Reductive Amination for LC–MS Signal Enhancement and Confirmation of the Presence of Caribbean Ciguatoxin-1 in Fish

Ciguatera poisoning is a global health concern caused by the consumption of seafood containing ciguatoxins (CTXs). Detection of CTXs poses significant analytical challenges due to their low abundance even in highly toxic fish, the diverse and in-part unclarified structures of many CTX congeners, and the lack of reference standards. Selective detection of CTXs requires methods such as liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) or high-resolution MS (LC–HRMS). While HRMS data can provide greatly improved resolution, it is typically less sensitive than targeted LC–MS/MS and does not reliably comply with the FDA guidance level of 0.1 µg/kg CTXs in fish tissue that was established for Caribbean CTX-1 (C-CTX-1). In this study, we provide a new chemical derivatization approach employing a fast and simple one-pot derivatization with Girard’s reagent T (GRT) that tags the C-56-ketone intermediate of the two equilibrating C-56 epimers of C-CTX-1 with a quaternary ammonium moiety. This derivatization improved the LC–MS/MS and LC–HRMS responses to C-CTX-1 by approximately 40- and 17-fold on average, respectively. These improvements in sensitivity to the GRT-derivative of C-CTX-1 are attributable to: the improved ionization efficiency caused by insertion of a quaternary ammonium ion; the absence of adduct-ions and water-loss peaks for the GRT derivative in the mass spectrometer, and; the prevention of on-column epimerization (at C-56 of C-CTX-1) by GRT derivatization, leading to much better chromatographic peak shapes. This C-CTX-1–GRT derivatization strategy mitigates many of the shortcomings of current LC–MS analyses for C-CTX-1 by improving instrument sensitivity, while at the same time adding selectivity due to the reactivity of GRT with ketones and aldehydes.     

Age-dependent alteration in muscle regeneration: the critical role of tissue niche

Although adult skeletal muscle is composed of fully differentiated fibers, it retains the capacity to regenerate in response to injury and to modify its contractile and metabolic properties in response to changing demands. The major role in the growth, remodeling and regeneration is played by satellite cells, a quiescent population of myogenic precursor cells that reside between the basal lamina and plasmalemma and that are rapidly activated in response to appropriate stimuli. However, in pathologic conditions or during aging, the complete regenerative program can be precluded by fibrotic tissue formation and resulting in functional impairment of the skeletal muscle. Our study, along with other studies, demonstrated that although the regenerative program can also be impaired by the limited proliferative capacity of satellite cells, this limit is not reached during normal aging, and it is more likely that the restricted muscle repair program in aging is presumably due to missing signals that usually render the damaged muscle a permissive environment for regenerative activity.     

Molecular characterization,serotyping, and antibiotic susceptibility profile of Leptospira interrogans serovar Copenhageni isolates from Brazil

Leptospira interrogans serogroup Icterohaemorrhagiae is the major serogroup infecting humans worldwide, and rodents and dogs are the most significant transmission sources in urban environments. Knowledge of the prevalent serovars and their maintenance hosts is essential to understand the epidemiology of leptospirosis. In this study, 20 Leptospira isolates were evaluated by pulsed-field gel electrophoresis (PFGE), variable number tandem-repeat analysis (VNTR), serotyping, and determination of antimicrobial resistance profile. Isolates, originated from bovine, canine, human, and rodent sources, were characterized by microscopic agglutination test with polyclonal and monoclonal antibodies and were identified as L. interrogans serogroup Icterohaemorrhagiae serovar Copenhageni. MICs of antimicrobials often used in veterinary medicine were determined by broth microdilution test. Most of tested antibiotics were effective against isolates, including penicillin, ampicillin, and ceftiofur. Higher MIC variability was observed for fluoroquinolones and neomycin; all isolates were resistant to trimethoprim/sulfamethoxazole and sulphadimethoxine. Isolates were genotyped by PFGE and VNTR; both techniques were unable to discriminate between serovars Copenhageni and Icterohaemorrhagiae, as expected. PFGE clustered all isolates in 1 pulsotype, indicating that these serovars can be transmitted between species and that bovine, rodent, and dogs can maintain them in the environment endangering the human population.     

Altered erythrocyte Na-K pump in anorectic patients

The status of the erythrocyte sodium pump was evaluated in a group of patients suffering from anorexia nervosa and a group of healthy female control subjects. Anorectic patients showed significantly higher mean values of digoxin-binding sites/cell (ie, the number of Na-K-ATPase units) with respect to control subjects while no differences were found in the specific 86Rb uptake (which reflects the Na-K-ATPase activity) between the two groups. A significant correlation was found between relative weight and the number of Na-K-ATPase pump units (r = -0.66; P less than 0.0001). Anorectic patients showed lower serum T3 concentrations (71.3 +/- 53 ng/dL) with respect to control subjects (100.8 +/- 4.7 ng/dL; P less than 0.0005) and a significant negative correlation between T3 levels and the number of pump units (r = -0.52; P less than 0.003) was found. Our study therefore shows that the erythrocyte Na-K pump may be altered in several anorectic patients. We suggest that this feature could be interrelated with the degree of underweight and/or malnutrition.     

Measurement of gastric emptying time by real-time ultrasonography

This paper describes an ultrasound method of assessing gastric emptying time based on measurements of the gastric antrum, which is visible in almost all subjects before and after meals. A total of 54 subjects were examined including 18 normal subjects and 36 subjects with idiopathic functional dyspepsia. The emptying time was determined in all subjects by measuring the changes in the cross-sectional area of the gastric antrum. In a subgroup of 34 subjects the volume of the whole antropyloric region was also considered. Measurements were taken by the same observer after fasting and at regular 30-min intervals after a standard 800-cal meal. Final emptying time (calculated in relation to the start of the meal) was considered to be the time at which the antral area or volume returned to basal value. Final emptying time (mean +/- SD) was 248 +/- 39 min in normal subjects and 359 +/- 64 min in patients with functional dyspepsia (p less than 0.001). A significantly higher degree of dilatation of the gastric antrum was found in dyspeptic patients than in control subjects. Barium x-ray of the stomach in 19 subjects always confirmed the ultrasound finding on the presence or absence of contents within the stomach. We conclude that this kind of ultrasound study of the antropyloric region allows accurate determination of total gastric emptying time.