Development of an oxidative stress sensor in live bacteria using the optimized HyPer2 protein.

Oxidative stress is a key regulator in many cellular processes but also an important burden for living organisms. The source of oxidative damage usually is difficult to measure and assess with analytical tools or chemical indicators. One major limitation is to discriminate the presence of secondary oxidant molecules derived from the cellular metabolism after exposure to the oxidant or the scavenging capacity of reactive oxygen species by cells. Using a whole-cell reporter system based on an optimized HyPer2 protein for Escherichia coli expression, we demonstrate that, as previously shown for eukaryotic organisms, the effect at the transcriptional level of hydrogen peroxide can be monitored in vivo using flow cytometry of bacterial cells without the need of a direct analytical measurement. In this approach, we generated two different HyPer2 expression systems, one that is induced by IPTG and a second one that is induced by oxidative stress responsive promoters to control the expression of the HyPer2 protein and the exposure of higher H2O2 concentrations that has been shown to activate oxidative response genes. Both systems showed that the pathway that leads to the generation of H2O2 in vivo can be traced from H2O2 exposure. Our results indicate that hydrogen peroxide pulses can be readily detected in E. coli cells by a defined fluorescence signature that is H2O2 concentration-dependent. Our findings indicate that although less sensitive than purified protein or expressed in eukaryotic cells, HyPer2 is a good bacterial sensor for H2O2. As proof of concept, this system was used to trace the oxidative capacity of Toluidine Blue O showing that oxidative stress and redox imbalance is generated inside the cell. This system is expanding the repertoire of whole cell probes available for tracing cellular stress in bacteria.

Contribution of neutral sphingomyelinases to in vitro virulence of Entamoeba histolytica.

Amoebiasis is a worldwide health problem caused by the pathogen Entamoeba histolytica. Several virulence factors have been implicated in host invasion, immune evasion, and tissue damage. There are still new factors that remain to be elucidated and characterized. In this work, we obtained amoebic transfectants overexpressing three of the neutral sphingomyelinase enzymes encoded in the E. histolytica genome. The EhnSM3 overexpression induced an increase in hemolytic and cytotoxic activities, besides an increase in gene expression of amoebapore A, B, and C. Meanwhile the EhnSM1 and EhnSM2 overexpression caused an increase in cytopathic activity. In all the neutral sphingomyelinases overexpressing strains, the gene expression levels for cysteine proteinase 5, adhesin 112 and, heavy and light Gal/GalNAc lectin subunits were not affected. We propose that the increase of cytotoxic and lytic effect of EhnSM3 overexpressed strain can be related to the sum of the effect of EhnSM3 plus amoebapores, in a process cell contact-dependent or as mediator by inducing the gene expression of amoebapores enabling a link between EhnSM3 with the virulence phenotype in E. histolytica. Our results suggest a differential role for neutral sphingomyelinases in E. histolytica virulence.

Self-medication practice in pregnant women from central Mexico.

Self-medication during pregnancy represents a serious threat for mother and child health. The objective of this study was to evaluate the prevalence and the factors associated with self-medication among Mexican women living in the central region of Mexico. This is a descriptive interview-study of 1798 pregnant women or women who were pregnant no more than 3 years ago, when the interview was carried out. Data analysis was carried out with chi-square analysis and odds ratio. The prevalence of self-medication (allopathic drugs, medicinal plants, and other products, including vitamins, food supplements, among others) was 21.9%. The factors associated (p < 0.05) with self-medication were: higher education (college and postgraduate), smoking, and consumption of alcohol. Smoking was the strongest factor (OR: 2.536; 1.46-4.42) associated to self-medication during pregnancy, followed by consumption of alcohol (OR: 2.06; 1.38-3.08), and higher education (OR: 1.607; 1.18-2.19). Medicinal plant consumption was associated with nausea, constipation, migraine, and cold (p < 0.05), whereas he self-medication of allopathy was associated with gastritis and migraine (p < 0.05). Self-medication was influenced mainly by a relative or friend, who recommended the use of herbal medicine/allopathic medication. Two of the most common medicinal plants (arnica and ruda) here informed are reported to induce abortion or toxicity during pregnancy. The findings showed that self-medication (medicinal plants and allopathic medication) is a common practice among pregnant women from central Mexico. Adequate counselling of pregnant women by healthcare professionals about the potential risks of self-medication with herbal medicine and allopathic drugs during pregnancy is strongly warranted.

Attenuation of In Vitro and In Vivo Virulence Is Associated with Repression of Gene Expression of AIG1 Gene in Entamoeba histolytica.

Entamoeba histolytica virulence results from complex host-parasite interactions implicating multiple amoebic components (e.g., Gal/GalNAc lectin, cysteine proteinases, and amoebapores) and host factors (microbiota and immune response). UG10 is a strain derived from E. histolytica virulent HM-1:IMSS strain that has lost its virulence in vitro and in vivo as determined by a decrease of hemolytic, cytopathic, and cytotoxic activities, increased susceptibility to human complement, and its inability to form liver abscesses in hamsters. We compared the transcriptome of nonvirulent UG10 and its parental HM-1:IMSS strain. No differences in gene expression of the classical virulence factors were observed. Genes downregulated in the UG10 trophozoites encode for proteins that belong to small GTPases, such as Rab and AIG1. Several protein-coding genes, including iron-sulfur flavoproteins and heat shock protein 70, were also upregulated in UG10. Overexpression of the EhAIG1 gene (EHI_180390) in nonvirulent UG10 trophozoites resulted in augmented virulence in vitro and in vivo. Cocultivation of HM-1:IMSS with E. coli O55 bacteria cells reduced virulence in vitro, and the EhAIG1 gene expression was downregulated. In contrast, virulence was increased in the monoxenic strain UG10, and the EhAIG1 gene expression was upregulated. Therefore, the EhAIG1 gene (EHI_180390) represents a novel virulence determinant in E. histolytica.

Entamoeba histolytica: molecular cloning and characterization of a novel neutral sphingomyelinase.

A novel neutral sphingomyelinase (nSMase) was characterized in Entamoeba histolytica trophozoites. SMase, a sphingomyelin-specific form of phospholipase C, catalyzes the hydrolysis of sphingomyelin to ceramide and phosphorylcholine. Three amebic putative nSMase genes were found to be actively transcribed. Mg(2+)-independent nSMase activity in the soluble fraction of the trophozoites was stimulated by Mn(2+) and partially inhibited by Zn(2+). nSMase activity of the recombinant protein EhnSM1, increased 4.5-fold in the presence of 0.5mM Mn(2+), and abolished by 5mM Zn(2+). A dose-dependent inhibition of rEhnSM1 was observed with scyphostatin, a specific inhibitor of nSMases. The EhnSM1 and EhnSM3 were detected in the soluble fraction of the amebic lysate as 35-37kDa proteins by western blot analysis. Immunofluorescence assay showed that the overexpressed HA-tagged EhnSM1 and EhnSM3 were localized to the cytosol. The biological role of these novel E. histolytica nSMases described in this work remains to be determined.

Entamoeba histolytica: effect on virulence, growth and gene expression in response to monoxenic culture with Escherichia coli 055.

Monoxenic cultivation of pathogenic Entamoeba histolytica trophozoites with Escherichia coli serotype 055 which binds strongly to the Gal/GalNAc amoebic lectin, markedly improved the growth of E. histolytica and produced a significant decrease in cysteine proteinase activity and a lower cytopathic activity on monolayer cells after 3 months of monoxenic culture. However, after long term monoxenic culture (12 months) the proteolytic and cytopathic activities were recovered and the amoebic growth reached the maximum yield. Employing the GeneFishing(R) technology and DNA macroarrays we detected differentially gene expression related to the amoebic interaction with bacteria. A number of differentially expressed genes encoding metabolic enzymes, ribosomal proteins, virulence factors and proteins related with cytoskeletal and vesicle trafficking were found. These results suggest that E. coli 055 has a nutritional role that strongly supports the amoebic growth, and is also able to modulate some biological activities related with amoebic virulence.

Self-treatment with herbal products for weight-loss among overweight and obese subjects from central Mexico.

ETHNOPHARMACOLOGICAL RELEVANCE: Mexico ranks second in the world for obesity prevalence. In Mexico, obese and overweight subjects commonly seek alternative treatments for weight-loss, including the use of herbal products. AIM OF THE STUDY: The main objective of this study was to evaluate the prevalence of self-medication with herbal products for weight-loss among overweight and obese subjects residing in four states (Guanajuato, San Luis Potosi, State of Mexico, and Mexico City) from central Mexico. In addition, the factors related to self-medication among patients were studied. MATERIALS AND METHODS: A total of 1404 overweight and obese subjects were interviewed. A chi-square test examined associations between socio-demographic and socio-economic information, and self-medication with herbal products for weight-loss. RESULTS: The prevalence of self-medication was 42.9% among the participants who used herbal products for weight-loss. The female gender was the strongest factor (OR: 2.20 (1.75-2.77) associated with self-medication for weight-loss, followed by a low educational level (elementary and middle school) [OR: 1.80 (1.31-2.44)], and a middle-socioeconomic status [OR: 1.75 (1.21-2.52)]. The main herbal products used for weight-loss were based on: i) green tea, Camellia sinensis (12.7% of frequency), ii) aceitilla, Bidens odorata (6.6%), and iii) soybean, Glycine max (5.3%). In addition, 65% of the respondents considered herbal products ineffective for weight-loss after 6 months of use. CONCLUSION: Due to the high incidence of overweight and obesity in Mexico, there is a high prevalence (42.9%) of self-medication using natural products for weight-loss, particularly in women from Central Mexico. This study indicates the important need to educate patients about the harmful effects of consuming these products.

Chemical characterization, pharmacological effects, and toxicity of an ethanol extract of Celtis pallida Torr. (Cannabaceae) aerial parts.

ETHNOPHARMACOLOGICAL RELEVANCE: Celtis pallida Torr (Cannabaceae) is employed as a folk medicine for the treatment of inflammation, pain, skin infections, and diarrhea, among other diseases. AIM OF THE STUDY: The purpose of this work was to assess the chemical composition, the in vitro and in vivo toxicity, the antimicrobial, anti-inflammatory, antidiarrheal, antinociceptive, locomotor, and sedative effects of an ethanolic extract obtained from Celtis pallida aerial parts (CPE). MATERIALS AND METHODS: The composition of CPE was carried out by GC-MS. The in vitro and in vivo toxic activity of CPE was estimated with the comet assay (10-1000 µg/ml) for 5 h in peripheral blood mononuclear cells, and the acute toxicity test (500-5000 mg/kg p.o.), for 14 days, respectively. The antimicrobial effect of CPE was evaluated using the minimum inhibitory concentration (MIC) assay, whereas the antidiarrheal activity (10-200 mg/kg p.o.) was calculated using the castor oil test. The antinociceptive effects of CPE (50-200 mg/kg p.o.) were estimated with the acetic acid and formalin tests, as well as the hot plate test. The sedative and locomotor activities of CPE (50-200 mg/kg p.o.) were assessed with the pentobarbital-induced sleeping time test and the rotarod test, respectively. RESULTS: The main compound found in CPE was the triterpene ursolic acid (22% of the extract). CPE at concentrations of 100 µg/ml or higher induced genotoxicity in vitro and showed low in vivo toxicity (LD50 > 5000 mg/kg p.o.). Additionally, CPE lacked (MIC > 400 µg/ml) antimicrobial activity but exerts antinociceptive (ED50 = 12.5 ±â€¯1.5 mg/kg) and antidiarrheal effects (ED50 = 2.8 mg/kg), without inducing sedative effects or altering the locomotor activity. The antinociceptive activity of CPE suggests the participation of adrenoceptors, as well as the nitric oxide/cyclic guanosine monophosphate (cGMP) pathway. CONCLUSION: C. pallida exerts its antinociceptive effects probably mediated by the nitric oxide/cyclic guanosine monophosphate (cGMP) pathway.