Low-level quantification of melamine and cyanuric acid in limited samples of rat serum by UPLC-electrospray tandem mass spectrometry

This paper reports the development and validation of a methodology for the low-level quantification of melamine and cyanuric acid in limited samples of rat serum. The methodology, based upon ion-exchange solid phase extraction (SPE) and ultra-performance liquid chromatography (UPLC) coupled with electrospray tandem mass spectrometry (MS/MS) in multiple reaction monitoring (MRM) mode, relies on the use of stable isotope-labeled internal standards and requires only 15 μL samples of serum. The method provides a recovery of 80-110% of melamine with a signal suppression of ca. 55%, and a recovery of 50-90% of cyanuric acid with a signal suppression ca. 40-60%, affording lower limits of quantification (LLOQ) for melamine or cyanuric acid of, respectively, 5 ppb (mean accuracy 109%; CV=4.9%) and 10 ppb (mean accuracy 96%; CV=8.6%). The small sample requirements, excellent sensitivity, accuracy and precision, and high-throughput (5 min of instrument run time) make this methodology optimal for toxicokinetic or exposure assessments studies.     

High sero-prevalence of caseous lymphadenitis identified in slaughterhouse samples as a consequence of deficiencies in sheep farm management in the state of Minas Gerais, Brazil

Background

Multiple congenital ocular anomalies (MCOA) syndrome is a hereditary congenital eye defect that was first described in Silver colored Rocky Mountain horses. The mutation causing this disease is located within a defined chromosomal interval, which also contains the gene and mutation that is associated with the Silver coat color (PMEL17, exon 11). Horses that are homozygous for the disease-causing allele have multiple defects (MCOA-phenotype), whilst the heterozygous horses predominantly have cysts of the iris, ciliary body or retina (Cyst-phenotype). It has been argued that these ocular defects are caused by a recent mutation that is restricted to horses that are related to the Rocky Mountain Horse breed. For that reason we have examined another horse breed, the Icelandic horse, which is historically quite divergent from Rocky Mountain horses.

Results

We examined 24 Icelandic horses and established that the MCOA syndrome is present in this breed. Four of these horses were categorised as having the MCOA-phenotype and were genotyped as being homozygous for the PMEL17 mutation. The most common clinical signs included megaloglobus, iris stromal hypoplasia, abnormal pectinate ligaments, iridociliary cysts occasionally extending into the peripheral retina and cataracts. The cysts and pectinate ligament abnormalities were observed in the temporal quadrant of the eyes. Fourteen horses were heterozygous for the PMEL17 mutation and were characterized as having the Cyst-phenotype with cysts and occasionally curvilinear streaks in the peripheral retina. Three additional horses were genotyped as PMEL17 heterozygotes, but in these horses we were unable to detect cysts or other forms of anomalies. One eye of a severely vision-impaired 18 month-old stallion, homozygous for the PMEL17 mutation was examined by light microscopy. Redundant duplication of non-pigmented ciliary body epithelium, sometimes forming cysts bulging into the posterior chamber and localized areas of atrophy in the peripheral retina were seen.

Conclusions

The MCOA syndrome is segregating with the PMEL17 mutation in the Icelandic Horse population. This needs to be taken into consideration in breeding decisions and highlights the fact that MCOA syndrome is present in a breed that are more ancient and not closely related to the Rocky Mountain Horse breed.     

Diversity of mesophilic clostridia in Costa Rican soils

Costa Rica is located in the Tropic, one of the most biologically diverse regions of the world; its soil is an important epicenter of biodiversity and Clostridium spp. are among the most frequent bacteria. The diversity of clostridia in Costa Rican soils and its possible association with geographic zone, pH or type of soil was studied in 117 soil samples: 18 from the Atlantic Zone, 30 from the Central Plateau, 30 from the Dry Pacific, 13 from the North Zone, and 26 from the South Pacific. The pH and the mesophilic clostridia species were determined for each sample. For bacterial isolation, a selective methodology for spores and pre-reduced anaerobically sterilized media were used. A total of 1945 strains of clostridia were isolated, 98% were identified and corresponded to 54 species; the most frequent species were C. subterminale (56%), C. oceanicum (51%), C. bifermentans and C. glycolicum (50%, each), C. sporogenes (49%), and C. sordellii (42%). An average of 7.1 species per sample was obtained; the Atlantic Zone showed the greatest diversity: 8.6 species per sample and a total of 45 species. Except for C. chauvoei, all described toxigenic clostridia species were isolated; C. sordellii (42%) and C. perfringens (38%) were the most frequent. No statistical relation could be established between geographic zone or type of soil and any species, showing that clostridia had a high adaptation capability to grow in different soil conditions; only some clostridia were isolated from very acidic samples while others from soils with a wide range of pH. In general, a uniform distribution of most species and a high variety of clostridia in Costa Rican soils were observed, in agreement with the high biodiversity described for other living beings in this country.     

Nucleotidic variations of two captive groups of tepezcuintle, Agouti paca (Rodentia: Agoutidae), from two sites in Yucatan, Mexico

The objective of this work was to estimate the nucleotidic variation between two groups of tepezcuintles (Agouti paca) from the states of Campeche and Quintana Roo, Mexico and within members of each group. Blood samples were collected from eleven A. paca kept in captivity. DNA from leukocytic cells was used for Ramdom Amplification of DNA Polimorphism (RAPD). The primers three 5'-d(GTAGACCCGT)- 3' and six 5'-d(CCCGTCAGCA)- 3' were selected from de Amersham kit (Ready.To.Go. RAPD Analysis Beads, Amersham Pharmacia Biotech), because they produced an adequate number of bands. The electrophoretic pattern of bands obtained was analyzed using software for phylogenetic analysis based on the UPGMA method, to estimate the units of nucleotidic variation. The phylogenetic tree obtained with primer three reveals a dicotomic grouping between the animals from both states in the Yucatan Peninsula showing a divergent value of 1.983 nucleotides per hundred. Animals from Quintana Roo show a grouping with primer six; an additional grouping was observed with animals from Campeche. Nucleotidic variation between both groups was 2.118 nucleotides per hundred. The nucleotidic variation for the two primers within the groups from both states, showed fluctuating values from 0.46 to 1.68 nucleotides per hundred, which indicates that nucleotidic variation between the two groups of animals is around two nucleotides per hundred and, within the groups, less than 1.7 nucleotides per hundred.     

A method for obtaining Schwann cell cultures from adult rabbit nerve based on "in vitro" pre-degeneration and neuregulin treatment

Schwann cells (SCs) are basic elements for cell therapy and tissue engineering in the central and peripheral nervous system. Therefore, the development of a reliable method to obtain SC cultures is required. For possible therapeutic applications the cultures need to produce a sufficiently large number of SCs with a high level of purity in a relatively short period of time. To increase SC yield and purity we pre-degenerated pieces of 1-2 mm of adult rabbit sciatic nerves by incubating them for seven days in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal bovine serum, penicillin/streptomycin and NRG1-β1. Following pre-degeneration the nerve pieces were dissociated and then cultured for 6 or 15 days in the same culture medium. After 6 days of culture we obtained around 9.5x103 cells/mg with approximately 94% SCs (S-100 positive) purity. After 15 days of culture the yield was about 80x103 cells/mg and the purity was approximately 75%. Pre-degeneration and subsequent culture of small pieces of adult nerve with NRG1-β1 supplemented medium increased the number of SCs and restricted the overgrowth of fibroblast-like cells.     

Graph Based Study of Allergen Cross-Reactivity of Plant Lipid Transfer Proteins (LTPs) Using Microarray in a Multicenter Study

The study of cross-reactivity in allergy is key to both understanding. the allergic response of many patients and providing them with a rational treatment In the present study, protein microarrays and a co-sensitization graph approach were used in conjunction with an allergen microarray immunoassay. This enabled us to include a wide number of proteins and a large number of patients, and to study sensitization profiles among members of the LTP family. Fourteen LTPs from the most frequent plant food-induced allergies in the geographical area studied were printed into a microarray specifically designed for this research. 212 patients with fruit allergy and 117 food-tolerant pollen allergic subjects were recruited from seven regions of Spain with different pollen profiles, and their sera were tested with allergen microarray. This approach has proven itself to be a good tool to study cross-reactivity between members of LTP family, and could become a useful strategy to analyze other families of allergens.     

Optimization of antimalarial,and anticancer activities of (E)-methyl 2-(7-chloroquinolin-4-ylthio)-3-(4-hydroxyphenyl) acrylate

Chemically modified versions of bioactive substances, are particularly useful in overcoming barriers associated with drug formulation, drug delivery and poor pharmacokinetic properties. In this study, a series of fourteen (E)-methyl 2-(7-chloroquinolin-4-ylthio)-3-(4-hydroxyphenyl) acrylate (2–15) were prepared by using a one step synthesis from 1 previously described by us as potential antimalarial and antitumor agent. Molecules were evaluated as inhibitors of β-hematin formation, where most of them showed a significant inhibition value (%?>?70). The best inhibitors were tested in vivo as potential antimalarials in mice infected with P. berghei ANKA, chloroquine susceptible strain. Three of them (5, 6, and 15) displayed antimalarial activity comparable to that of chloroquine. Also, molecules were evaluated for their cytotoxic activity against two human cancer cell lines (Jurkat E6.1 and HL60) and primary culture of human lymphocytes. Most of the synthesized compounds, except for analogs 2–6, 8, and 10–12, displayed cytotoxicity against cancer cell lines without affecting normal cells. The potency of the compounds was 15???1, and 14?>?7, 9, and 13. Flow cytometry analysis demonstrated an increase in apoptotic cell death after 24?h. The compounds may affect tumor cell autophagy and consequently increase cell apoptosis.     

Comparison of the metabolic activities of four wild-type <Emphasis Type="Italic">Clostridium perfringens</Emphasis> strains with their gatifloxacin-selected resistant mutants

The production of short-chain fatty acids, reductive enzymes, and hydrolytic enzymes by four gatifloxacin-selected, fluoroquinolone-resistant, mutant strains of C. perfringens, with stable mutations either in DNA gyrase or in both DNA gyrase and topoisomerase IV, was compared with that produced by the wild-type parent strains to investigate the effect of mutations associated with the selection of gatifloxacin resistance on bacterial metabolic activities. The mutants differed from their respective wild-type parent strains in the enzymatic activities of azoreductase, nitroreductase, and β-glucosidase and in the ratio of butyric acid to acetic acid production. Microarray analysis of one wild type and the corresponding mutant revealed different levels of mRNA expression for the enzymes involved in short-chain fatty acid (SCFA) synthesis and for β-glucosidase and oxidoreductases. In addition to mutations in the target genes, selection of resistance to gatifloxacin resulted in strain-specific physiological changes in the resistant mutants of C. perfringens that affected their metabolic activities.