Impact of Helicobacter pylori virulence markers on clinical outcomes in adult populations

BACKGROUND In recent years,associations between specific virulence markers of Helicobacter pylori(H.pylori)and gastrointestinal disorders have been suggested.AIM To investigate the presence of virulence factors including vacuolating cytotoxin A genotypes(s1m1,s1m2,s2m1,and s2m2),cytotoxin-associated gene A(CagA),and urease activity in H.pylori strains isolated from Arab and Jewish populations in northern Israel and to assess associations between these factors and patients’demographics and clinical outcomes.METHODS Patients(n=108)who underwent gastroscopy at the Baruch Padeh Medical Center,Poriya due to symptomatic gastroduodenal pathologies as part of H.pylori diagnosis were enrolled in the study.Gastric biopsy specimens were collected from the antrum of the stomach.Clinical condition was assessed by clinical pathology tests.Bacteria were isolated on modified BD Helicobacter Agar(BD Diagnostics,Sparks,MD,United States).Bacterial DNA was extracted,and PCR was performed to detect CagA and vacuolating cytotoxin A genes.Urease activity was assessed using a rapid urease test.RESULTS A significant correlation was found between disease severity and patient ethnicity(P=0.002).A significant correlation was found between CagA presence and the s1m1 genotype(P=0.02),which is considered the most virulent genotype.Further,a higher level of urease activity was associated with isolates originating from the Jewish population.Moreover,higher urease activity levels were measured among CagA-/s1m1 and CagA-/s2m2 isolates.CONCLUSION Our study highlights the importance of incorporating molecular methods for detection of virulence markers of H.pylori in order to tailor optimal treatments for each patient.Further investigation should be performed regarding associations between H.pylori virulence factors and ethnicity.

Restoration of Mitochondrial Structure and Function within Helicobacter pylori VacA Intoxicated Cells

The Helicobacter pylori vacuolating cytotoxin (VacA) is an intracellular, mitochondrial-targeting exotoxin that rapidly causes mitochondrial dysfunction and fragmentation. Although VacA targeting of mitochondria has been reported to alter overall cellular metabolism, there is little known about the consequences of extended exposure to the toxin. Here, we describe studies to address this gap in knowledge, which have revealed that mitochondrial dysfunction and fragmentation are followed by a time-dependent recovery of mitochondrial structure, mitochondrial transmembrane potential, and cellular ATP levels. Cells exposed to VacA also initially demonstrated a reduction in oxidative phosphorylation, as well as increase in compensatory aerobic glycolysis. These metabolic alterations were reversed in cells with limited toxin exposure, congruent with the recovery of mitochondrial transmembrane potential and the absence of cytochrome c release from the mitochondria. Taken together, these results are consistent with a model that mitochondrial structure and function are restored in VacA-intoxicated cells.

Preliminary pharmacological evaluation of Alocasia indica Schott tuber

To elucidate potential antioxidant, antidiarrheal, cytotoxic, and antibacterial activities of the ethanol extract of A/ocasia indica Schott tuber in different experimental models established in vitro and in vivo. METHODS： In vitro antioxidant activity was evaluated by 2, 2-diphenyl-l-picrylhydrazyl （DPPH） radical-scavenging assay. Phenolic content was estimated by using Folin-Ciocalteu＇s reagent while reducing ability was measured by ferric reducing power assay./n vivo antidiarrheal studies were carried out in mice, and the activity was evaluated in castor oil and magnesium sulfate- induced diarrhea. Disk diffusion assay was utilized to determine antibacterial activity against a number of pathogenic bacterial strains. Acute toxicity test was carried out to measure the safe doses for the extract. RESULTS： In DPPH radical-scavenging assay, the extract exhibited strong radical-scavenging activity with the 50% inhibitory concentration value of 42.66 IJg/mL. Total phenolic content was found to be 542.26 mg gallic acid equivalent per 100 g of dried tuber extract, whereas flavonoid content was found to be 4.30 mg quercetin equivalent/g of dried tuber extract. In reducing power assay, the extract showed strong reducing power in a concentration-dependent manner. The extract significantly （P 〈 0.01） enhanced the latent period and decreased defecation in both castor oil- and magnesium sulfate-induced diarrhea. The extract also lessened gastrointestinal motility in mice. Potential antibacterial activity was exhibited by the extract against all the tested bacterial strains in disk diffusion assay. The 50% lethal concentration against brine shrimp nauplii was 81.09 μg/mL. CONCLUSION： The results demonstrated that the ethanol extract of A. inc/ica has potential antioxidant, antidiarrheal, cytotoxic, and antibacterial activity.

Therapeutic potential of snake venom in cancer therapy:current perspectives

Many active secretions produced by animals have been employed in the development of new drugs to treat diseases such as hypertension and cancer.Snake venom toxins coutributed significantly to the treatment of many medical conditions.There are many published studies describing and elucidating the anti-cancer potential of snake venom.Cancer therapy is one of the main areas for the use of protein peptides and enzymes originating from animals of different species.Some of these proteins or peptides and enzymes from snake venom when isolated and evaluated may bind specifically to cancer cell membranes,affecting the migration and proliferation of these cells.Some of substances found in the snake venom present a great potential as anti-tumor agent.In this review,we presented the main results of recent years of research involving the active compounds of snake venom that have anticancer activity.

Cryptotanshinone inhibits cytotoxin-associated gene A-associated development of gastric cancer and mucosal erosions

BACKGROUND Approximately 90%of new cases of noncardiac gastric cancer(GC)are related to Helicobacter pylori(H.pylori),and cytotoxin-associated gene A(CagA)is one of the main pathogenic factors.Recent studies have shown that the pharmacological effects of cryptotanshinone(CTS)can be used to treat a variety of tumors.However,the effects of CTS on H.pylori,especially CagA+strain-induced gastric mucosal lesions,on the development of GC is unknown.AIM To assess the role of CTS in CagA-induced proliferation and metastasis of GC cells,and determine if CagA+H.pylori strains causes pathological changes in the gastric mucosa of mice.METHODS The effects of CTS on the proliferation of GC cells were assessed using the Cell Counting Kit-8(CCK-8)assay,and the abnormal growth,migration and invasion caused by CagA were detected by CCK-8 and transwell assays.After transfection with pSR-HA-CagA and treatment with CTS,proliferation and metastasis were evaluated by CCK-8 and transwell assays,respectively,and the expression of Src homology 2(SH2)domain–containing phosphatase 2(SHP2)and phosphorylated SHP2(p-SHP2)was detected using western blotting in AGS cells.The enzymelinked immunosorbent assay was used to determine the immunoglobulin G(IgG)level against CagA in patient serum.Mice were divided into four groups and administered H.pylori strains(CagA+or CagA-)and CTS(or PBS)intragastrically,and establishment of the chronic infection model was verified using polymerase chain reaction and sequencing of isolated strains.Hematoxylin and eosin staining was used to assess mucosal erosion in the stomach and toxicity to the liver and kidney.RESULTS CTS inhibited the growth of GC cells in dose-and time-dependent manners.Overexpression of CagA promoted the growth,migration,and invasion of GC cells.Importantly,we demonstrated that CTS significantly inhibited the CagAinduced abnormal proliferation,migration,and invasion of GC cells.Moreover,the expression of p-SHP2 protein in tumor tissue was related to the expression of IgG against CagA in the serum of GC patients.Additionally,CTS suppressed the protein expression levels of both SHP2 and p-SHP2 in GC cells.CTS suppressed CagA+H.pylori strain-induced mucosal erosion in the stomach of mice but had no obvious effects on the CagA-H.pylori strain group.CONCLUSION CTS inhibited CagA-induced proliferation and the epithelial-mesenchymal transition of GC cells in vitro,and CagA+H.pylori strains caused mucosal erosions of the stomach in vivo by decreasing the protein expression of SHP2.

Study on the Synthesis and Antitumor Activity of New Indolocarbazole Compounds

A series of new antitumor compounds having indolecarbazole structures were designed and synthesized. The methoxy substituted indolecarbazole parent nucleus, which was firstly synthesized, is condensed with bromine substituted amino acid methyl ester to produce the target compounds. The target compounds were performed with methylthiazolyldiphenyl-tetrazolium bromide（MTT） in vitro cytotoxin activity test and the results showed that compounds CZ-1, CZ-3 and CZ-6 have higher activity against human colon cancer（HT-29） and（HCT-8）, hepatocellular carcinoma（Bel-7402）, NSCLC（A549） and breast cancer（MCF-7） cells as compared to the positive control JDC-108.

Role of nickel-regulated small RNA in modulation of Helicobacter pylori virulence factors

Helicobacter pylori(H.pylori)is a Gram-negative bacterium that infects about half of the world's population.H.pylori infection prevails by several mechanisms of adaptation of the bacteria and by its virulence factors including the cytotoxin associated antigen A(CagA).CagA is an oncoprotein that is the protagonist of gastric carcinogenesis associated with prolonged H.pylori infection.In this sense,small regulatory RNAs(sRNAs)are important macromolecules capable of inhibiting and activating gene expression.This function allows sRNAs to act in adjusting to unstable environmental conditions and in responding to cellular stresses in bacterial infections.Recent discoveries have shown that nickelregulated small RNA(NikS)is a post-transcriptional regulator of virulence properties of H.pylori,including the oncoprotein CagA.Notably,high concentrations of nickel cause the reduction of NikS expression and consequently this increases the levels of CagA.In addition,NikS expression appears to be lower in clinical isolates from patients with gastric cancer when compared to patients without.With that in mind,this minireview approaches,in an accessible way,the most important and current aspects about the role of NikS in the control of virulence factors of H.pylori and the potential clinical repercussions of this modulation.

Study on the Synthesis and Antitumor Activity of Indolecarbazole Compounds Modified by Amino Acid and Piperidine

A series of antitumor compounds with indolecarbazole structure modified by amino acid and piperidine were designed and synthesized. The indolecarbazole parent nucleus was firstly synthesized, condensed with bromine substituted amino acid methyl ester, then hydrolyzed and condensed with piperidine to produce the target compounds. In vitro cytotoxin activity test was performed against 7 target compounds with methylthiazolyldiphenyl-tetrazolium bromide（MTT）, and the results showed that compounds CZ-2, CZ-3 and CZ-5 have higher activity against human colon cancers（HT-29） and（HCT-8）, hepatocellular carcinoma（Bel-7402）, NSCLC（A549） and breast cancer（MCF-7） cells as compared with the positive control JDC-108.

Virulence properties of a peptide hemolysin produced by Enterococcus faecalis

The characterization and prevalence of virulence factors associated with enterococcal invasiveness and severity of disease are important areas to be investigated. Recently, we described the production of a heat-stable hemolysin by clinical isolates of Enterococcus faecalis cultived in BHI-GA (BHI with glucose and L-arginine). Now, we purified the hemolysin from the culture supernatant by ultra-filtration (PM-10 membrane) and ethanol extraction followed by chromatography in a mBondapak C18 and Superdex Peptide columns. The hemolytic activity was not affected by the proteolytic enzymes. Cholesterol, phospholipids, EDTA and also bivalent ions did not inhibit the hemolytic activity. Among the various carbohydrates, only dextran 4 protected the erythrocytes against lyse. Scanning electron microscopy showed that lyse of erythrocytes occured at once after the exposure to the hemolysin. The mito-chondrial activity and the cell membrane integrity were significantly affected by the hemolysis, within 20 min of exposure and caused apoptosis after 12 h incubation, 51.92% in HeLa and 68% in HEp-2 cells, analyzed by flow cytometry. These results suggest that the heat-stable pore forming hemolysin might be a putative virulence factor in enterococci infections.

Medicinal potential of Passiflora foetida L. plant extracts: biological and pharmacological activities

OBJECTIVE： To investigate analgesic, antidiarrhoeal and cytotoxic activities of the ethanol extract of Passiflora foetida L. （Passifloraceae） by three experimental methods. METHODS： Analgesic activity of the ethanol extract of Passiflora foetida L. （EEPF） was carried out using acetic acid-induced writhing inhibition in mice. The method of castor oil-induced diarrhoea in mice was utilized to evaluate antidiarrhoeal activity. The cytotoxic activity of EEPF was explored with a brine shrimp lethality bioassay. RESULTS： The extract showed 68.75% and 30.00% inhibition of writhe at the doses of 500 and 250 mg/kg body weight, respectively. The extract increased the mean latent period prior to diarrhoeal onset to about 1.55 h and 1.17 h, and decreased the mean number of stools to 4.4 and 5.6 at the doses of 500 and 250 mg/kg body weight. The extract also demonstrated cytotoxic activity in the brine shrimp lethality assay, and the median lethal concentration for brine shrimp nauplii was 80 pg/mL. CONCLUSION： The results suggest that the plant extract has analgesic and antidiarrhoeal activities, supporting its uses in traditional medicine. The results also demonstrate that the plant extract possesses cytotoxic activities.

An ethanolic extract of Bailian(Radix Ampelopsis Japonicae):demonstration of colorectal cancer treatment efficacy via inhibition of β-catenin signaling in vitro

OBJECTIVE:To investigate the underlying mechanism of Bailian (Radix Ampelopsis Japonicae,BL) extract action on colorectal cancer (CRC).METHODS:We explored the involvement of β-catenin signaling on the anti-CRC effects of an BL ethanolic extract (BLE) in cell models by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay,immunofluorescent staining,luciferase assay,Western blot analysis and real-time quantitative polymerase chain reaction analysis.Anti-CRC compounds were quantified by high performance liquid chromatography.RESULTS:The contents of gallic acid,catechin,and epicatechin in the BLE were 0.23,1.25,and 0.18 g/kg,respectively.BLE-mediated cytotoxic and apoptotic effects were accompanied by lowered β-catenin/Tcf transcriptional activity,reduced β-catenin nuclear localization,and downregulated protein and mRNA levels of both β-catenin and molecules regulated by β-catenin.CONCLUSION:The mechanism underpinning the anti-CRC effects of BLE may involve inhibition of β-catenin signaling.Further studies are necessary to establish the role of β-catenin signaling in the action of BLE-mediated anti-CRC effects.

Microbiostatic, antioxidative and cytotoxic potentiation of some grasses of Bahawalpur, Pakistan

OBJECTIVE: To evaluate biological potential of methanol and n-hexane extracts of aerial parts in seven species of family Poaceae.METHODS: Qualitative phytochemical tests were done by using standard protocols. In vitro antioxidant activity was performed via different assays and antimicrobial potential was observed via disc diffusion method. Cytotoxic activity was carried out using brine shrimps’ assay.RESULTS: Phytochemical studies revealed the presence of alkaloids, flavonoids, glycosides, phenols,steroids, saponins, tannins, anthocyanins and coumarins in most of the plant extracts. Maximum antioxidant and antimicrobial potential were observed in Cymbopogon citratus methanol extract and Cymbopogon citratus n-hexane extract along with significant number of total flavonoids and phenols contents. However, Polypogon monspeliensis methanol extract and Polypogon monspeliensis n-hexane extract showed minimum antioxidant as well as antimicrobial potential. Moreover, methanol extracts showed a cytotoxic effect with their effectiveness ranked Polypogon monspeliensis methanol extract > Cymbopogon citratus methanol extract > Phalaris minor n-hexane extract > Aristida funiculata methanol extract > Stipagrostis plumosa methanol extract > Cenchrus ciliaris methanol extract > Panicum antidotale methanol extract. Similarly, n-hexane plant extracts revealed cytotoxic activity in decreasing order Cenchrus ciliaris n-hexane extract > Stipagrostis plumosa n-hexane extract > Phalaris minor n-hexane extract > Aristida funiculata n-hexane extract > Polypogon monspeliensis n-hexane extract > Panicum antidotale n-hexane extract > Cymbopogon citratus n-hexane extract respectively.CONCLUSION: Methanol extracts exhibit significant antioxidant and antimicrobial potential which can be correlated to their medicinal values. The observed brine shrimp’s lethality of the plant extracts revealed the presence of potent cytotoxic components in these plants.

Rumex dentatus could be a potent alternative to treatment of microbial infections and of breast cancer

OBJECTIVE:To investigate the phytochemicals and in vitro antioxidant,antimicrobial and cytotoxic potential of Rumex dentatus(R.dentatus)leaf extracts.METHODS:The total phenolics and flavonoids content of R.dentatus extracts were evaluated by the Folin-Ciocalteu and aluminum chloride colorimetric methods respectively.Antioxidant potential of studied plant extracts was assessed through 2,2-diphenyl-1-picrylhydrazyl radical scavenging capacity,total reducing power and total antioxidant methods.Moreover,antibacterial and antifungal capacity was also evaluated by disc diffusion method against six clinically isolated multi-drug resistant bacterial strains as well as six fungal isolates.Further,cell cytotoxicity was also evaluated through3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay.RESULTS:Ethanol extract showed highest total phenolic[(38.9±1.5)μg gallic acid equivalent/mg]and total flavonoids[(17.2±1.9)μg quercetin equivalent/mg]contents.Antioxidant assays indicated that ethanol and methanol extracts possess potent antioxidant potential.Moreover,it was observed that ethanol and hexane extracts have the potential to inhibit most of the tested multi-drug resistant bacterial strains while methanol,chloroform and hexane extracts could inhibit the growth of pathogenic fungal strains successfully.Among all the studied extracts,ethanolic extract showed highest cytotoxicity against MCF-7 cell line then Hep-2 and DU-145 cell lines by MTT assay with lowest IC50 of 47.3μg/m L.CONCLUSION:These results suggest that R.dentatus could be a potent alternative candidate for treatment of microbial infections and for breast cancer treatment.

Phytochemical investigation of medicinally important plants of the Pothohar region of Pakistan

OBJECTIVE:To report the phytochemical composition(flavonoids,phenolics,saponins,tannins,and terpenoids)and biological activities(antioxidant,phytotoxic,cytotoxic,antifungal activities)of medicinally important plants of Pothohar region.METHODS:In the current study various qualitative and quantitative phytochemical tests were conducted to investigate the phytochemical composition of plant extracts prepared in benzene,chloroform and methanol.The same extracts were further utilized in different biological activities i.e.,antioxidant[total antioxidant capacity,total reducing power,and 2,2-diphenyl-1-picrylhydrazyl(DPPH)scavenging assay],phytotoxicity(Radish seed bioassay),brine shrimp cytotoxicity and antifungal activities(against four pathogenic fungal strains).RESULTS:Phytochemical analysis indicate flavonoids,phenolics,saponins,tannins and terpenoids were present in almost all plants however,maximum amount of total phenolic and flavonoid contents was reported in of Malva parviflora and Lepidium pinnatifidum.Extracts of Malva parviflora and Lepidium pinnatifidum also depicted the superlative antioxidant activity(total antioxidant capacity,total reducing power,and DPPH scavenging activity).Maximum fungal growth inhibition was demonstrated by extracts of Centaurea calcitropa,Vicia sativa and Malva parviflora.The maximum cytotoxicity was shown by the extracts of Centaurea calcitropa.Moreover,for phytotoxicity assay the highest allelopathic potential was exhibited by extracts of Lepidium pinnatifidum.Solvents prejudiced results;the best results were obtained by Methanol followed by Chloroform and Benzene.CONCLUSION:Centaurea calcitropa,Vicia sativa,Malva parviflora and Lepidium pinnatifidum are most promising medicinal plants of Pothohar region for future studies.

Biological evaluation of some important medicinal plants from Poonch valley, Azad Kashmir, Pakistan

OBJECTIVE:To estimate the phytochemical composition,antioxidant,antifungal,cytotoxic and phytotoxic activities of three important medicinal plants of Poonch valley,Azad Kashmir Pakistan.METHODS:Phytochemicals were investigated for both qualitative(phenols,flavonoids,alkaloids and saponins)and quantitative(total phenolic and flavonoid contents)analysis of plant extracts prepared in ethanol,ethyl acetate and benzene.Moreover,antioxidant potential was investigated by screening botanical extracts against 2,2-diphenyl-1-picrylhydrazyl scavenging assay,phosphomolybdenum and total reducing power assay.While,disc diffusion method was used for determination of antifungal potential of all extracts against four pathogenic strains.Furthermore,brine shrimp cytotoxicity assay was conducted for cytotoxic potential and radish seed germination assay was carried out for estimation of allelopathic potential of plant extracts.RESULTS:Phytochemical evaluation showed the presence of phenolics,flavonoids,alkaloids and saponins in almost all plants.In addition to significant antioxidant potential,Rubia cordifolia(ethanolic extract)revealed highest amount of phenolics and flavonoids.The said plant also exhibited good cytotoxic activity.Among plant extracts highest antifungal activity and allelopathic potential was recorded by Oxalis latifolia(ethyl acetate extract).Ethanol and ethyl acetate were observed to be the best choice of solvents.The best solvents-based results were obtained by Ethanol followed by Ethyl acetate and Benzene.CONCLUSION:For further studies,Rubia cordifolia and Oxalis latifolia are the most promising medicinal plants of Poonch valley,Azad Kashmir Pakistan at molecular level in order to isolate the compounds concerning with pronounced activities.

Toxin(s), Other Than Cholera Toxin, Produced by Environmental Non O1 Non O139 Vibrio cholerae

A total of 39 Vibrio cholerae non O1 non O139 strains were isolated from surface waters of different parts of Dhaka City, Bangladesh. All these strains showed lack of ctx or zot gene, as demonstrated by the PCR analysis. Eighteen representative strains were tested for enterotoxin production using a rabbit ileal loop model, of which live cells of 8 strains and culture filtrates of 6 strains produced fluid accumulation in ileal loops. However, none of them produced heat stable toxin （ST）, as detected by suckling mouse assay. On the other hand, 15% of isolates produced cytotoxin as detected by the Chinese Hamster Ovary （CHO） cell assay. Fifty times concentrated culture filtrates of the representative strains did not give any precipitin band against the anti-cholera toxin, suggesting the strains produced an enterotoxin, which is antigenically different from known cholera toxin （CT）. Eighty percent of the total isolates were found to be positive for heat labile haemolysin detected by tube method, whereas, 39% were found positive by the Christie-Atkins-Munch-Petersen （CAMP） method. However, 87% of the isolates were positive for haemagglutinin/protease and all of the strains were positive for mannose-sensitive-haemagglutinin assay.

Purification and partial amino acid sequences of a new presynaplic toxin and a cytotoxin from venom of pit veper Agkistrodon blomhoffii brevicaudus

The technique of the reverse-phase performance liquid chromatography (RP-HPLC) was employed to separate and purify the toxic proteins from the venom of Agkistrodon blomhoffii brevicaudus collected in China 3 toxic proteins marked as AgTx-1, AgTx-2 and AgTx-3 consisting of about 122 amino acid residues were screened The toxicities (LD50,) of the AgTx-1, AgTx-2 and AgTx-3 were 0.075, 0.51 and 6.6 mg per kg weight of mice respectively. Toxicological experiment in the chick biventer cervicis nerve-muscle preparation showed that the acetylcholine (Ach) sensitivity of the preparation was unchanged after the total failure of the indirect contraction caused by AgTx-1 and AgTx-2. suggesting that they were presynaptic blockers, namely β-type of snake toxins. However, the amplitude of indirect contraction of the preparation was gradually reduced due to its incomplete relaxation caused by AgTx-3, indicating that it should belong to the category of cytotoxins. The partial amino acid sequences of 3 toxins have