[Determination of 18 caine anesthetics in animal meat using solid phase extraction combined with ultra-performance liquid chromatography-tandem mass spectrometry].

Because of the widespread application of anesthetic drugs in the fields of animal breeding and transportation, demand for the rapid, sensitive detection of anesthetic drugs in animal meat is increasing. The complex animal meat matrix contains various interfering substances, such as proteins, fats, and phospholipids, along with anesthetic drug residues at very low concentrations. Therefore, adopting appropriate pretreatment methods is necessary to improve the sensitivity of detection. In this study, a rapid, accurate analytical method based on ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and solid phase extraction (SPE) was established to determine the contents of 18 caines in animal meat. The MS parameters, such as the collision energies of 18 caines, were optimized. Furthermore, the chromatographic separation conditions and response intensities of the caine in different mobile phases were compared. The effects of different pretreatment conditions on the extraction efficiencies of the 18 caines in meat samples and those of different purification conditions, such as extraction solvent, SPE column, and dimethylsulfoxide (DMSO) dosage, on their recoveries were investigated. Combined with the external standard method, the 18 caines in meat were successfully quantified. Sample pretreatment is a three-step process. First, in ultrasound-assisted extraction, 2.0 g samples were added to 2.0 mL water and extracted using 10 mL 0.1% (v/v) formic acid in acetonitrile under ultrasound conditions for 10 min. SPE was then performed using an Oasis PRIME HLB column. Finally, DMSO-assisted concentration was employed: the organic layer was collected and dried at 40 ℃ under a stream of N2 gas with the addition of 100 µL DMSO. Acetonitrile-water (1∶9, v/v) was added to the residue to yield a final volume of 1.0 mL for use in UPLC-MS/MS. The 18 caines were separated using an HSS T3 (100 mm×2.1 mm, 1.8 µm) column with 0.1% (v/v) formic acid in water (containing 0.02 mmol/L ammonium acetate) and methanol as mobile phases. Samples were detected using an electrospray ion source (ESI) in the positive ion and multiple reaction monitoring (MRM) modes during UPLC-MS/MS. Under the optimized conditions, the 18 target caine anesthetics displayed good linearities in the range of 1.00-50.0 µg/L, and the correlation coefficients (R2) were >0.999. The respective limits of detection (LODs) and quantification (LOQs) were 0.2-0.5 µg/kg, and 0.6-1.5 µg/kg. In pork, beef, and mutton samples, the recoveries obtained at three spiked levels were 83.4%-100.4% with relative standard deviations (RSDs) of 3.1%-8.5%. This simple, rapid, sensitive method may be applied in the detection of 18 caine anesthetics in animal meat and may provide technical support to the food safety department in China in monitoring the residues of caine anesthetics in animal meat.

Automated Bright Field Segmentation of Cells and Vacuoles Using Image Processing Technique.

Understanding the mechanisms and other variants of programmed cell death will help provide deeper insight into various disease processes. Although complex procedures are required to distinguish each type of cell death, the formation of vacuoles is one of the important features in some process of cell death under different conditions. Thus, monitoring and counting the number of vacuoles and the ratio of cells with vacuoles is a commonly used method to indicate and quantify the efficacy of the therapy. Several studies have shown that image processing can provide a quick, convenient and precise mean of performing cell detection. Hence, this study uses an image processing technique to detect and quantify vacuolated cells without the need for dyes. The system both counts the number of vacuolated cells and determines the ratio of cells with vacuoles. The performance of the proposed image processing system was evaluated using 38 images. It has been shown that a strong correlation exists between the automated counts and the manual counts. Furthermore, the absolute percentage errors between automated counts and manual counts for cell detection and vacuolated cell detection using data pooled from all images are 3.61 and 3.33%, respectively. A user-friendly graphical user interface (GUI) is also developed and freely available for download, providing researchers in biomedicine with a more convenient instrument for vacuolization analysis.

Developmental staging of male murine embryonic gonad by SAGE analysis.

Despite the identification of key genes such as Sry integral to embryonic gonadal development, the genomic classification and identification of chromosomal activation of this process is still poorly understood. To better understand the genetic regulation of gonadal development, we performed Serial Analysis of Gene Expression (SAGE) to profile the genes and novel transcripts, and an average of 152,000 tags from male embryonic gonads at E10.5 (embryonic day 10.5), E11.5, E12.5, E13.5, E15.5 and E17.5 were analyzed. A total of 275,583 non-singleton tags that do not map to any annotated sequence were identified in the six gonad libraries, and 47,255 tags were mapped to 24,975 annotated sequences, among which 987 sequences were uncharacterized. Utilizing an unsupervised pattern identification technique, we established molecular staging of male gonadal development. Rather than providing a static descriptive analysis, we developed algorithms to cluster the SAGE data and assign SAGE tags to a corresponding chromosomal position; these data are displayed in chromosome graphic format. A prominent increase in global genomic activity from E10.5 to E17.5 was observed. Important chromosomal regions related to the developmental processes were identified and validated based on established mouse models with developmental disorders. These regions may represent markers for early diagnosis for disorders of male gonad development as well as potential treatment targets.

Variable presentation of precocious puberty associated with the D564G mutation of the LHCGR gene in children with testotoxicosis.

We report on a family with familial male-limited precocious puberty (FMPP) due to a D564G mutation of the LHCGR gene. Family members show a varied phenotypic expression from severe precocity unresponsive to therapy with compromise of the predicted final height in some members, to attainment of tall final stature in other members who never received medical treatment. DNA amplification and sequencing of exon 11 of the LHCGR gene was done for the three affected male members and their mother. DNA analysis revealed a D564G mutation in the third cytoplasmic loop of the LHCGR receptor. All three males had precocious puberty with elevated testosterone levels. The index case developed central precocious puberty and evidence of compromised final height while on therapy. In contrast, the untreated older siblings attained a tall final height. This report underscores the possibility that the effects of the mutant luteinizing hormone/choriogonadotropin receptor on phenotypic expression of FMPP, such as adult final height, are modified by other factors.

The complexity of antisense transcription revealed by the study of developing male germ cells.

Computational analyses have identified the widespread occurrence of antisense transcripts in the human and the mouse genome. However, the structure and the origin of the majority of the antisense transcripts are unknown. The presence of antisense transcripts for 19 of 64 differentially expressed genes during mouse spermatogenesis was demonstrated with orientation-specific RT-PCR. These antisense transcripts were derived from a wide variety of origins, including processed sense transcripts, intronic and exonic sequences of a single gene or multiple genes, intergenic sequences, and pseudogenes. They underwent normal and alternative splicing, 5' capping, and 3' polyadenylation, similar to the sense transcripts. There were also antisense transcripts that were not capped and/or polyadenylated. The testicular levels of the sense transcripts were higher than those of the antisense transcripts in all cases, while the relative expression in nontesticular tissues was variable. Thus antisense transcripts have complex origins and structures and the sense and antisense transcripts can be regulated independently.

Transcriptome analyses of male germ cells with serial analysis of gene expression (SAGE).

Serial analysis of gene expression (SAGE) provides an alternative with additional advantages to microarrays for studying gene expression during spermatogenesis. The digitized transcriptome provided by SAGE of purified mouse germ cells identified 27,504 species of transcripts expressed in type A spermatogonia, pachytene spermatocytes, and round spermatids. Over 2700 of these transcripts were novel. Computational analyses allowed the identification of clusters of co-regulated genes, cell-specific promoter modules, cell-specific biological processes, as well as "preferential" biological networks in different cell types. These analyses provided potential drug targets for interference of specific pathways at different stages of spermatogenesis. Analyses of the transcriptomes revealed the prominent role of cytochrome c oxidase in germ cells and suggest a novel role for this enzyme in cytochrome c-mediated apoptosis in spermatogonia. A number of genes were shown to undergo differential splicing during spermatogenesis giving rise to cell-specific splice variants.

Analysis of mouse germ-cell transcriptome at different stages of spermatogenesis by SAGE: biological significance.

The transcriptomes of mouse type A spermatogonia (Spga), pachytene spermatocytes (Spcy), and round spermatids (Sptd) were determined by sequencing the respective SAGE (Serial Analysis of Gene Expression) libraries. A total of 444,015 tags derived from one Spga, two Spcy, and one Sptd library were analyzed, and 34,619 different species of transcripts were identified, 5279 of which were novel. Results indicated the germ-cell transcriptome comprises of more than 30,000 transcripts. Virtual subtraction showed that cell-specific transcripts constitute 12-19.5% of the transcriptome. Components of the protein biosynthetic machinery are highly expressed in Spga. In Spcy transcription factors are abundantly expressed while transcripts encoding proteins involved in chromosome remodeling and testis-specific transcripts are prominent in Sptd. The databases generated by this work provide very useful resources for cellular localization of genes in silico. They are also extremely useful as sources for identification of splice variants of genes in germ cells.

A novel missense homozygous inactivating mutation in the fourth transmembrane helix of the luteinizing hormone receptor in leydig cell hypoplasia.

Loss-of-function mutations/inactivating mutations of the human chorionic gonadotropin/luteinizing hormone receptor (hCG/LHR), a G-protein coupled receptor, lead to impaired Leydig cell differentiation. Leydig cell hypoplasia/agenesis/dysplasia (LCH) is one of the causes of male pseudohermaphroditism (MPH). We studied a 19-year-old MPH patient with female phenotype and 46,XY karyotype. Testicular histology and hormonal profile of the patient is typical of LCH. Nucleotide sequencing of exon 11 of hLHR identified a novel T1505C transversion mutation. The mutation is homozygous in the patient and is heterozygous in both parents. The single base mutation caused the substitution of a conserved leucine at 502 position to proline in transmembrane helix (TM) IV of the hLHR. This is the first LCH causing mutation identified in TM IV of the hLHR. Expression study of the mutated hLHR in human embryonic kidney (HEK)293 cells showed reduced cAMP production and ligand binding. Receptor trafficking was not affected by the mutation when the green fluorescence protein conjugated mutated receptor was expressed in HEK293 cells. The mutation caused inactivation of the hLHR and resulted in LCH in the patient.