Whole cell screen for inhibitors of pH homeostasis in Mycobacterium tuberculosis

Bacterial pathogens like Mycobacterium tuberculosis (Mtb) encounter acidic microenvironments in the host and must maintain their acid-base homeostasis to survive. A genetic screen identified two Mtb strains that cannot control intrabacterial pH (pHIB) in an acidic environment; infection with either strain led to severe attenuation in mice. To search for additional proteins that Mtb requires to survive at low pH, we introduced a whole-cell screen for compounds that disrupt pHIB, along with counter-screens that identify ionophores and membrane perturbors. Application of these methods to a natural product library identified four compounds of interest, one of which may inhibit novel pathway(s). This approach yields compounds that may lead to the identification of pathways that allow Mtb to survive in acidic environments, a setting in which Mtb is resistant to most of the drugs currently used to treat tuberculosis

Criterion and Construct Validity of the CogState Schizophrenia Battery in Japanese Patients with Schizophrenia

BACKGROUND: The CogState Schizophrenia Battery (CSB), a computerized cognitive battery, covers all the same cognitive domains as the Measurement and Treatment Research to Improve Cognition in Schizophrenia (MATRICS) Consensus Cognitive Battery but is briefer to conduct. The aim of the present study was to evaluate the criterion and construct validity of the Japanese language version of the CSB (CSB-J) in Japanese patients with schizophrenia. METHODOLOGY/PRINCIPAL FINDINGS: Forty Japanese patients with schizophrenia and 40 Japanese healthy controls with matching age, gender, and premorbid intelligence quotient were enrolled. The CSB-J and the Brief Assessment of Cognition in Schizophrenia, Japanese-language version (BACS-J) were performed once. The structure of the CSB-J was also evaluated by a factor analysis. Similar to the BACS-J, the CSB-J was sensitive to cognitive impairment in Japanese patients with schizophrenia. Furthermore, there was a significant positive correlation between the CSB-J composite score and the BACS-J composite score. A factor analysis showed a three-factor model consisting of memory, speed, and social cognition factors. CONCLUSIONS/SIGNIFICANCE: This study suggests that the CSB-J is a useful and rapid automatically administered computerized battery for assessing broad cognitive domains in Japanese patients with schizophrenia

Nitazoxanide Disrupts Membrane Potential and Intrabacterial pH Homeostasis of <i>Mycobacterium tuberculosis</i>

Nitazoxanide (Alinia), a nitro-thiazolyl antiparasitic drug, kills diverse microorganisms by unknown mechanisms. Here we identified two actions of nitazoxanide against <i>Mycobacterium tuberculosis</i> (Mtb): disruption of Mtb’s membrane potential and pH homeostasis. Both actions were shared by a structurally related antimycobacterial compound, niclosamide. Reactive nitrogen intermediates were reported to synergize with nitazoxanide and its deacetylated derivative tizoxanide in killing Mtb. Herein, however, we could not attribute this to increased uptake of nitazoxanide or tizoxanide as monitored by targeted metabolomics, nor to increased impact of nitazoxanide on Mtb’s membrane potential or intrabacterial pH. Thus, further mechanisms of action of nitazoxanide or tizoxanide may await discovery. The multiple mechanisms of action may contribute to Mtb’s ultralow frequency of resistance against nitazoxanide

Lactococcus Lactis Subsp. cremoris Is an Efficacious Beneficial Bacterium that Limits Tissue Injury in the Intestine

Summary: The use of beneficial bacteria to promote health is widely practiced. However, experimental evidence corroborating the efficacy of bacteria promoted with such claims remains limited. We address this gap by identifying a beneficial bacterium that protects against tissue damage and injury-induced inflammation in the gut. We first employed the Drosophila animal model to screen for the capacity of candidate beneficial bacteria to protect the fly gut against injury. From this screen, we identified Lactococcus lactis subsp. cremoris as a bacterium that elicited potent cytoprotective activity. Then, in a murine model, we demonstrated that the same strain confers powerful cytoprotective influences against radiological damage, as well as anti-inflammatory activity in a gut colitis model. In summary, we demonstrate the positive salutary effects of a beneficial bacterium, namely, L. lactis subsp. cremoris on intestinal tissue and propose the use of this strain as a therapeutic to promote intestinal health. : Biological Sciences; Microbiology; Cell Biology Subject Areas: Biological Sciences, Microbiology, Cell Biolog

Effect of 1G9 on pH_IB, survival, and membrane potential of <i>Mtb</i> and on Vero green monkey kidney cells.

<p>(A) Structure of 1G9. (B) pH_IB was measured following exposure of H37Rv-pHGFP to increasing concentrations of 1G9 for two days at 37°C. (C and D) Survival of H37Rv-pHGFP following exposure to increasing concentrations of 1G9 at pH 4.5 (C) and pH 7.4 (D) for two and six days. Means ± SEM of triplicate samples from two experiments are shown. (E) Vero cells were exposed to varying concentrations of 1G9 for two days at 37°C and viability assessed microscopically and by a tetrazolium (MTS) reduction assay. Results represent two independent experiments, each performed in triplicate. Mean ± standard deviations are shown. (F) H37Rv treated with DMSO, CCCP, RIF (0.4 µg/mL), or 1G9 at pH 7.4 or 4.5 was exposed to the membrane potential sensitive dye, DiOC₂. Results represent means ± standard deviations for two independent experiments, each performed in triplicate. The p value was determined using an unpaired t test: ****p<0.0001.</p

Effect of 17D7 and 1048 on pH_IB, survival, membrane potential of <i>Mtb</i> and on toxicity to Vero green monkey kidney cells.

<p>(A) Structure of 17D7 and 1048. (B) pH_IB was measured following exposure of H37Rv-pHGFP to increasing concentrations of 1048 at pH 4.5 for two days at 37°C. (C and D) Survival of H37Rv-pHGFP following exposure to increasing concentrations of 1048 at pH 4.5 (C) and pH 7.4 (D) for two and six days. Means ± SEM of triplicate samples from two experiments are shown. (E) Vero cells were exposed to varying concentrations of 1048 for two days at 37°C and viability assessed microscopically and by a tetrazolium (MTS) reduction assay. Results represent means and standard deviations for two independent experiments, each performed in triplicate. (F) H37Rv treated with DMSO, CCCP, RIF (0.4 µg/mL), or 1048 at pH 7.4 or 4.5 was exposed to the membrane potential sensitive dye, DiOC₂. Results represent means and standard deviations for two independent experiments, each performed in triplicate. The p value was determined using an unpaired t test: **p = 0.0045; *p = 0.029.</p

Secondary screens and summary of HTS results.

<p>(A) Liposome assay: large unilamellar vesicles were loaded with fluorescein-5-(and-6-)-sulfonic acid, trisodium salt at pH 7.4 and exposed to pH 6.4 with or without compound. Fluorescence was measured over a 5 second time course, and numbers are adjusted for DMSO control. (B) Gramicidin channel assay: ANTS-loaded large unilamellar vesicles were mixed with quenching buffer and compound (10 µM) and the rate of fluorescence quenching was measured. <i>n = </i>7–8 for each compound and 33 for control (DMSO only). *denotes that the <i>p</i> value relative to control is less than 0.001. (C) Human red blood cells were exposed to compound for 1 hour and absorbance was read in a spectrophotometer at 560 nm to evaluate heme release. Dotted black line represents 5% lysis cutoff. Results show means ± SEM of two independent experiments, each performed in duplicate. (D) Summary of screening results.</p