[Internal quality control products for computer-assisted sperm analysis: Research and application].

OBJECTIVE: To investigate the application of the self-made semen quality control (QC) product in internal QC of computer-assisted sperm analysis (CASA). METHODS: CASA was calibrated with high- and low-concentration commercially available semen QC product and meanwhile 15 samples of self-made mixed semen QC product were placed in 75 cryotubes containing liquid nitrogen, followed by CASA of the concentration, motility, curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), linearity (LIN), wobble (WOB) and straightness (STR) of the sperm using standard procedures and 50 days of continuous monitoring. The Makler counting plate was used to measure the concentration and motility of the self-made sperm. RESULTS: The coefficients of variation (CV) of the commercially available semen QC product at high and low concentrations were 6.18% and 7.85%, respectively. CASA showed that the concentration of the self-made QC product was (25.97 ± 1.41) ×106/ml, with a CV of 5.42%, and the sperm motility, VCL, VSL, VAP, LIN, WOB and STR were (22.15 ± 1.75)% (CV = 7.9%), (59.18 ± 2.05) µm/s (CV = 3.46%), (26.79 ± 1.2) µm/s (CV = 4.48%), (34.98 ± 1.4) µm/s (CV = 4.01%), 46.81 ± 1.55 (CV = 3.3%), 60.52 ± 1.3 (CV = 2.15%) and 76.46 ± 1.98 (CV = 2.59%), respectively. The concentration and motility of the self-made sperm detected with the Makler counting plate were (34.39 ± 2.37) ×106/ml (CV = 6.89%) and (38.04 ± 1.69)% (CV = 4.44%), respectively. Levey-Jennings QC charts were plotted for the indicators using the means and standard deviation. CONCLUSIONS: The self-made internal QC product by liquid nitrogen cryopreservation is feasible and effective for monitoring the accuracy and precision of CASA-derived sperm concentration and motion parameters, and it has a smaller CV than the commercially available QC product in measuring sperm concentration.

[Recognition of water-injected meat based on visible/near-infrared spectrum and sparse representation].

The present paper proposed a new nondestructive method based on visible/near infrared spectrum (Vis/NIRS) and sparse representation to rapidly and accurately discriminate between raw meat and water-injected meat. Water-injected meat model was built by injecting water into non-destructed meat samples comprising pigskin, fat layer and muscle layer. Vis/NIRS data were collected from raw meat and six scales of water-injected meat with spectrometers. To reduce the redundant information in the spectrum and improve the difference between the samples,. some preprocessing steps were performed for the spectral data, including light modulation and normalization. Effective spectral bands were extracted from the preprocessed spectral data. The meat samples were classified as raw meat and water-injected meat, and further, water-injected meat with different water injection rates. All the training samples were used to compose an atom dictionary, and test samples were represented by the sparsest linear combinations of these atoms via l1-minimization. Projection errors of test samples with respect to each category were calculated. A test sample was classified to the category with the minimum projection error, and leave-one-out cross-validation was conducted. The recognition performance from sparse representation was compared with that from support vector machine (SVM).. Experimental results showed that the overall recognition accuracy of sparse representation for raw meat and water-injected meat was more than 90%, which was higher than that of SVM. For water-injected meat samples with different water injection rates, the recognition accuracy presented a positive correlation with the water injection rate difference. Spare representation-based classifier eliminates the need for the training and feature extraction steps required by conventional pattern recognition models, and is suitable for processing data of high dimensionality and small sample size. Furthermore, it has a low computational cost. In this paper, spare representation is employed for the first time to identify water-injected meat based on Vis/NIRS, with a promising recognition accuracy. The experimental results demonstrate that the proposed method can be effectively used for discriminating water-injected meat from raw meat.

Analysis of 2,297 expressed sequence tags (ESTs) from a cDNA library of flax (Linum ustitatissimum L.) bark tissue.

Bast fibre crops are the second most important natural fibre crops following cotton. Of these, flax (Linum ustitatissimum L.) is the most widely planted in the world, with its fibre used for high quality linen textile. A cDNA library of flax bark tissues was constructed with the purpose of identifying genes involved in the Bast fibre development. A total of 2,297 unigene sequences were obtained from 3,200 randomly selected clones of the cDNA library. These sequences were grouped into 155 clusters and 2,142 singletons, which have been submitted to the GenBank databases. By putative functional annotation, 23.3% of these sequences were similar to known proteins in GenBank, 44.0% of these sequences were similar to unknown proteins, and 32.7% of these sequences showed no significant similarity to any other protein sequences in existing databases. Classified by the Gene Ontology, 24.8, 23.1 and 14.3% were assigned to molecular function, biological process, and cellular component GO terms, respectively. By further bioinformatics approaches, about 110 ESTs matched cell wall related genes in the MAIZEWALL database, representing 16 functional categories of all 19 categories, of which, the most abundant category was protein synthesis. Based on the PlantTFDB database, 39 of the 64 transcription factor families in the Arabidopsis thaliana genome were identified as being involved in flax cell wall formation. The sequences and bioinformatics analysis data generated in this paper will be useful for gene expression, cloning and genetic engineering studies to characterize bast fibre development and improve the properties of the bast fibres.

Gene expression patterns in gastric cancer.

OBJECTIVE: To identify gene expression patterns in distinct stages of intestinal-type gastric cancer(GC). METHODS: Gene expression patterns of distinct stages of intestinal-type GC samples from 3 patients were compared with cDNA microarray, which contained 576 genes. There were 506 target genes, which included 51 genes identified from our previous experiment with suppression subtractive hybridization(SSH) and other 455 genes chosen for their important roles in cancers. Hierarchical clustering was performed to clarify genes in association with distinct stages of GC. RESULTS: One hundred and eighty-one differentially expressed genes with average Cy5:Cy3 ratios higher than 2.0 or lower than 0.5 in at least one stage of GC were identified by cDNA microarray. Among them, 48 genes were up-regulated and 133 down-regulated. Hierarchical clustering analysis separated the differentially expressed genes in different stages of GC into 5 main characteristic groups. Some important differentially expressed genes in different stages of GC were identified, such as SEC23IP, LIPF, ES(BQ291520), SLC5A1, PG(encoding similar to pepsin A precursor), CXCR4, DICER1, SH3GL2, and IGF2R. CONCLUSION: The differentially expressed gene patterns and some important genes were identified, which might be useful in further study on carcinogenesis, progression and metastasis of intestinal-type GC.

[Screening and analysis of associated genes in the carcinogenesis and progression of gastric cancer].

OBJECTIVE: To screen and analyze the important associated genes in different stages of gastric cancer. METHODS: Using suppression subtractive hybridization (SSH) to screen differentially expressed genes; detecting the expression of genes in different stages of gastric cancer with dot blot hybridization; and verifying the results with semi-quantitative reverse transcriptase-polymerase chain reaction(RT-PCR). RESULTS: Twenty-six differentially expressed gene fragments were obtained by means of SSH. Among them,24 were known genes, 1 was a new expressed sequence tags(EST), and 1 was a hypothetical gene. The results of dot blot hybridization demonstrated that the expressions of Annexin A2, RPS29, RPS12 etc. in dysplasia were higher than those in normal mucosa; the expressions of RPS12 etc.in early cancer were higher than those in normal mucosa;the expressions of cytochromosome C oxidase II, ferritin light chain, RPS12 etc. in advanced gastric cancer and lymph node metastases were consistently higher than those in normal mucosa. The expression of proteasome 26S subunit gene in advanced gastric cancer was higher than that in normal mucosa. The expression of RPS12 was consistently higher in different stages of gastric cancer. It was demonstrated by RT-PCR that the expression of RPS12 in gastric cancer was higher than that in normal mucosa. CONCLUSION: The authors have identified some important genes that might be involved in the carcinogenesis and progression of gastric cancer, and RPS12 may play more important roles in gastric cancer.

Expression of MTLC gene in gastric carcinoma.

AIM: To investigate the expression of c-myc target from laryngeal cancer cells (MTLC) gene in gastric carcinoma (GC) tissues and the effect of MTLC over-expression on gastric carcinoma cell line BGC823. METHODS: RT-PCR was performed to determine the expression of MTLC mRNA in GC and matched control tissues. BGC823 cells were transfected with an expression vector pcDNA3.1-MTLC by liposome and screened by G418. Growth of cells expressing MTLC was observed daily by manual counting. Apoptotic cells were determined by TdT-mediated dUTP nick-end labeling (TUNEL) assay. RESULTS: The expression of MTLC mRNAs was down-regulated in 9(60%) of 15 cases of GC tissues. The growth rates of the BGC823 cells expressing MTLC were indistinguishable from that of control cells. A marked acceleration of apoptosis was observed in MTLC-expressing cells. CONCLUSION: MTLC was down-regulated in the majority of GC tissues and could promote apoptosis of GC cell lines, which suggests that MTLC may play an important role in the carcinogenesis of gastric carcinoma.

[Studies of promoter methylation status and protein expression of E-cadherin gene in associated progression stages of gastric cancer].

Gastric cancer, like all cancers, is considered to result in part from the accumulation of multiple genetic alterations leading to oncogene overexpression and tumour suppressor loss. More recently, the role of epigenetic change as a distinct and crucial mechanism to silence a variety of methylated tissue-specific and imprinted genes has emerged in many cancer types. The study of DNA methylation changes in gastric cancer has now provided additional clues into the pathogenesis of the disease. E-cadherin as a metastases suppressor is mutationally inactivated in both familial and sporadic forms of gastric cancers. Evidence now suggests that the transcriptional silencing of E-cadherin gene by promotor methylation plays a crucial role in the development and progression of gastric malignancies. In order to further analyze the role of E-cadherin gene promotor methylation in gastric carcinogenesis and progression, we performed the studies of promoter methylation status and protein expression of E-cadherin gene in associated progression stages of gastric cancer. DNA were extracted from the paraffin embedded gastric specimens of dysplasia(23 cases), early cancer (20 cases) and advanced cancer (20 cases). Methylation specific PCR and immunohistochemistry were used to analyze the promoter methylation status and the protein expression level of E-cadherin gene. Our results showed that E-cadherin promoter methylation occurred in all stages of gastric precancerous lesion and carcinogenesis, which suggests E-cadherin promotor methylation is an important event during gastric carcinogenesis and progression. The positive rate of E-cadherin promotor methylation in dysplasia, early gastric cancer and advanced gastric cancer was 78.3%, 80% and 90% respectively. There were significant differences between experimental groups and control group(30%), P < 0.05, but no significant differences among experimental groups, P > 0.05. All of advanced gastric cancer examined were completely E-cadherin protein-negative by immunohistochemistry. Fourteen of 20 early gastric cancer were E-cadherin-negative. And 23 dysplasia were all E-cadherin-positive. Thirty-one of 34(91%) of the E-cadherin-negative tumours had promotor methylation. This result indicated the downregulation expression of E-cadherin was associated with promotor methylation in early and advanced gastric cancer (P < 0.01).

[Protein splicing and its application in protein engineering].

Protein splicing, which is an intein mediated posttranslational processing, involves a series of intramolecular cleavage-ligation reactions. Intein is an intervening polypeptide which can catalytic self-cleavage from a pre-protein accompanied by the concomitant joining of the two flanking polypeptides (the extein) through a peptide bond. Protein splicing not only enriches genetic theory of posttranslational processing, but also have wide application prospect.

[The I1307K mutation and protein expression of APC gene in gastric cancer].

In order to explore the correlation of the abnormalities of tumor suppressor gene APC with the carcinogenesis and progression of gastric cancer. The I1307K mutation of APC gene in gastric cancer was analysed using Amplification Refractory Mutation System PCR(ARMS ,PCR),also the expression of APC protein in gastric cancer of different stages was detected by immunohistochemical method. We found that there wasn't I1307K mutation of APC gene in 62 cases of blood samples of susceptible population in high incidence areas of gastric cancer and 45 cases of gastric cancer tissues. The positive rates of APC protein in gastric cancer (both early and progressive gastric cancer) were significantly lower than that in normal mucosa,the positive rates of APC protein in progressive gastric cancer were significantly lower than that in early gastric cancer,the positive rates of APC protein in gastric cancer with lymph node metastasis were significantly lower than that in gastric cancer without lymph node metastasis. So it was thought that there might be no correlation between the I1307K mutation of APC gene and carcinogenesis of gastric cancer in China,but the decreased expression of APC protein was closely related to the carcinogenesis,progression and lymph node metastasis of gastric cancer.

[Cloning associated genes using microdissection-cDNA PCR-SSH in gastric dysplasia].

OBJECTIVE: To construct cDNA subtracted libraries from gastric dysplasia and further screen differentially expressed genes. METHODS: Relatively pure dysplasia and normal tissue were procured by manual microdissection, and amplified by cDNA-PCR, which was used to carry on for suppression subtractive hybridization (SSH). Subtracted cDNA fragments were linked with vector, cloned, screened, sequenced, and made homologous search. Differentially expressed fragments were verified by dot hybridization. RESULTS: Two subtracted cDNA libraries were constructed. Among 26 sequenced clones, 15 fragments corresponded to known genes, 3 fragments were known EST and 8 fragments were unknown EST (GenBank BQ164614-BQ164616, BQ291516-BQ291520). Fifteen fragments were verified to be differentially expressed in gastric dysplasia. CONCLUSIONS: Subtracted cDNA libraries from gastric dysplasia are constructed using combination of microdissection-cDNA PCR and SSH setup in our laboratory. Some fragments have been screened and verified to help to search for novel associated genes with gastric carcinogenesis.

[Effect of electromagnetic radiation on discharge activity of neurons in the hippocampus CA1 in rats].

OBJECTIVE: In order to explore effect of electromagnetic radiation on learning and memory ability of hippocampus neuron in rats, the changes in discharge patterns and overall electrical activity of hippocampus neuron after electromagnetic radiation were observed. METHODS: Rat neurons discharge was recorded with glass electrode extracellular recording technology and a polygraph respectively. Radiation frequency of electromagnetic wave was 900 MHZ and the power was 10 W/m2. In glass electrode extracellular recording, the rats were separately irradiated for 10, 20, 30, 40, 50 and 60 min, every points repeated 10 times and updated interval of 1h, observing the changes in neuron discharge and spontaneous discharge patterns after electromagnetic radiation. In polygraph recording experiments, irradiation group rats for five days a week, 6 hours per day, repeatedly for 10 weeks, memory electrical changes in control group and irradiation group rats when they were feeding were repeatedly monitored by the implanted electrodes, observing the changes in peak electric digits and the largest amplitude in hippocampal CA1 area, and taking some electromagnetic radiation sampling sequence for correlation analysis. RESULTS: (1) Electromagnetic radiation had an inhibitory role on discharge frequency of the hippocampus CA1 region neurons. After electromagnetic radiation, discharge frequency of the hippocampus CA1 region neurons was reduced, but the changes in scale was not obvious. (2) Electromagnetic radiation might change the spontaneous discharge patterns of hippocampus CA1 region neurons, which made the explosive discharge pattern increased obviously. (3) Peak potential total number within 5 min in irradiation group was significantly reduced, the largest amplitude was less than that of control group. (4) Using mathematical method to make the correlation analysis of the electromagnetic radiation sampling sequence, that of irradiation group was less than that of control group, indicating that there was a tending to be inhibitory connection between neurons in irradiation group after electromagnetic radiation. CONCLUSION: Electromagnetic radiation may cause structure and function changes of transfer synaptic in global, make hippocampal CA1 area neurons change in the overall discharge characteristic and discharge patterns, thus lead to decrease in the ability of learning and memory.