using lucigenin- and luminol-derived chemiluminescence. After 2 h of hypoglycemia, superoxide generation was 60% higher and hydrogen peroxide generation was two-fold higher in mitochondria from hypoglycemia animals than in controls (p < 0.005). These data confirm that the ability of the mitochondria to produce ROS is increased after hypoglycemia in immature brain, and are, to our knowledge, the first evidence that ROS may play a role in brain injury due to neonatal hypoglycemia. Increased mitochondrial ROS production could result in alterations in brain structure and function due to oxidant injury to mitochondrial proteins and DNA or changes in oxidant-sensitive signal transduction pathways in brain.

beta(2)-Adrenergic stimulation attenuates left ventricular remodeling, decreases apoptosis, and improves calcium homeostasis in a rodent model of ischemic cardiomyopathy.

The benefit of the beta(2)-adrenergic agonist, clenbuterol, in left ventricular assist device patients with dilated cardiomyopathy has been reported, but its effect on ischemic heart failure (HF) is unknown. We investigated whether clenbuterol improves left ventricular remodeling, myocardial apoptosis and has synergy with a beta(1) antagonist, metoprolol, in a model of ischemic HF. Rats were randomized to: 1) HF only; 2) HF + clenbuterol; 3) HF + metoprolol; 4) HF + clenbuterol + metoprolol; and 5) rats with sham surgery. HF was induced by left anterior descending artery (LAD) artery ligation and confirmed by decreased left ventricular fractional shortening, decreased maximum left ventricular dP/dt (dP/dt(max)), and elevated left ventricular end-diastolic pressure (LVEDP) compared with sham rats (p < 0.01). After 9 weeks of oral therapy, echocardiographic, hemodynamic, and ex vivo end-diastolic pressure-volume relationship (EDPVR) measurements were obtained. Immunohistochemistry was performed for myocardial apoptosis and DNA damage markers. Levels of calcium-handling proteins were assessed by Western blot analysis. Clenbuterol-treated HF rats had increased weight gain and heart weights versus HF rats (p < 0.05). EDPVR curves revealed a leftward shift in clenbuterol rats versus metoprolol and HF rats (p < 0.05). The metoprolol-treated group had a lower LVEDP and higher dP/dt(max) versus the HF group (p < 0.05). Clenbuterol and metoprolol groups had decreased myocardial apoptosis and DNA damage markers and increased DNA repair markers versus HF rats (all p < 0.01). Protein levels of the ryanodine receptor and sarcoplasmic reticulum calcium-ATPase were improved in clenbuterol-, metoprolol-, and clenbuterol+metoprolol-treated groups versus HF rats. However, as a combination therapy, there were no synergistic effects of clenbuterol+metoprolol treatment. We conclude that clenbuterol ameliorates EDPVR, apoptosis, and calcium homeostasis but does not have synergy with metoprolol in our model of ischemic HF.

Cardiac myocyte apoptosis is associated with increased DNA damage and decreased survival in murine models of obesity.

Disruption of leptin signaling is associated with obesity, heart failure, and cardiac hypertrophy, but the role of leptin in cardiac myocyte apoptosis is unknown. We tested the hypothesis that apoptosis increases in leptin-deficient ob/ob and leptin-resistant db/db mice and is associated with aging and left ventricular hypertrophy, increased DNA damage, and decreased survival. We studied young (2- to 3-month-old) and old (12- to 14-month-old) ob/ob and db/db mice and wild-type (WT) controls (n=2 to 4 per group). As expected, ventricular wall thickness and heart weights were similar among young ob/ob, db/db, and WT mice, but higher in old ob/ob and db/db versus old WT. Young ob/ob and db/db showed markedly elevated apoptosis by TUNEL staining and caspase 3 levels compared with WT. Differences in apoptosis were further accentuated with age. Leptin treatment significantly reduced apoptosis in ob/ob mice both in intact hearts and isolated myocytes. Tissue triglycerides were increased in ob/ob hearts, returning to WT levels after leptin repletion. Furthermore, the DNA damage marker, 8oxoG (8-oxo-7,8-dihydroguanidine), was increased, whereas the DNA repair marker, MYH glycosylase, was decreased in old ob/ob and db/db compared with old WT mice. Both ob/ob and db/db mice had decreased survival compared with WT mice. We conclude that leptin-deficient and leptin-resistant mice demonstrate increased apoptosis, DNA damage, and mortality compared with WT mice, suggesting that normal leptin signaling is necessary to prevent excess age-associated DNA damage and premature mortality. These data offer novel insights into potential mechanisms of myocardial dysfunction and early mortality in obesity.

pKa of acetate in water: a computational study.

Several computational methods including the conductor-like polarizable continuum model, CPCM with both UAKS and UAHF cavities, Cramer and Truhlar's generalized Born solvation model, SM5.4(AM1), SM5.4(PM3), and SM5.43R(mPW1PW91/6-31+G(d)), and mixed QM/MM-Ewald simulations were used to calculate the pK(a) values of acetate and bicarbonate anions in aqueous solution. This work provided a critical and comprehensive assessment of the quality of these theoretical models in the calculation of aqueous solvation free energies for the singly charged acetate and bicarbonate ions, as well as the doubly charged acetate dianion and carbonate dianion. It was shown that QM/MM-Ewald simulations could give an accurate and consistent evaluation of the pK(a) values of acetate and bicarbonate based on both the relative and absolute pK(a) formulas, while other methods could yield satisfactory results only for certain calculations. However, this does not mean that the current QM/MM-Ewald protocol is superior to other methods. The useful information obtained in this investigation is that both the absolute and relative pK(a) formulas should better be tested in accurate calculations of pK(a) values based on any methods.

Oxidative DNA damage and DNA repair enzyme expression are inversely related in murine models of fatty liver disease.

Mitochondrial generation of reactive oxygen species (ROS) is increased in mice with fatty livers induced by genetic obesity, chronic consumption of ethanol, or methionine/choline-deficient diets. Both nuclear and mitochondrial (mt) DNA are targets for ROS-induced damage and accumulate hydroxylated bases, such as 8-hydroxy-2'-deoxyguanosine (8-oxoG) and base substitution of adenine with 8-oxoG (A*8-oxoG), that introduce mutations that promote cancer as well as cell death. The mammalian homolog of the bacterial DNA mismatch repair enzyme MutY (MYH) removes A*8-oxoG from nuclear and mtDNA, reduces 8-oxoG accumulation, and restores genomic stability after ROS exposure. Cumulative damage to mtDNA occurs as fatty liver disease progresses. Therefore, differences in hepatic MYH activity may influence the severity of fatty liver disease. To evaluate this hypothesis, we compared mtH2O2 production, MYH expression, oxidative DNA damage, and hepatocyte death in healthy mice and different mouse models of fatty liver disease. The results show that diverse causes of steatohepatitis increase mtROS production, limit repair of mtDNA, and oxidatively damage DNA. However, there are important differences in the DNA repair response to oxidant stress among mouse models of fatty liver disease. Independent of the degree of mtROS generation, models with the least MYH exhibit the greatest accumulation of 8-oxoG and the most hepatocyte death. These findings raise the intriguing possibility that inherited or acquired differences in DNA repair enzyme activity may underlie some of the interindividual differences in fatty liver disease outcomes.