Detection of diarrhetic shellfish poisoning toxins from tropical shellfish using liquid chromatography-selected reaction monitoring mass spectrometry

A negative mode liquid chromatography-selected reaction monitoring mass spectrometry (LC-SRM MS) method was developed to detect low concentrations of the diarrhetic shellfish poisoning (DSP) toxins okadaic acid and dinophysistoxin-1 (DTX-1). Detection relies upon monitoring the transition of negative precursor ions [M - H]- to a common fragment ion of m/z 255. Our limit of detection for okadaic acid with this method is 0.5 pg on column. LC-SRM MS has allowed us to detect persistent, low concentrations of DSP toxins from Singapore shellfish.     

Effects of thalidomide on reproductive function and early embryonic development in male and female New Zealand white rabbits

BACKGROUND : The present work was performed to determine the effect of thalidomide exposure on reproductive function and early embryonic development. METHODS : Twenty‐five female New Zealand White rabbits were orally gavaged with 0, 10, 50, or 100 mg/kg/day thalidomide 14 days prior to mating through to gestation day 7 for a total of 22 days. Treated females were Caesarean‐sectioned approximately 29 days after the date of attempted mating. Following mating with treated females, male rabbits (25/dose) were gavaged with 0, 30, 150, or 500 mg/kg/day beginning 14 days prior to mating with a group of untreated females (25/dose). Doses were administered through mating until the day before sacrifice for a minimum of 56 days. Untreated females were Caesarean‐sectioned 29 days after the last attempted mating. Comprehensive necropsy and histopathology of the reproductive system were performed. RESULTS : Treated females had reduction in body weight gain during gestation. Mating and pregnancy parameters were unaffected by thalidomide. At 100 m/kg, litter averages for corpora lutea, implantations, litter sizes, does with viable fetuses and live fetuses decreased and the number of early resorptions, does with any resorptions, does with all conceptuses resorbed, and the percent resorbed conceptuses per litter increased. The number of early resorptions, the average number of early resorptions per litter, and the percent resorbed conceptuses per litter increased at 10 and 50 mg/kg. There were no thalidomide‐related external fetal malformations. Mating and fertility in male rabbits were unaffected by thalidomide. There was an increased incidence of flaccid testes at 150 and 500 mg/kg and of bilateral small testes in all treated groups. At 500 mg/kg, there was degeneration of the germinal epithelium of the testicles with an increase in multinucleated giant cells in seminiferous tubule and a loss of round and elongating spermatids. CONCLUSIONS : Thalidomide had no adverse effects on mating and fertility in male and female rabbits dosed up to 500 and 100 mg/kg/day, respectively, for 14 days prior to mating. After 56 day of dosing, histopathologic changes with no associated sperm abnormalities were observed in the testicles. Embryonic development NOAEL for treated females mated to untreated males was <10 mg/kg. Corresponding fertility NOAEL for treated males mated to untreated females was 500 mg/kg. Birth Defects Res B 71:1–16, 2004. © 2004 Wiley‐Liss, Inc.     

Metabolism of thalidomide in human microsomes, cloned human cytochrome P-450 isozymes, and Hansen's disease patients

Previous in vitro studies in rat microsomal preparations suggested that thalidomide is metabolized by the cytochrome P450 system (CYP). In this study, we examined the extent of thalidomide metabolism by preparations of pooled human microsomes, microsomes containing cloned human CYP isozymes (CYPIA2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4), and Hansen's disease patients. Results indicated that thalidomide was a poor substrate for CYP isozymes. Alteration of incubation buffer, pH, incubation time, and microsome and thalidomide concentrations did not increase the production of any metabolites. Thalidomide also did not inhibit metabolism of CYP-specific substrates and therefore any interactions with other drugs that are metabolized by the same enzyme system are unlikely. Hansen's patients were given a single oral dose of thalidomide (400 mg), and their blood and urine were collected at time points up to 72 hours, processed, and analyzed by tandem mass spectrometry. Although thalidomide was present in the plasma and urine, no metabolites were found in the plasma and very low amounts of the 5-OH thalidomide metabolite were present in the urine. These results suggest that thalidomide does not undergo significant metabolism by human CYP and that clinically important interactions between thalidomide and drugs that are also metabolized by this enzyme system are unlikely. The major route of thalidomide breakdown in humans and animals is through spontaneous hydrolysis with subsequent elimination in the urine.     

OsGL1-3 is Involved in Cuticular Wax Biosynthesis and Tolerance to Water Deficit in Rice

Cuticular wax covers aerial organs of plants and functions as the outermost barrier against non-stomatal water loss. We reported here the functional characterization of the Glossy1(GL1)-homologous gene OsGL1-3 in rice using overexpression and RNAi transgenic rice plants. OsGL1-3 gene was ubiquitously expressed at different level in rice plants except root and its expression was up-regulated under ABA and PEG treatments. The transient expression of OsGL1-3–GFP fusion protein indicated that OsGL1-3 is mainly localized in the plasma membrane. Compared to the wild type, overexpression rice plants exhibited stunted growth, more wax crystallization on leaf surface, and significantly increased total cuticular wax load due to the prominent changes of C₃₀–C₃₂ aldehydes and C₃₀ primary alcohols. While the RNAi knockdown mutant of OsGL1-3 exhibited no significant difference in plant height, but less wax crystallization and decreased total cuticular wax accumulation on leaf surface. All these evidences, together with the effects of OsGL1-3 on the expression of some wax synthesis related genes, suggest that OsGL1-3 is involved in cuticular wax biosynthesis. Overexpression of OsGL1-3 decreased chlorophyll leaching and water loss rate whereas increased tolerance to water deficit at both seedling and late-tillering stages, suggesting an important role of OsGL1-3 in drought tolerance.     

Invariant NKT Cells Act as an Adjuvant to Enhance Th2 Inflammatory Response in an OVA-Induced Mouse Model of Asthma

Background

Invariant natural killer T cells (iNKT cells) are a unique subset of T lymphocytes and are considered to play an important role in the development of allergic bronchial asthma. Recently, iNKT cells were shown to play an immunoregulatory role in CD4⁺ and CD8⁺ T cell-mediated adaptive immune response. Allergen-specific Th2 inflammatory responses are an important part of the adaptive immune response in asthma. However, the regulatory functions of the Th2 inflammatory response in asthma have not been studied in detail.

Method

In this study, we have investigated the regulatory functions of iNKT cells on the Th2 inflammatory response in an ovalbumin (OVA)-induced murine model of asthma.

Results

Our results demonstrate that α-Galactosylceramide (α-GalCer) administration activated iNKT cells but could not induce the Th2 inflammatory response in wild-type (WT) mice. In the OVA-induced asthma model, α-GalCer administration and adoptive transfer of iNKT cells significantly augmented the Th2 inflammatory responses, including elevated inflammatory cell infiltration in the lung and bronchoalveolar lavage fluid (BALF); increased levels of IL-4, IL-5, and IL-13 in the BALF and splenocyte culture supernatant; and increased serum levels of OVA-specific IgE and IgG1. In addition, the Th2 inflammatory response was reduced, but not completely abrogated in CD1d^-/- mice immunized and challenged with OVA, compared with WT mice.

Conclusion

These results suggest that iNKT cells may serve as an adjuvant to enhance Th2 inflammatory response in an OVA-induced murine model of asthma.     

Retinal Microvascular Abnormalities and Risk of Renal Failure in Asian Populations

BackgroundRetinal microvascular signs may provide insights into the structure and function of small vessels that are associated with renal disease. We examined the relationship of retinal microvascular signs with both prevalent and incident end-stage renal disease (ESRD) in a multi-ethnic Asian population.MethodsA total of 5763 subjects (aged ≥40 years) from two prospective population-based studies (the Singapore Malay Eye Study and the Singapore Prospective Study) were included for the current analysis. Retinopathy was graded using the modified Airlie House classification system. Retinal vascular parameters were measured using computer-assisted programs to quantify the retinal vessel widths (arteriolar and venular caliber) and retinal vascular network (fractal dimension). Data on ESRD was obtained by record linkage with the ESRD cases registered by National Registry of Diseases Office, Singapore. Multi-variable adjusted regression analyses were performed to assess the associations of baseline retinal vascular parameters and prevalent and incident ESRD.ResultsAt baseline, 21(0.36%) persons had prevalent ESRD. During a median follow-up of 4.3 years, 33 (0.57%) subjects developed ESRD. In our analyses, retinopathy was associated with prevalent ESRD (multi-variable adjusted odds ratio [OR], 3.21, 95% confidence interval [CI]: 1.28–8.05) and incident ESRD (multi-variable adjusted hazard ratio [HR], 2.51, 95%CI: 1.14–5.54). This association was largely seen in person with diabetes (HR, 2.60, 95%CI: 1.01–6.66) and not present in persons without diabetes (HR, 1.65, 95%CI: 0.14–18.98). Retinal arteriolar caliber, retinal venular caliber and retinal vascular fractal dimension were not associated with ESRD.ConclusionRetinopathy signs in persons with diabetes are related to an increased risk of ESRD; however, other microvascular changes in the retina are not associated with ESRD.     

Using an Adenosine Triphosphate Bioluminescent Assay to Determine Effective Antibiotic Combinations against Carbapenem-Resistant Gram Negative Bacteria within 24 Hours

Background

Current in vitro combination testing methods involve enumeration by bacterial plating, which is labor-intensive and time-consuming. Measurement of bioluminescence, released when bacterial adenosine triphosphate binds to firefly luciferin-luciferase, has been proposed as a surrogate for bacterial counts. We developed an ATP bioluminescent combination testing assay with a rapid turnaround time of 24h to determine effective antibiotic combinations.

Methods

100 strains of carbapenem-resistant (CR) GNB [30 Acinetobacter baumannii (AB), 30 Pseudomonas aeruginosa (PA) and 40 Klebsiella pneumoniae (KP)] were used. Bacterial suspensions (10⁵ CFU/ml) were added to 96-well plates containing clinically achievable concentrations of multiple single and two-antibiotic combinations. At 24h, the luminescence intensity of each well was measured. Receiver operator characteristic curves were plotted to determine optimal luminescence threshold (T_RLU) to discriminate between inhibitory/non-inhibitory combinations when compared to viable plating. The unweighted accuracy (UA) [(sensitivity + specificity)/2] of T_RLU values was determined. External validation was further done using 50 additional CR-GNB.

Results

Predictive accuracies of T_RLU were high for when all antibiotic combinations and species were collectively analyzed (T_RLU = 0.81, UA = 89%). When individual thresholds for each species were determined, UA remained high. Predictive accuracy was highest for KP (T_RLU = 0.81, UA = 91%), and lowest for AB (T_RLU = 0.83, UA = 87%). Upon external validation, high overall accuracy (91%) was observed. The assay distinguished inhibitory/non-inhibitory combinations with UA of 80%, 94% and 93% for AB, PA and KP respectively.

Conclusion

We developed an assay that is robust at identifying useful combinations with a rapid turn-around time of 24h, and may be employed to guide the timely selection of effective antibiotic combinations.