Antimicrobial screening involving Helicobacter pylori of nano-therapeutic compounds based on the amoxicillin antibiotic drug.

Nano-structure Cu(II) complex [Cu(AMAB)2 ]Cl2 with Schiff base (AMAB) derived from the condensation between 4-(dimethylamino)benzaldehyde and amoxicillin trihydrate was prepared. (AMAB) Schiff base and its Cu(II) complex were identified and confirmed by different physicochemical techniques. The Schiff base (AMAB) was coordinated to copper ion through carbonyl oxygen and imine nitrogen donor sites. X-ray powder diffraction shows a cubic crystal system of the Cu(II) complex. The density functional theory was used to optimize the structure geometries of the investigated compounds. The molecular docking of the active amino acids of the investigated proteins' interactions with the tested compounds was evaluated. The bactericidal or bacteriostatic effect of the compounds was screened against some bacterial strains. The activity of Cu-chelate against Gram-negative bacteria was mainly more effective than its (AMAB) ligand and vice versa in the case of Gram-positive bacteria. The biological activity of the prepared compounds with biomolecules calf thymus DNA (CT-DNA) was determined by electronic absorption spectra and DNA gel electrophoresis technique. All studies revealed that the Cu-chelate derivative exhibited better binding affinity to both CT-DNA than the AMAB and amoxicillin itself. The anti-inflammatory effect of the designed compounds was determined by testing their protein denaturation inhibitory activity spectrophotometrically. All obtained data supported that the designed nano-Cu(II) complex with Schiff base (AMAB) is a potent bactericide against H. pylori, and exhibits anti-inflammatory activity. The dual inhibition effects of the designed compound represent a modern therapeutic approach with extended spectrum of action. Therefore, it can act as good drug target in antimicrobial and anti-inflammtory therapies. Finally, H. pylori resistance to amoxicillin is absent or rare in many countries, thus amoxicillin nanoparticles may be beneficial for countries where amoxicillin resistance is reported.

Aminofutalosine Deaminase in the Menaquinone Pathway of Helicobacter pylori.

Helicobacter pylori is a Gram-negative bacterium that is responsible for gastric and duodenal ulcers. H. pylori uses the unusual mqn pathway with aminofutalosine (AFL) as an intermediate for menaquinone biosynthesis. Previous reports indicate that hydrolysis of AFL by 5'-methylthioadenosine nucleosidase (HpMTAN) is the direct path for producing downstream metabolites in the mqn pathway. However, genomic analysis indicates jhp0252 is a candidate for encoding AFL deaminase (AFLDA), an activity for deaminating aminofutolasine. The product, futalosine, is not a known substrate for bacterial MTANs. Recombinant jhp0252 was expressed and characterized as an AFL deaminase (HpAFLDA). Its catalytic specificity includes AFL, 5'-methylthioadenosine, 5'-deoxyadenosine, adenosine, and S-adenosylhomocysteine. The kcat/Km value for AFL is 6.8 × 104 M-1 s-1, 26-fold greater than that for adenosine. 5'-Methylthiocoformycin (MTCF) is a slow-onset inhibitor for HpAFLDA and demonstrated inhibitory effects on H. pylori growth. Supplementation with futalosine partially restored H. pylori growth under MTCF treatment, suggesting AFL deamination is significant for cell growth. The crystal structures of apo-HpAFLDA and with MTCF at the catalytic sites show a catalytic site Zn2+ or Fe2+ as the water-activating group. With bound MTCF, the metal ion is 2.0 Å from the sp3 hydroxyl group of the transition state analogue. Metabolomics analysis revealed that HpAFLDA has intracellular activity and is inhibited by MTCF. The mqn pathway in H. pylori bifurcates at aminofutalosine with HpMTAN producing adenine and depurinated futalosine and HpAFLDA producing futalosine. Inhibition of cellular HpMTAN or HpAFLDA decreased the cellular content of menaquinone-6, supporting roles for both enzymes in the pathway.

Targeting the Protein Tunnels of the Urease Accessory Complex: A Theoretical Investigation.

Urease is a nickel-containing enzyme that is essential for the survival of several and often deadly pathogenic bacterial strains, including Helicobacter pylori. Notwithstanding several attempts, the development of direct urease inhibitors without side effects for the human host remains, to date, elusive. The recently solved X-ray structure of the HpUreDFG accessory complex involved in the activation of urease opens new perspectives for structure-based drug discovery. In particular, the quaternary assembly and the presence of internal tunnels for nickel translocation offer an intriguing possibility to target the HpUreDFG complex in the search of indirect urease inhibitors. In this work, we adopted a theoretical framework to investigate such a hypothesis. Specifically, we searched for putative binding sites located at the protein-protein interfaces on the HpUreDFG complex, and we challenged their druggability through structure-based virtual screening. We show that, by virtue of the presence of tunnels, some protein-protein interfaces on the HpUreDFG complex are intrinsically well suited for hosting small molecules, and, as such, they possess good potential for future drug design endeavors.

A novel FRET peptide assay reveals efficient Helicobacter pylori HtrA inhibition through zinc and copper binding.

Helicobacter pylori (H. pylori) secretes the chaperone and serine protease high temperature requirement A (HtrA) that cleaves gastric epithelial cell surface proteins to disrupt the epithelial integrity and barrier function. First inhibitory lead structures have demonstrated the essential role of HtrA in H. pylori physiology and pathogenesis. Comprehensive drug discovery techniques allowing high-throughput screening are now required to develop effective compounds. Here, we designed a novel fluorescence resonance energy transfer (FRET) peptide derived from a gel-based label-free proteomic approach (direct in-gel profiling of protease specificity) as a valuable substrate for H. pylori HtrA. Since serine proteases are often sensitive to metal ions, we investigated the influence of different divalent ions on the activity of HtrA. We identified Zn++ and Cu++ ions as inhibitors of H. pylori HtrA activity, as monitored by in vitro cleavage experiments using casein or E-cadherin as substrates and in the FRET peptide assay. Putative binding sites for Zn++ and Cu++ were then analyzed in thermal shift and microscale thermophoresis assays. The findings of this study will contribute to the development of novel metal ion-dependent protease inhibitors, which might help to fight bacterial infections.

Smart drug delivery against Helicobacter pylori: pectin-coated, mucoadhesive liposomes with antiadhesive activity and antibiotic cargo.

The first step in the development of Helicobacter pylori pathogenicity is the receptor-mediated adhesion to the gastric epithelium. Inhibition of outer membrane proteins of H. pylori (e.g. BabA) by antiadhesive drugs will contribute to reduced recolonization and infection. Pectin from apple inhibits the BabA and LPS-mediated adhesion of H. pylori to human stomach cells. Pectin-coated liposomes with encapsulated amoxicillin were characterized for polydispersity, zeta potential, encapsulation efficiency, stability, and amoxicillin release. Coated liposomes did not influence the viability of AGS and HT29-MTX cells up to 100 µg/mL but exert cytotoxicity against H. pylori at 10 µg/mL. Pectin-coating of liposomes provoked direct interaction and subsequent binding of the particles to surface structures of H. pylori, and interaction with mucus from porcine stomach and mucus secreted by HT29-MTX cells. Laser scanning microscopy of H. pylori and AGS cells together with liposomes indicated co-aggregation. The mucoadhesive effect seems interesting as stomach cells are covered by a mucus layer. H. pylori is able to penetrate and cross the mucin rapidly to reach pH-neutral epithelium to escape the acidic environment, followed by interaction with epithelial cells. In summary, all experimental evidence is consistent with a specific interaction of pectin-coated liposomes with mucins and surface structures of H. pylori. As the coated liposomes show mucoadhesion to the negatively charged mucins, docking to stomach mucin, mucus penetration, and recognition of and adhesion to H. pylori, they can be considered a novel type of multifunctional drug carriers for local antibiotic therapy against H. pylori. KEY POINTS: • Smart, multifunctional mucoadhesive liposomes • Specific targeting against BabA/LPS of Helicobacter pylori • Inhibition of bacterial adhesion of H. pylori to human host cells • Release of antibiotic cargo.

Curcuma wenyujin Y. H. Chen et C. Ling n-Butyl Alcohol Extract Inhibits AGS Cell Helicobacter pyloriCagA+VacA+ Promoted Invasiveness by Down-Regulating Caudal Type Homeobox Transcription Factor and Claudin-2 Expression.

OBJECTIVE: To investigate the effects and possible mechanisms of action of Curcuma wenyujin Y. H. Chen et C. Ling n-Butyl alcohol extract (CWNAE) on repression of human gastric cancer (GC) AGS cell invasion induced by co-culturing with Helicobacter pylori (HP). METHODS: AGS cells were cultured with HP of positive or negative cytotoxin-associated gene A (CagA) and vacuolating cytotoxin gene A (VacA) expression (CagA+/- or VacA+/-) and divided into 5 group. Group A was cultured without HP as a control, Group B with HPCagA+VacA+, Group C with HPCagA-VacA-, Group D with HPCagA+VacA+ and CWNAE, and Group E with HPCagA-VacA- and CWNAE. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) and tumor invasion assays, examinations of morphology and ultramicroscopic structures, quantitative real-time polymerase chain reaction and Western blots were performed to measure the effects and uncover the mechanisms behind these effects of HPCagA+VacA+ and CWNAE on the epithelial-mesenchymal transition (EMT) of AGS cells. RESULTS: The 10% inhibitory concentration of CWNAE against AGS cells after a 48 h incubation was 19.73±1.30 µg/mL. More AGS cells were elongated after co-culturing with HPCagA+VacA+ than after culturing with HPCagA-VacA-. In tumor invasion assays, HPCagA+VacA+ significantly enhanced the invasiveness of AGS cells compared to the other experimental groups (all P value <0.05), and this effect was inhibited by CWNAE. Treatment with CWNAE normalized tight junctions and reduced the number of pseudopodia of AGS cells co-cultured with HPCagA+VacA+. HPCagA+VacA+ up-regulated zincfinger ebox binding homeobox 1 (ZEB1) in AGS cells after co-culturing for 24 h. Expression of caudal type homeobox transcription factor (CDX-2) and claudin-2 was significantly increased by HPCagA+VacA+ (P<0.05), but not by HPCagA-VacA-. CONCLUSION: HPCagA+VacA+ promoted the invasiveness of AGS cells through up-regulation of ZEB1 transcription and claudin-2 and CDX-2 expression. CWNAE inhibited these effects of HPCagA+VacA+ on AGS cells by down-regulating ZEB1 transcription, and CDX-2 and claudin-2 expression.

The influence of the Chuyou Yuyang granule on the Toll-like receptor/nuclear factor-κB signal pathway in Helicobacter pylori-positive peptic ulcer patients.

BACKGROUND: The cure rate of Helicobacter pylori (HP)-positive peptic ulcer has appeared to downward trend, and the resistance of the ulcer relapse has become a hot issue. METHODS: Hematoxylin and eosin staining was used to detect the repair of the damaged tissues in patients after treatment with the Chuyou Yuyang granule (CYYY). Elisa was used to analyze the expression of cytokine interleukin 18 (IL-18) and tumor necrosis factor α (TNF-α) in the patients' serum. Western blot analysis was used to explore the mechanism of the CYYY. Reverse-transcription polymerase chain reaction (RT-PCR) was used to detect the expression of microRNA-155a (miR-155a) and miR-146a in the blood of the patients and to confirm whether CYYY could cure peptic ulcer through regulation of miR-155a and miR-146a. RESULTS: The damaged gastric mucosal tissues of ulcer patients were significantly repaired by treating with CYYY. The pro-inflammatory cytokine IL18 and TNF-α were notably repressed after treating with CYYY. In addition, CYYY played a key role in regulation of the Toll-like receptor (TLR4)/nuclear factor-κB (NF-κB) signal pathway and the expression of miR-155a and miR-146a. CONCLUSION: CYYY was a highly effective therapeutic method for peptic ulcer patients by inhibiting the activation of the TLR4/NF-κB signal pathway and suppressing the expression of miR-155a and miR-146a.

Baicalin, Baicalein, and Lactobacillus Rhamnosus JB3 Alleviated Helicobacter pylori Infections in Vitro and in Vivo.

Helicobacter pylori infection is associated with chronic gastritis, peptic ulcers, and gastric cancer. The flavonoid compounds baicalin and baicalein found in many medicinal plants exhibit an anti-inflammatory effect. The administration of Lactobacillus strains reducing the risk of H. pylori infection is well accepted. In this study, the therapeutic effects against H. pylori infection of baicalin, baicalein, and L. rhamnosus JB3 (LR-JB3), isolated from a dairy product, were investigated. Compared to baicalin, baicalein exhibited stronger anti-H. pylori activity and cytotoxicity on human gastric cancer epithelial AGS cells. Baicalin and baicalein both suppressed the vacA gene expression of H. pylori and interfered with the adhesion and invasion ability of H. pylori to AGS cells, as well as decreased H. pylori-induced interleukin (IL)-8 expression. In the mice infection model, high dosages of baicalin and baicalein inhibited H. pylori growth in the mice stomachs. Serum IL-1ß levels and H. pylori-specific serum IgM and IgA levels in mice treated with baicalin and baicalein were decreased. Moreover, a synergistic therapeutic effect of baicalein and LR-JB3 on eradicating H. pylori infections was observed. Thus, administrating baicalin, baicalein, or LR-JB3 for an H. pylori infection could offer similar therapeutic effects to administering antibiotics while not disturbing the balance of gut microbiota. This study revealed the effects of baicalin, baicalein, and LR-JB3 on attenuating the virulence of H. pylori. The synergistic effect with baicalein and LR-JB3 provides the experimental rationale for testing the reliability, safety, and efficacy of this approach in higher animals and perhaps ultimately in humans to eradicate H. pylori infections. PRACTICAL APPLICATION: Baicalin and baicalein exert health promotion and avoidance of H. pylori infections by interfering with H. pylori growth and virulence. Lactobacillus rhamnosus JB3 was used to reduce the gastric inflammation caused by H. pylori infection.

[The Efficacy of Bismuth-containing Quadruple Therapy after Moxifloxacin-based Sequential Therapy Failure in Helicobacter pylori Eradication].

Background/Aims: Moxifloxacin-based sequential therapy showed an excellent eradication rate as the first line treatment of Helicobacter pylori (H. pylori) infection. However, to the best of our knowledge, there were only a few studies on the treatment of those with failed moxifloxacin-based sequential therapy. Hence, this study was to investigate the efficacy of bismuth-containing quadruple therapy in those with failed moxifloxacin-based sequential or reverse sequential therapy for H. pylori eradication. Methods: Between January 2013 and March 2016, we retrospectively analyzed patients who failed to eradicate H. pylori using moxifloxacin-based sequential (rabeprazole 20 mg bid and amoxicillin 1 g bid for 5-7 days, followed by rabeprazole 20 mg bid, metronidazole 500 mg bid, and moxifloxacin 400 mg qd for 5-7 days) and 10 days moxifloxacin-based reverse sequential therapy as the first line treatment. Then we investigated the eradication rates of bismuth-containing quadruple therapy as the second line treatment. All subjects had no history of H. pylori eradication before. Eradication rates were described as intention-to-treat (ITT) and per-protocol (PP) analyses. H. pylori status was evaluated by ¹³C-urea breath test 6 weeks after the end of the treatment. Moreover, we examined any side effects that caused discontinuation of therapy. Results: Twenty-three patients received bismuth-containing quadruple therapy as the second line treatment. The overall eradication rates by ITT and PP analyses were 60.87% (n=14/23) and 73.68% (n=14/19). All the patients showed good compliance, and there were no serious adverse events. Conclusions: Bismuth-containing quadruple therapy is insufficient as the second line eradication treatment after a failed attempt of moxifloxacin-based sequential or reverse sequential therapy. Large-scale clinical trials should be performed to establish better clinical evidence.

Gastric Mucosal Oxidative Stress Markers in Children with Helicobacter Pylori Infection.

BACKGROUND: 'Helicobacter pylori' "H. pylori" is one of the most common infections that colonizes human gastric mucosa and generates reactive oxygen and nitrogen species. The aim of this study was to evaluate the oxidative stress markers in the gastric mucosa of "H. pylori"- infected children. PATIENTS AND METHODS: The present study was carried out on 60 children infected with "H. pylori" including 28 males, 32 females with their age ranging from 7-10 years and mean age value of 8.5 ± 1.65 years (Group I). This study included also 60 healthy children as a control group including 26 males and 34 females with their age ranging from 7-11 years and mean age value of 8.99 ± 1.63 years (Group II). All children were subjected to full history taking, thorough clinical examination, diagnosis of "H. pylori" infection through "H. pylori" stool antigen testing using enzyme immunoassay kit (Group I and II) and gastric antrum mucosal biopsies which were tested for urease activity using Campylobacter like organism test (CLO test) (Group I only) and measurement of gastric mucosal oxidative stress markers including Malondialdehyde (MDA), Superoxide dismutase (SOD), reduced glutathione (GSH), Catalase and nitric oxide (NO) [The sum of Nitrite (NO2 -) and Nitrate (NO3 -)]. RESULTS: The main clinical presentations in studied patients and controls were recurrent abdominal pain, recurrent vomiting, dyspepsia and hematemesis with no significant difference between patients and controls as regard abdominal pain, vomiting or dyspepsia but hematemesis was found only in patients. There were significant differences between patients and controls as regard site and duration of abdominal pain with epigastrium being the most common site of pain in patients versus diffuse abdominal pain in control group with significantly longer duration of abdominal pain in patients compared with controls. "H. pylori" infected children has significantly lower gastric mucosal nitric oxide and reduced glutathione and significantly higher gastric mucosal MDA, catalase and SOD compared to controls (nitric oxide was 85.68 ± 23.16 nmol/gm in patients versus 106.423±2.111 nmol/gm in controls, reduced glutathione in patients was 1.83 ± 0.16 nmol/gm versus 2.44 ± 0.07 nmol/gm in controls, MDA in patients was 189.15 ± 6.14 nmol/gm versus 166.21 ± 3.13 nmol/gm in controls, catalase was 57.38 ± 19.85 unit/gm in patients versus 36.51 ± 2.34 unit/gm in controls and SOD in patients was 375.52 ± 26.51 unit/gm versus 318.51 ± 32.06 unit/gm in controls. CONCLUSION: "H. pylori" infection is associated with gastric mucosal oxidative stress with significantly lower gastric mucosal nitric oxide and reduced glutathione and significantly higher gastric mucosal MDA, Catalase and SOD in patients compared to controls. RECOMMENDATIONS: Antioxidants may be an important adjuvant therapy for "H. pylori" infection as this infection is associated with gastric mucosal oxidative stress.

[The Efficacy of Moxifloxacin-containing Triple Therapy after Hybrid Therapy Failure in Helicobacter pylori Eradication].

BACKGROUND/AIMS: Hybrid therapy was successful in eradicating Helicobacter pylori (H. pylori) according to previous reports. However, to the best of our knowledge, there have only been a few studies evaluating the optimal choice after hybrid failure. Hence, we aimed to evaluate the efficacy of moxifloxacin-containing triple therapy after hybrid therapy failure in H. pylori eradication. METHODS: Between January 2013 and March 2016, we retrospectively reviewed patients who underwent failed hybrid therapy, as first line treatment, in eradicating H. pylori (rabeprazole and amoxicillin b.i.d for 14 days, in addition to clarithromycin and metronidazole b.i.d for final 7 days). Then, we investigated the eradication rates of moxifloxacin-containing triple therapy (rabeprazole, amoxicillin b.i.d and moxifloxacin qd) as the second line of treatment. Intention-to-treat (ITT) and per-protocol (PP) analyses were used to determine the eradication rate. We evaluated the status of H. pylori by using 13C-urea breath test 6 weeks after the final treatment. Moreover, compliance and adverse effects of each patient were analyzed. RESULTS: Among those who failed the initial hybrid therapy, 11 patients received moxifloxacin-containing triple therapy. The overall eradication rates, as determined by ITT and PP, were 72.7% (n=8/11) and 80% (n=8/10), respectively. The compliance rate was 100%, and there were no serious adverse effects. CONCLUSIONS: Moxifloxacin-containing triple therapy can be used as a second line therapy in case of hybrid therapy failure. A large scale study is necessary to confirm the findings of this study and establish clinical evidence.

Narrow-spectrum inhibitors targeting an alternative menaquinone biosynthetic pathway of Helicobacter pylori.

We aimed to identify narrow-spectrum natural compounds that specifically inhibit an alternative menaquinone (MK; vitamin K2) biosynthetic pathway (the futalosine pathway) of Helicobacter pylori. Culture broth samples of 6183 microbes were examined using the paper disc method with different combinations of 2 of the following 3 indicator microorganisms: Bacillus halodurans C-125 and Kitasatospora setae KM-6054(T), which have only the futalosine pathway of MK biosynthesis, and Bacillus subtilis H17, which has only the canonical MK biosynthetic pathway. Most of the active compounds isolated from culture broth samples were from the families of polyunsaturated fatty acids (PUFAs). Only one compound isolated from the culture broth of Streptomyces sp. K12-1112, siamycin I (a 21-residue lasso peptide antibiotic), targeted the futalosine pathway. The inhibitory activities of representative PUFAs and siamycin I against the growth of B. halodurans or K. setae were abrogated by supplementation with MK. Thereafter, the growth of H. pylori strains SS1 and TN2GF4 in broth cultures was dose-dependently suppressed by eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), or siamycin I, and these inhibitory effects were reduced by supplementation with MK. Daily administration of EPA (100 µM), DHA (100 µM), or siamycin I (2.5 µM) in drinking water reduced the H. pylori SS1 colonization in the gastric mucosa of C57BL/6 mice by 96%, 78%, and 68%, respectively. These data suggest that EPA, DHA, and siamycin I prevented H. pylori infection by inhibiting the futalosine pathway of MK biosynthesis.

Carbon Fixation Driven by Molecular Hydrogen Results in Chemolithoautotrophically Enhanced Growth of Helicobacter pylori.

UNLABELLED: A molecular hydrogen (H2)-stimulated, chemolithoautotrophic growth mode for the gastric pathogen Helicobacter pylori is reported. In a culture medium containing peptides and amino acids, H2-supplied cells consistently achieved 40 to 60% greater growth yield in 16 h and accumulated 3-fold more carbon from [(14)C]bicarbonate (on a per cell basis) in a 10-h period than cells without H2 Global proteomic comparisons of cells supplied with different atmospheric conditions revealed that addition of H2 led to increased amounts of hydrogenase and the biotin carboxylase subunit of acetyl coenzyme A (acetyl-CoA) carboxylase (ACC), as well as other proteins involved in various cellular functions, including amino acid metabolism, heme synthesis, or protein degradation. In agreement with this result, H2-supplied cells contained 3-fold more ACC activity than cells without H2 Other possible carbon dioxide (CO2) fixation enzymes were not up-expressed under the H2-containing atmosphere. As the gastric mucus is limited in carbon and energy sources and the bacterium lacks mucinase, this new growth mode may contribute to the persistence of the pathogen in vivo This is the first time that chemolithoautotrophic growth is described for a pathogen. IMPORTANCE: Many pathogens must survive within host areas that are poorly supplied with carbon and energy sources, and the gastric pathogen Helicobacter pylori resides almost exclusively in the nutritionally stringent mucus barrier of its host. Although this bacterium is already known to be highly adaptable to gastric niches, a new aspect of its metabolic flexibility, whereby molecular hydrogen use (energy) is coupled to carbon dioxide fixation (carbon acquisition) via a described carbon fixation enzyme, is shown here. This growth mode, which supplements heterotrophy, is termed chemolithoautotrophy and has not been previously reported for a pathogen.

Aegle marmelos fruit extract attenuates Helicobacter pylori Lipopolysaccharide induced oxidative stress in Sprague Dawley rats.

BACKGROUND: Bael (Aegle marmelos (L.) Corr.) has been widely used in indigenous systems of Indian medicine to exploit its medicinal properties including astringent, antidiarrheal, antidysenteric, demulcent, antipyretic, antiulcer, anti-inflammatory and anti cancer activities. The present study aims to evaluate the antioxidative and antiulcer effect of methanolic extract of unripe fruit of Aegle marmelos (MEAM) against Helicobacter pylori-Lipopolysaccharide (HP-LPS) induced gastric ulcer in Sprague Dawley (SD) rats. METHODS: Dose and duration of HP-LPS and MEAM were fixed based on ulcer index of gastric tissue of experimental animals. Various gastric secretory parameters such as volume of gastric juice, free and total acidity, acid output, pepsin concentration were analyzed. The activities of enzymatic antioxidants (superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glutathione transferase), non-enzymatic antioxidants (reduced glutathione, vitamin C and vitamin E) and the levels of lipid peroxidation products were measured. Histological analysis was performed to evaluate the effect of Aegle marmelos on HP-LPS induced gastric ulcer. RESULTS: Oral administration of HP-LPS (50 µg per animal) for four consecutive days resulted in induction of ulcer with the increase in gastric secretory parameters such as volume of gastric juice, free and total acidity, acid output, pepsin concentration. Oral administration of methanolic extract of Aegle marmelos fruit (MEAM) (25, 50, 100, 250 and 500 mg/kg) reduced the gastric ulcer by 2.8 %, 52.4 %, 73 %, 93 % and 93.98 %, respectively, compared to 89.2 % reduction by sucralfate (100 mg/kg). MEAM treatment significantly (p < 0.05) inhibited the increase in gastric secretory parameters in ulcerated rats, and it also prevented the reduction of enzymatic (superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glutathione transferase) and non-enzymatic antioxidants (reduced glutathione, vitamin C and vitamin E) after HP-LPS induction. In addition, lipid peroxidation was inhibited by MEAM in HP-LPS induced rats. Results of histological analysis correlated well with biochemical parameters. CONCLUSION: These observations explored the antioxidant properties of MEAM contributing to the gastroprotective effect in HP-LPS induced gastric ulcer model.

Prediction of protein-protein interactions with clustered amino acids and weighted sparse representation.

With the completion of the Human Genome Project, bioscience has entered into the era of the genome and proteome. Therefore, protein-protein interactions (PPIs) research is becoming more and more important. Life activities and the protein-protein interactions are inseparable, such as DNA synthesis, gene transcription activation, protein translation, etc. Though many methods based on biological experiments and machine learning have been proposed, they all spent a long time to learn and obtained an imprecise accuracy. How to efficiently and accurately predict PPIs is still a big challenge. To take up such a challenge, we developed a new predictor by incorporating the reduced amino acid alphabet (RAAA) information into the general form of pseudo-amino acid composition (PseAAC) and with the weighted sparse representation-based classification (WSRC). The remarkable advantages of introducing the reduced amino acid alphabet is being able to avoid the notorious dimensionality disaster or overfitting problem in statistical prediction. Additionally, experiments have proven that our method achieved good performance in both a low- and high-dimensional feature space. Among all of the experiments performed on the PPIs data of Saccharomyces cerevisiae, the best one achieved 90.91% accuracy, 94.17% sensitivity, 87.22% precision and a 83.43% Matthews correlation coefficient (MCC) value. In order to evaluate the prediction ability of our method, extensive experiments are performed to compare with the state-of-the-art technique, support vector machine (SVM). The achieved results show that the proposed approach is very promising for predicting PPIs, and it can be a helpful supplement for PPIs prediction.

Inhibitory effects of emodin, baicalin, schizandrin and berberine on hefA gene: treatment of Helicobacter pylori-induced multidrug resistance.

AIM: To investigate the inhibitory effects of emodin, baicalin, etc. on the hefA gene of multidrug resistance (MDR) in Helicobacter pylori (H. pylori). METHODS: The double dilution method was used to screen MDR H. pylori strains and determine the minimum inhibitory concentrations (MICs) of emodin, baicalin, schizandrin, berberine, clarithromycin, metronidazole, tetracycline, amoxicillin and levofloxacin against H. pylori strains. After the screened MDR stains were treated with emodin, baicalin, schizandrin or berberine at a 1/2 MIC concentration for 48 h, changes in MICs of amoxicillin, tetracycline, levofloxacin, metronidazole and clarithromycin were determined. MDR strains with reduced MICs of amoxicillin were selected to detect the hefA mRNA expression by real-time quantitative PCR. RESULTS: A total of four MDR H. pylori strains were screened. Treatment with emodin, baicalin, schizandrin and berberine significantly decreased the MICs of amoxicillin and tetracycline against some strains, decreased by 1 to 2 times, but did not significantly change the MICs of clarithromycin, levofloxacin, and metronidazole against MDR strains. In the majority of strains with reduced MICs of amoxicillin, hefA mRNA expression was decreased; one-way ANOVA (SPSS 12.0) used for comparative analysis, P < 0.05. CONCLUSION: Emodin, baicalin, schizandrin and berberine significantly decreased the MICs of amoxicillin and tetracycline against some H. pylori strains, possibly by mechanisms associated with decreasing hefA mRNA expression.

Nickel-responsive regulation of two novel Helicobacter pylori NikR-targeted genes.

Nickel is an essential transition metal for the survival of Helicobacter pylori in the acidic human stomach. The nickel-responsive transcriptional regulator HpNikR is important for maintaining healthy cytosolic nickel concentrations through the regulation of multiple genes, but its complete regulon and role in nickel homeostasis are not well understood. To investigate potential gene targets of HpNikR, ChIP sequencing was performed using H. pylori grown at neutral pH in nickel-supplemented media and this experiment identified HPG27_866 (frpB2) and HPG27_1499 (ceuE). These two genes are annotated to encode a putative iron transporter and a nickel-binding, periplasmic component of an ABC transporter, respectively. In vitro DNA-binding assays revealed that HpNikR binds both gene promoter sequences in a nickel-responsive manner with affinities on the order of ∼10(-7) M. The recognition sites of HpNikR were identified and loosely correlate with the HpNikR pseudo-consensus sequence (TATTATT-N11-AATAATA). Quantitative PCR experiments revealed that HPG27_866 and HPG27_1499 are transcriptionally repressed following growth of H. pylori G27 in nickel-supplemented media, and that this response is dependent on HpNikR. In contrast, iron supplementation results in activation of HPG27_1499, but no impact on the expression of HPG27_866 was observed. Metal analysis of the Δ866 strain revealed that HPG27_866 has an impact on nickel accumulation. These studies demonstrate that HPG27_866 and HPG27_1499 are both direct targets of HpNikR and that HPG27_866 influences nickel uptake in H. pylori.

Medicinal plant activity on Helicobacter pylori related diseases.

More than 50% of the world population is infected with Helicobacter pylori (H. pylori). The bacterium highly links to peptic ulcer diseases and duodenal ulcer, which was classified as a group I carcinogen in 1994 by the WHO. The pathogenesis of H. pylori is contributed by its virulence factors including urease, flagella, vacuolating cytotoxin A (VacA), cytotoxin-associated gene antigen (Cag A), and others. Of those virulence factors, VacA and CagA play the key roles. Infection with H. pylori vacA-positive strains can lead to vacuolation and apoptosis, whereas infection with cagA-positive strains might result in severe gastric inflammation and gastric cancer. Numerous medicinal plants have been reported for their anti-H. pylori activity, and the relevant active compounds including polyphenols, flavonoids, quinones, coumarins, terpenoids, and alkaloids have been studied. The anti-H. pylori action mechanisms, including inhibition of enzymatic (urease, DNA gyrase, dihydrofolate reductase, N-acetyltransferase, and myeloperoxidase) and adhesive activities, high redox potential, and hydrophilic/hydrophobic natures of compounds, have also been discussed in detail. H. pylori-induced gastric inflammation may progress to superficial gastritis, atrophic gastritis, and finally gastric cancer. Many natural products have anti-H. pylori-induced inflammation activity and the relevant mechanisms include suppression of nuclear factor-κB and mitogen-activated protein kinase pathway activation and inhibition of oxidative stress. Anti-H. pylori induced gastric inflammatory effects of plant products, including quercetin, apigenin, carotenoids-rich algae, tea product, garlic extract, apple peel polyphenol, and finger-root extract, have been documented. In conclusion, many medicinal plant products possess anti-H. pylori activity as well as an anti-H. pylori-induced gastric inflammatory effect. Those plant products have showed great potential as pharmaceutical candidates for H. pylori eradication and H. pylori induced related gastric disease prevention.

Preclinical studies of amixicile, a systemic therapeutic developed for treatment of Clostridium difficile infections that also shows efficacy against Helicobacter pylori.

Amixicile shows efficacy in the treatment of Clostridium difficile infections (CDI) in a mouse model, with no recurrence of CDI. Since amixicile selectively inhibits the action of a B vitamin (thiamine pyrophosphate) cofactor of pyruvate:ferredoxin oxidoreductase (PFOR), it may both escape mutation-based drug resistance and spare beneficial probiotic gut bacteria that do not express this enzyme. Amixicile is a water-soluble derivative of nitazoxanide (NTZ), an antiparasitic therapeutic that also shows efficacy against CDI in humans. In comparative studies, amixicile showed no toxicity to hepatocytes at 200 µM (NTZ was toxic above 10 µM); was not metabolized by human, dog, or rat liver microsomes; showed equivalence or superiority to NTZ in cytochrome P450 assays; and did not activate efflux pumps (breast cancer resistance protein, P glycoprotein). A maximum dose (300 mg/kg) of amixicile given by the oral or intraperitoneal route was well tolerated by mice and rats. Plasma exposure (rats) based on the area under the plasma concentration-time curve was 79.3 h · µg/ml (30 mg/kg dose) to 328 h · µg/ml (100 mg/kg dose), the maximum concentration of the drug in serum was 20 µg/ml, the time to the maximum concentration of the drug in serum was 0.5 to 1 h, and the half-life was 5.6 h. Amixicile did not concentrate in mouse feces or adversely affect gut populations of Bacteroides species, Firmicutes, segmented filamentous bacteria, or Lactobacillus species. Systemic bioavailability was demonstrated through eradication of Helicobacter pylori in a mouse infection model. In summary, the efficacy of amixicile in treating CDI and other infections, together with low toxicity, an absence of mutation-based drug resistance, and excellent drug metabolism and pharmacokinetic metrics, suggests a potential for broad application in the treatment of infections caused by PFOR-expressing microbial pathogens in addition to CDI.

High-dose extended-release lansoprazole (dexlansoprazole) and amoxicillin dual therapy for Helicobacter pylori infections.

BACKGROUND: Helicobacter pylori infections have become increasingly difficult to treat. AIM: To examine whether amoxicillin and high-dose dexlansoprazole would reliably achieve an H. pylori eradication rate of ≥90%. METHODS: An open-label prospective pilot study of H. pylori eradication in treatment-naïve subjects with active H. pylori infection (positive by two tests). THERAPY: amoxicillin 1 g and dexlansoprazole 120 mg each twice a day at approximately 12-hour intervals for 14 days. Success was accessed by urea breath test. An effective therapy was defined as a per-protocol treatment success of 90% or greater; treatment success of 80% or less was prespecified as an unacceptable result. RESULTS: After 13 subjects were entered (12 men, one woman; average age of 54 years), the prespecified stopping rule of six treatment failures was achieved (i.e., the 95% confidence interval excluded achieving the required 90% success rate even if the proposed study of 50 completed patients were entered) and enrollment was stopped. Per-protocol and intention-to-treat treatment success were both 53.8%; (7/13); 95% CI = 25-80%. Compliance was 100%. Three patients (23%) reported side effects, all of which were mild and none interrupted therapy. CONCLUSION: Theoretically, dual PPI plus amoxicillin should reliably eradicate H. pylori provided nearly neutral intragastric pH can be maintained. Clearly, dexlansoprazole, despite being administered at high dose and twice a day (i.e., total daily dose 240 mg), failed to achieve an intragastric milieu consistent with dual PPI plus amoxicillin therapy being an effective anti-H. pylori regimen.