Nonsteroidal anti-inflammatory drug (NSAID)-induced oxidative stress plays a critical role in gastric mucosal cell apoptosis and gastropathy. NSAIDs induce the generation of hydroxyl radical ((*)OH) through the release of free iron, which plays an important role in developing gastropathy. Thus, molecules having both iron-chelating and antiapoptotic properties will be beneficial in preventing NSAID-induced gastropathy. Gallic acid (GA), a polyphenolic natural product, has the capacity to chelate free iron. Here, we report that GA significantly prevents, as well as heals, NSAID-induced gastropathy. In vivo, GA blocks NSAID-mediated mitochondrial oxidative stress by preventing mitochondrial protein carbonyl formation, lipid peroxidation, and thiol depletion. In vitro, GA scavenges free radicals and blocks (*)OH-mediated oxidative damage. GA also attenuates gastric mucosal cell apoptosis in vivo as well as in vitro in cultured gastric mucosal cells as evident from the TUNEL assay. GA prevents NSAID-induced activation of caspase-9, a marker for the mitochondrial pathway of apoptosis, and restores NSAID-mediated collapse of the mitochondrial transmembrane potential and dehydrogenase activity. Thus, the inhibition of mitochondrial oxidative stress by GA is associated with the inhibition of NSAID-induced mitochondrial dysfunction and activation of apoptosis in gastric mucosal cells, which are responsible for gastric injury or gastropathy.

A peroxidase (30 kDa) has been purified from the human malaria parasite Plasmodium falciparum to its homogeneity. The protein is a dimer of 15 kDa subunit as evident from SDS-PAGE and MALDI-TOF mass analysis. The antibodies developed against the purified protein cross-react selectively with this protein present in parasite lysate. It is a heme containing peroxidase [R/Z value (A408/A278)=2.33] showing characteristic heme spectra with Soret peak at 408 nm and visible peaks at 536 and 572 nm. Analysis of Soret spectra in presence or absence of cyanide or azide reveals that iron of heme is in Fe-III state. Circular dichroism spectral analysis establishes that this protein contains mainly alpha-helix (60-70%). H2O2 interacts with the heme moiety of the enzyme as evidenced by optical difference spectroscopy and spectral studies indicate the formation of catalytically active peroxidase-H2O2 complex (Soret peak at 413 nm) to exhibit peroxidase activity. During the erythrocytic stages of its life cycle, the parasite is exposed to oxidative stress. As the parasite is susceptible to oxidative stress, this peroxidase may offer antioxidant role by scavenging endogenous H2O2.

Intracellular protein trafficking is crucial for survival of cell and proper functioning of the organelles; however, these pathways are not well studied in the malaria parasite. Its unique cellular architecture and organellar composition raise an interesting question to investigate.

The alarming rise of multidrug-resistant Gram-positive bacteria has precipitated a healthcare crisis, necessitating the development of new antimicrobial therapies. Here we describe a new class of antibiotics based on a ring-fused 2-pyridone backbone, which are active against vancomycin-resistant enterococci (VRE), a serious threat as classified by the Centers for Disease Control and Prevention, and other multidrug-resistant Gram-positive bacteria. Ring-fused 2-pyridone antibiotics have bacteriostatic activity against actively dividing exponential phase enterococcal cells and bactericidal activity against nondividing stationary phase enterococcal cells. The molecular mechanism of drug-induced killing of stationary phase cells mimics aspects of fratricide observed in enterococcal biofilms, where both are mediated by the Atn autolysin and the GelE protease. In addition, combinations of sublethal concentrations of ring-fused 2-pyridones and standard-of-care antibiotics, such as vancomycin, were found to synergize to kill clinical strains of VRE. Furthermore, a broad range of antibiotic resistant Gram-positive pathogens, including those responsible for the increasing incidence of antibiotic resistant healthcare-associated infections, are susceptible to this new class of 2-pyridone antibiotics. Given the broad antibacterial activities of ring-fused 2-pyridone compounds against Gram-positive (GmP) bacteria we term these compounds GmPcides, which hold promise in combating the rising tide of antibiotic resistant Gram-positive pathogens.

Psychological stress, depression and anxiety lead to multiple organ dysfunctions wherein stress-related mucosal disease (SRMD) is common to people experiencing stress and also occur as a side effect in patients admitted to intensive care units; however the underlying molecular aetiology is still obscure. We report that in rat-SRMD model, cold restraint-stress severely damaged gut mitochondrial functions to generate superoxide anion (O2•-), depleted ATP and shifted mitochondrial fission-fusion dynamics towards enhanced fission to induce mucosal injury. Activation of mitophagy to clear damaged and fragmented mitochondria was evident from mitochondrial translocation of Parkin and PINK1 along with enhanced mitochondrial proteome ubiquitination, depletion of mitochondrial DNA copy number and TOM 20. However, excess and sustained accumulation of O2•--generating defective mitochondria overpowered the mitophagic machinery, ultimately triggering Bax-dependent apoptosis and NF-κB-intervened pro-inflammatory mucosal injury. We further observed that stress-induced enhanced serum corticosterone stimulated mitochondrial recruitment of glucocorticoid receptor (GR), which contributed to gut mitochondrial dysfunctions as documented from reduced ETC complex 1 activity, mitochondrial O2•- accumulation, depolarization and hyper-fission. GR-antagonism by RU486 or specific scavenging of mitochondrial O2•- by a mitochondrially targeted antioxidant mitoTEMPO ameliorated stress-induced mucosal damage. Gut mitopathology and mucosal injury were also averted when the perception of mental stress was blocked by pre-treatment with a sedative or antipsychotic. Altogether, we suggest the role of mitochondrial GR-O2•--fission cohort in brain-mitochondria cross-talk during acute mental stress and advocate the utilization of this pathway as a potential target to prevent mitochondrial unrest and gastropathy bypassing central nervous system.

Macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine, is involved in the development of an array of inflammatory disorders including rheumatoid arthritis, inflammatory bowel disease, psoriasis, multiple sclerosis and sepsis. The synthesis of MIF-inhibitor is a rationale approach to develop novel anti-inflammatory agent to treat multitude of inflammatory diseases. In this work, we have synthesized and evaluated MIF-inhibitory activity of a series of small molecules containing isoxazoline skeleton. Mode of binding of this inhibitor to human MIF (huMIF) was determined by docking studies. The synthesized molecules inhibit tautomerase activity of huMIF. The anti-inflammatory activity of the most active inhibitor, 4-((3-(4-hydroxy-3-methoxyphenyl)-4, 5-dihydroisoxazol-5-yl) methoxy) benzaldehyde (4b) was evaluated against huMIF-induced inflammation in a cellular model (RAW 264.7 cell). Compound 4b significantly inhibits huMIF-mediated NF-κB translocation to the nucleus, up-regulation of inducible nitric oxide synthase and nitric oxide production in RAW 264.7 cell which are the markers for inflammation. The compound 4b is not cytotoxic as evident from cell viability assay. Hence, the compound 4b has potential to be a novel anti-inflammatory agent.

Listeria monocytogenes is a facultative Gram-positive bacterium that causes listeriosis, a severe foodborne disease. We previously discovered that ring-fused 2-pyridone compounds can decrease virulence factor expression in Listeria by binding and inactivating the PrfA virulence activator. In this study, we tested PS900, a highly substituted 2-pyridone that was recently discovered to be bactericidal to other Gram-positive pathogenic bacteria, such as Staphylococcus aureus and Enterococcus faecalis. We show that PS900 can interact with PrfA and reduce the expression of virulence factors. Unlike previous ring-fused 2-pyridones shown to inactivate PrfA, PS900 had an additional antibacterial activity and was found to potentiate sensitivity toward cholic acid. Two PS900-tolerant mutants able to grow in the presence of PS900 carried mutations in the brtA gene, encoding the BrtA repressor. In wild-type (WT) bacteria, cholic acid binds and inactivates BrtA, thereby alleviating the expression of the multidrug transporter MdrT. Interestingly, we found that PS900 also binds to BrtA and that this interaction causes BrtA to dissociate from its binding site in front of the mdrT gene. In addition, we observed that PS900 potentiated the effect of different osmolytes. We suggest that the increased potency of cholic acid and osmolytes to kill bacteria in the presence of PS900 is due to the ability of the latter to inhibit general efflux, through a yet-unknown mechanism. Our data indicate that thiazolino 2-pyridones constitute an attractive scaffold when designing new types of antibacterial agents. IMPORTANCE Bacteria resistant to one or several antibiotics are a very large problem, threatening not only treatment of infections but also surgery and cancer treatments. Thus, new types of antibacterial drugs are desperately needed. In this work, we show that a new generation of substituted ring-fused 2-pyridones not only inhibit Listeria monocytogenes virulence gene expression, presumably by inactivating the PrfA virulence regulator, but also potentiate the bactericidal effects of cholic acid and different osmolytes. We identified a multidrug repressor as a second target of 2-pyridones. The repressor-2-pyridone interaction displaces the repressor from DNA, thus increasing the expression of a multidrug transporter. In addition, our data suggest that the new class of ring-fused 2-pyridones are efficient efflux inhibitors, possibly explaining why the simultaneous addition of 2-pyridones together with cholic acid or osmolytes is detrimental for the bacterium. This work proves conclusively that 2-pyridones constitute a promising scaffold to build on for future antibacterial drug design.

A growing body of evidence has ascertained that apoptosis is not only restricted to metazoans but also exists in unicellular parasites. In Plasmodium falciparum, the presence of a putative gene having sequence homology with apoptosis related protein (PfARP) (Gene ID PFI0450c) has raised enormous interest to unravel the function of this unique protein in cell death of malaria parasite. To characterize this protein, the PfARP gene has been amplified from the P. falciparum transcriptome by RT-PCR and the amplified gene has been successfully cloned, over-expressed and purified to homogeneity. The purified PfARP exhibits minimum subunit MW of approximately 24kDa as evident from SDS-PAGE. CD analysis reveals that the alpha and beta content of the recombinant PfARP are 61% and 15%, respectively. Semiquantitative RT-PCR analysis indicates the expression of PfARP at both metabolically less active ring and highly active trophozoite stages of malaria parasite. Immunofluorescence microscopy further supports that PfARP expresses stage specifically with the highest expression at trophozite stage and very little in the schizont stage. PfARP is a cytosolic protein as evident from immunofluorescence microscopy. The role of this protein in P. falciparum cell death and stage progression is not yet known. The identification, purification and characterization would certainly be a step to initiate work on this protein to evaluate its role in P. falciparum growth, multiplication and stage progression.

Free heme is very toxic because it generates highly reactive hydroxyl radicals ((.)OH) to cause oxidative damage. Detoxification of free heme by the heme oxygenase (HO) system is a very common phenomenon by which free heme is catabolized to form bilirubin as an end product. Interestingly, the malaria parasite, Plasmodium falciparum, lacks an HO system, but it forms hemozoin, mainly to detoxify free heme. Here, we report that bilirubin significantly induces oxidative stress in the parasite as evident from the increased formation of lipid peroxide, decrease in glutathione content, and increased formation of H(2)O(2) and (.)OH. Bilirubin can effectively inhibit hemozoin formation also. Furthermore, results indicate that bilirubin inhibits parasite growth and induces caspase-like protease activity, up-regulates the expression of apoptosis-related protein (Gene ID PFI0450c), and reduces the mitochondrial membrane potential. (.)OH scavengers such as mannitol, as well as the spin trap alpha-phenyl-n-tert-butylnitrone, effectively protect the parasite from bilirubin-induced oxidative stress and growth inhibition. These findings suggest that bilirubin, through the development of oxidative stress, induces P. falciparum cell death and that the malaria parasite lacks an HO system probably to protect itself from bilirubin-induced cell death as a second line of defense.

Nonalcoholic fatty liver disease (NAFLD) is a progressive condition that includes steatosis (NAFL) and nonalcoholic steatohepatitis (NASH). In the U.S., Hispanics (HIS) are afflicted with NAFLD at a higher rate and severity compared to other ethnicities. To date, the mechanisms underlying this disparity have not been elucidated. In this pilot study, we compared untargeted plasma metabolomic profiles for primary metabolism, complex lipids, choline and related compounds between a group of HIS (n = 7) and White Caucasian (CAU, n = 8) subjects with obesity and biopsy-characterized NAFL to ethnicity-matched lean healthy controls (n = 14 HIS and 8 CAU). We also compared liver and plasma metabolomic profiles in a group of HIS and CAU subjects with obesity and NASH of comparable NAFLD Activity Scores, to BMI-matched NASH-free subjects in both ethnicities. Results highlight signs of metabolic dysregulation observed in HIS, independent of obesity, including higher plasma triglycerides, acylcarnitines, and free fatty acids. With NASH progression, there were ethnicity-related differences in the hepatic profile, including higher free fatty acids and lysophospholipids seen in HIS, suggesting lipotoxicity is involved in the progression of NASH. We also observed greater hepatic triglyceride content, higher plasma triglyceride concentrations and lower hepatic phospholipids with signs of impaired hepatic mitochondrial β-oxidation. These findings provide preliminary evidence indicating ethnicity-related variations that could potentially modulate the risk for progression of NALD to NASH.

Wilson disease (WD) is an autosomal recessive disease caused by mutations in ATP7B encoding a copper transporter. Consequent copper accumulation results in a variable WD clinical phenotype involving hepatic, neurologic, and psychiatric symptoms, without clear genotype-phenotype correlations. The goal of this study was to analyze alterations in DNA methylation at the whole-genome level in liver and blood from patients with WD to investigate epigenomic alterations associated with WD diagnosis and phenotype. We used whole-genome bisulfite sequencing (WGBS) to examine distinct cohorts of WD subjects to determine whether DNA methylation could differentiate patients from healthy subjects and subjects with other liver diseases and distinguish between different WD phenotypes.

Complex ganglioside expression is highly deregulated in several tumors which is further dependent on specific ganglioside synthase genes. Here, we designed and constructed a pair of highly specific transcription-activator like effector endonuclease (TALENs) to disrupt a particular genomic locus of mouse GM2-synthase, a region conserved in coding sequence of all four transcript variants of mouse GM2-synthase. Our designed TALENs effectively work in different mouse cell lines and TALEN induced mutation rate is over 45%. Clonal selection strategy is undertaken to generate stable GM2-synthase knockout cell line. We have also demonstrated non-homologous end joining (NHEJ) mediated integration of neomycin cassette into the TALEN targeted GM2-synthase locus. Functionally, clonally selected GM2-synthase knockout clones show reduced anchorage-independent growth (AIG), reduction in tumor growth and higher cellular adhesion as compared to wild type Renca-v cells. Insight into the mechanism shows that, reduced AIG is due to loss in anoikis resistance, as both knockout clones show increased sensitivity to detachment induced apoptosis. Therefore, TALEN mediated precise genome editing at GM2-synthase locus not only helps us in understanding the function of GM2-synthase gene and complex gangliosides in tumorigenicity but also holds tremendous potential to use TALENs in translational cancer research and therapeutics.

We have investigated the DNA-binding nature as well as the function of a putative Alba (Acetylation lowers binding affinity) family protein (PfAlba3) from Plasmodium falciparum. PfAlba3 possesses DNA-binding property like Alba family proteins. PfAlba3 binds to DNA sequence non-specifically at the minor groove and acetylation lowers its DNA-binding affinity. The protein is ubiquitously expressed in all the erythrocytic stages of P. falciparum and it exists predominantly in the acetylated form. PfAlba3 inhibits transcription in vitro by binding to DNA. Plasmodium falciparum Sir2 (PfSir2A), a nuclear localized deacetylase interacts with PfAlba3 and deacetylates the lysine residue of N-terminal peptide of PfAlba3 specific for DNA binding. PfAlba3 is localized with PfSir2A in the periphery of the nucleus. Fluorescence in situ hybridization studies revealed the presence of PfAlba3 in the telomeric and subtelomeric regions. ChIP and ChIP ReChIP analyses further confirmed that PfAlba3 binds to the telomeric and subtelomeric regions as well as to var gene promoter.

Translocation of various proteins to the subcellular organelles is an essential mechanism to regulate the metabolic pathways and often vacuolar protein sorting (VPS) proteins are involved in this transportation. Plasmodium falciparum VPS29 (PfVPS29) is predicted to be a functional component in the assembly of the retromer complex; however, so far detailed characterization of PfVPS29 in its native form is not yet done. We report the successful expression and purification of tag-free recombinant PfVPS29 with a yield of 5.6 mg from 1 L of Escherichia coli culture. PfVPS29 was purified by combined anion-exchange and size exclusion chromatography. The protein showed a single band in SDS-PAGE and it exhibited molecular mass of 21.7 kDa as measured by MALDI-TOF mass spectrometry. Secondary structure was elucidated by circular dichroism spectroscopy. It was found to be a monomeric protein in solution as evident from dynamic light scattering studies, chemical cross-linking experiments and size exclusion chromatography. Subsequently, polyclonal anti-PfVPS29 antibody was generated and used for evaluating protein expression by western blot and following subcellular localization in P. falciparum by confocal immunofluoroscence microscopy. PfVPS29 was found to be located in cytoplasm and expressed from early trophozoite to schizont stages with maximum expression in trophozoite stage. This study provides purification, biophysical characterization and subcellular localization of PfVPS29 in different asexual stages of P. falciparum.

Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are national and global epidemics. The disease is characterized by a spectrum of liver steatosis (fat deposition), inflammation (in NASH) and fibrosis. NAFLD and specifically NASH can lead to cirrhosis, which carry risks of progression to portal hypertension and hepatocellular carcinoma (HCC). NASH is also associated with higher mortality from cardiovascular causes. Most of the data for NAFLD has been obtained from the perspective of developed nations, although the disease is increasing and threatening to reach epidemic proportions across the world. Emerging data is notable for high prevalence of NAFLD in South Asian populations, presumably resulting from a combination of underlying genetic polymorphisms and changes in socio-economic status. It is also notable that an 'Asian Paradox' has been defined for NAFLD based upon the observation of lower than pre-defined body mass index (BMI), otherwise termed as "lean NAFLD", among this population. Yet, data remains limited in regards to the characteristics of NAFLD/NASH in this population. In this article, we present a review of the literature and discuss the prevalence, associated risk factors and burden of HCC in South Asians with NAFLD.

Plasmodium falciparum macrophage migration inhibitory factor (PfMIF) exhibits thioredoxin (Trx)-like oxidoreductase activity but the active site for this activity and its function have not been evaluated. A bioinformatics search revealed that the conserved CXXC motif, which is responsible for Trx-like oxidoreductase activity, is absent from PfMIF. In contrast, the adjacent N-terminal Cys-3 and Cys-4 are conserved in MIF across species of malarial parasites. Mutation of either vicinal Cys-3 or Cys-4 of PfMIF abolished the Trx-like activity, whereas the mutation of the remaining Cys-59 or Cys-103 did not affect it. PfMIF has an antioxidant function. It prevents reactive oxygen species-mediated lipid peroxidation and oxidative damage of DNA as evident from DNA nicking assay. Interestingly, chemical modification of the vicinal cysteines by phenylarsine oxide (PAO), a specific vicinal thiol modifier, significantly prevented this antioxidant activity. Modification of Cys-3 and Cys-4 was confirmed by MALDI-TOF mass spectroscopy of peptide fragments obtained after cyanogen bromide digestion of PAO-modified PfMIF. Furthermore, mutation of either Cys-3 or Cys-4 of PfMIF resulted in the loss of both Trx-like oxidoreductase and antioxidant activities of PfMIF. Altogether, our results suggest that the vicinal Cys-3 and Cys-4 play a critical role in the Trx-like oxidoreductase activity and antioxidant property of PfMIF.

Symptom burden, medical comorbidities, and functional well-being of patients with chronic hepatitis C virus (HCV) initiating direct acting antiviral (DAA) therapy in real-world clinical settings are not known. We characterized these patient-reported outcomes (PROs) among HCV-infected patients and explored associations with sociodemographic, liver disease, and psychiatric/substance abuse variables.

Mycobacterium tuberculosis (Mtb) drug resistance poses an alarming threat to global tuberculosis control. We previously reported that C10, a ring-fused thiazolo-2-pyridone, inhibits Mtb respiration, blocks biofilm formation, and restores the activity of the antibiotic isoniazid (INH) in INH-resistant Mtb isolates. This discovery revealed a new strategy to address INH resistance. Expanding upon this strategy, we identified C10 analogues with improved potency and drug-like properties. By exploring three heterocycle spacers (oxadiazole, 1,2,3-triazole, and isoxazole) on the ring-fused thiazolo-2-pyridone scaffold, we identified two novel isoxazoles, 17h and 17j. 17h and 17j inhibited Mtb respiration and biofilm formation more potently with a broader therapeutic window, were better potentiators of INH-mediated inhibition of an INH-resistant Mtb mutant, and more effectively inhibited intracellular Mtb replication than C10. The (-)17j enantiomer showed further enhanced activity compared to its enantiomer and the 17j racemic mixture. Our potent second-generation C10 analogues offer promise for therapeutic development against drug-resistant Mtb.

Generation of phosphocholine by choline kinase is important for phosphatidylcholine biosynthesis via Kennedy pathway and phosphatidylcholine biosynthesis is essential for intraerythrocytic growth of malaria parasite. A putative gene (Gene ID PF14_0020) in chromosome 14, having highest sequence homology with choline kinase, has been identified by BLAST searches from P. falciparum genome sequence database. This gene has been PCR amplified, cloned, over-expressed and characterized. Choline kinase activity of the recombinant protein (PfCK) was validated as it catalyzed the formation of phosphocholine from choline in presence of ATP. The K(m) values for choline and ATP are found to be 145+/-20 microM and 2.5+/-0.3 mM, respectively. PfCK can phosphorylate choline efficiently but not ethanolamine. Southern blotting indicates that PfCK is a single copy gene and it is a cytosolic protein as evidenced by Western immunoblotting and confocal microscopy. A model structure of PfCK was constructed based on the crystal structure of choline kinase of C. elegans to search the structural homology. Consistent with the homology modeling predictions, CD analysis indicates that the alpha and beta content of PfCK are 33% and 14%, respectively. Since choline kinase plays a vital role for growth and multiplication of P. falciparum during intraerythrocytic stages, we can suggest that this well characterized PfCK may be exploited in the screening of new choline kinase inhibitors to evaluate their antimalarial activity.

Following endocytosis, internalized molecules are found within intracellular vesicles and tubules that move along the cytoskeleton and undergo fission, as demonstrated here using primary cultured rat hepatocytes. Although the use of depolymerizing drugs has shown that the cytoskeleton is not required to segregate endocytic protein, many studies suggest that the cytoskeleton is involved in the segregation of protein in normal cells. To investigate whether cytoskeletal-based movement results in the segregation of protein, we tracked the contents of vesicles during in vitro microscopy assays. These studies showed that the addition of ATP causes fission of endocytic contents along microtubules, resulting in the segregation of proteins that are targeted for different cellular compartments. The plasma membrane proteins, sodium (Na+) taurocholate cotransporting polypeptide (ntcp) and transferrin receptor, segregated from asialoorosomucoid (ASOR), an endocytic ligand that is targeted for degradation. Epidermal growth factor receptor, which is degraded, and the asialoglycoprotein receptor, which remains partially bound to ASOR, segregated less efficiently from ASOR. Vesicles containing ntcp and transferrin receptor had reduced fission in the absence of ASOR, suggesting that fission is regulated to allow proteins to segregate. A single round of fission resulted in 6.5-fold purification of ntcp from ASOR, and 25% of the resulting vesicles were completely depleted of the endocytic ligand.