Identification and Validation of an Antivirulence Agent Targeting HlyU-Regulated Virulence in Vibrio vulnificus.

Antimicrobial resistance (AMR) in pathogens is the result of indiscriminate use of antibiotics and consequent metabolic/genetic modulation to evolve survival strategies and clonal-selection in AMR strains. As an alternative to antibiotic treatment, antivirulence strategies are being developed, not only to combat bacterial pathogenesis, but also to avoid emerging antibiotic resistance. Vibrio vulnificus is a foodborne pathogen that causes gastroenteritis, necrotizing wound infections, and sepsis with a high rate of mortality. Here, we developed an inhibitor-screening reporter platform to target HlyU, a master transcriptional regulator of virulence factors in V. vulnificus by assessing rtxA1 transcription under its control. The inhibitor-screening platform includes wild type and ΔhlyU mutant strains of V. vulnificus harboring the reporter construct P rtxA1::luxCDABE for desired luminescence signal detection and control background luminescence, respectively. Using the inhibitor-screening platform, we identified a small molecule, fursultiamine hydrochloride (FTH), that inhibits the transcription of the highly invasive repeat-in-toxin (rtxA1) and hemolysin (vvhA) along with other HlyU regulated virulence genes. FTH has no cytotoxic effects on either host cells or pathogen at the tested concentrations. FTH rescues host cells from the necrotic cell-death induced by RtxA1 and decreases the hemolytic activity under in vitro conditions. The most important point is that FTH treatment does not induce the antivirulence resistance. Current study validated the antivirulence strategy targeting the HlyU virulence transcription factor and toxin-network of V. vulnificus and demonstrated that FTH, exhibits a potential to inhibit the pathogenesis of deadly, opportunistic human pathogen, V. vulnificus without inducing AMR.

Comparison of the effects of pantethine and fursultiamine on plasma gastrointestinal peptide levels in healthy volunteers.

Pantethine and fursultiamine have been evaluated for their clinical usefulness in the treatment and prevention of uncomplicated postoperative adhesive intestinal obstruction. In recent years, the actions of drugs used to treat gastrointestinal diseases have been elucidated pharmacologically from the viewpoints of gastrointestinal peptide levels. We examined the effects of pantethine and fursultiamine on plasma levels of calcitonin gene-related peptide (CGRP)-, vasoactive intestinal polypeptide (VIP)-, motilin- and substance P (SP)-like immunoreactive substances (IS) in healthy subjects. An open-labeled study was conducted on five healthy volunteers. Each subject was administered a single oral dose of pantethine, fursultiamine and placebo at intervals of one month. Venous blood samples were collected before and at 20, 40, 60, 90, 120, 180 and 240 min after each administration. Plasma peptide levels were measured using a highly sensitive enzyme immunoassay. A single oral dose of pantethine resulted in significant increases of plasma CGRP- and VIP-IS levels compared to placebo. Furthermore, areas under the plasma concentration-time curves (AUC(0-240)) of CGRP- and VIP-IS were significantly higher after pantethine administration compared with placebo. On the other hand, fursultiamine had no effect on plasma levels and AUC(0-240) of CGRP-, VIP-, motilin- and SP-IS. This study demonstrated the different effects of pantethine and fursultiamine from the viewpoint of plasma gastrointestinal peptide changes. The pharmacological effects of pantethine may be closely related to the changes in plasma CGRP- and VIP-IS levels.

Human brain activation in response to olfactory stimulation by intravenous administration of odorants.

To identify the BOLD effects related to olfaction in humans, we recorded functional magnetic resonance imaging (fMRI) scans in response intravenously instilled thiamine propyl disulfide (TPD) and thiamine tetrahydrofurfuryl disulfide monohydrochloride (TTFD). TPD and TTFD evoked a strong and weak odor sensation, respectively. Since we did not spray the odor stimuli directly, this method is expected to reduce the effect caused by direct stimulation of the trigeminal nerve. For the analysis of fMRI data, statistical parametric mapping (SPM2) was employed and the areas significantly activated during olfactory processing were located. Both strong and weak odorants induced brain activities mainly in the orbitofrontal gyrus (Brodmann's area: BA 11) in the left hemisphere. TPD (a strong odorant) induced activity in the subthalamic nucleus in the left hemisphere and the precentral gyrus (BA 6) and insula in the right hemisphere. TTFD (a weak odorant) induced activity in the superior frontal gyrus (BA 11) in the right hemisphere. In both circumstances, there was an increase in blood flow at the secondary olfactory cortex (SOC) but not the primary olfactory cortex (POC), probably due to a habituation effect in the POC. From the present results, we found brain activity in not only odor-specific regions but also regions whose levels of activity were changed by an intensity difference of odor stimuli.

The effect of a thiamin derivative on exercise performance.

The purpose of this study was to investigate the effect of a thiamin derivative, thiamin tetrahydrofurfuryl disulfide (TTFD), on oxygen uptake (VO2), lactate accumulation and cycling performance during exercise to exhaustion. Using a randomized, double-blind, cross-over design with a 10-day washout between trials, 14 subjects ingested either 1 g.day-1 of TTFD or a placebo (PL) for 4 days. On day 3, subjects performed a progressive exercise-test to exhaustion on a cycle ergometer for the determination of VO2submax, VO2peak, lactate concentration ([La-]), lactate threshold (ThLa) and heart rate (fc). On day 4, subjects performed a maximal 2000-m time trial on a cycle ergometer. A one-way analysis of variance (ANOVA) with repeated measures was used to determine significant differences between trials. There were no significant differences detected between trials for serial measures of VO2submax, [La-] or fc. Likewise, VO2peak [PL 4.06 (0.19) TTFD 4.12 (0.19) l.min-1, P = 0.83], ThLa [PL 2.47 (0.17), TTFD 2.43 (0.16) l.min-1, P = 0.86] and 2000-m performance time [PL 204.5 (5.5), TTFD 200.9 (4.3).s, P = 0.61] were not significantly different between trials. The results of this study suggest that thiamin derivative supplementation does not influence high-intensity exercise performance.

Effects of thiamine supplementation on exercise-induced fatigue.

High-dose thiamine (vitamin B1) supplementation (100 mg/day) may be helpful in preventing or accelerating recovery from exercise-induced fatigue. Sixteen volunteer male athletes volunteer, 8 with a blood thiamine level of 40 ng/ml or more (normal thiamine group) and 8 with levels below that level (low thiamine group) were selected as subjects. They exercised on a bicycle ergometer and the effects of thiamine supplementation were compared with placebo. Blood thiamine level markedly increased following supplementation of thiamine for 3 days before exercise. Exercise-induced changes in hemodynamic parameters and cardiopulmonary function indicated the onset of fatigue. Thiamine supplementation significantly suppressed the increase in blood glucose in the normal thiamine group and significantly decreased the number of complaints shortly after exercise in the subjective fatigue assessment of 30 items.