Tumor gene mutations and messenger RNA expression: correlation with clinical response to icotinib hydrochloride in non-small cell lung cancer.

BACKGROUND: Molecular targeted drugs is now widely used in non-small cell lung cancer (NSCLC) clinical treatment. Icotinib hydrochloride is a new type of oral epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKIs). In this study, we examined the role of EGFR, K-RAS, B-RAF somatic mutations and EGFR mRNA expression in tumor specimens from advanced NSCLC patients as predicators of the efficacy of icotinib hydrochloride. METHODS: We analyzed tumor paraffin-embedded specimens, which were obtained from 14 of 40 patients with advanced NSCLC who enrolled in the stage I clinical trial of icotinib hydrochloride. Somatic mutations were evaluated by mutant-enriched liquidchip (MEL) technology, and EGFR mRNA expression was measured by branched DNA liquidchip (MBL) technology. RESULTS: In the 14 specimens, seven patients showed EGFR mutations, exon 19 deletion (3/7) and exon 21 point mutation (4/7); and two patients showed K-RAS mutation. No mutations in EGFR exon 20 or B-RAF were detected. In patients with EGFR mutation, one patient developed progress disease (PD), three patients had stable disease (SD), two patients had partial responses (PR) and one patient had a complete response (CR). In patients with wild-type EGFR, four patients had PD, three patients acquired SD, and none had PR/CR (P = 0.0407). EGFR mutations were associated with better progress-free survival (PFS) (141 days vs. 61 days) but without a statistically significant difference (P = 0.8597), and median overall survival (OS) (≥ 449 days vs. 140 days). EGFR mRNA expression levels were evaluated (three high, eight moderate, one low, and two that can not be measured due to insufficient tumor tissue) and no statistically significant relationships was observed with response, PFS or OS. CONCLUSIONS: The EGFR mutation rate was consistent with that reported in the Asian population, so the MEL technology is reliable for measuring EGFR mutation with high throughput and rapidity. EGFR exon 19 deletions and exon 21 point mutation are predictive biomarkers for response to icotinib hydrochloride as second line treatment or above.

[Clinical efficacy of immediate implants restoration in anterior zone].

PURPOSE: To elevate the clinical efficacy of immediate loading in anterior implants. METHODS: Forty eight implants were placed in 33 patients and the implants were immediately loaded to provide support for fixed provisional prosthesis within 3-6 months. Then, metal-ceramic crowns were restored. The patients were revisited every 12 months after restoration. RESULTS: The follow-up period was 3 to 50 months. All cases achieved good clinical efficacy. CONCLUSION: Clinical studies on the placement of immediate restoration in anterior teeth have revealed predictable results.

[Clinical study of maxillary sinus floor elevation with simultaneous placement of implants from the top of alveoli].

PURPOSE: To evaluate the clinical results of maxillary sinus floor elevation technique with bone condensers on implantation. METHODS: 104 patients underwent maxillary sinus floor elevation and 126 implants were inserted and restored. Clinical examination and radiographs were conducted. Spiral CT scanning and three-dimensional reconstruction was performed among 30 cases during follow-up. RESULTS: All the patients with 9.16 mm (5-11 mm) height of residual bone in the posterior maxilla underwent sinus floor elevation with simultaneous implant placement using bone condensers. The elevation height was 2-6 mm. The follow-up time was 16-82 months. No sinus complication was observed during the follow-up period. The survival rate was 100%. Spiral CT scanning and three-dimensional reconstruction showed that dental implants were submerged in the alveoli around, their tops were covered with bone. CONCLUSIONS: With bone condensers, maxillary sinus floor elevation with simultaneous placement of implants from the top of alveoli was a predictable and safe technique. There were no implants loose or lost, no clinical complaint of maxillary sinus area, and X-ray examination showed well osseointegration.

Demilune cell and parotid protein from murine oviductal epithelium stimulates preimplantation embryo development.

In mammals, fertilization and early preimplantation embryo development occur in the oviduct. We hypothesized that interaction exists between the developing embryos and the maternal genital tract, such that the embryos modulate the physiology and gene expression of the oviduct so that it is conducive to their development. By comparing the gene expression patterns in mouse oviducts containing transferred preimplantation embryos with those of oviducts containing oocytes, we report here the characterization of demilune cell and parotid protein (Dcpp), which was up-regulated in the embryo-containing oviduct. Dcpp mRNA was highly expressed in the oviductal epithelium at the estrus stage. The Dcpp gene codes for a protein of 150 amino acids and contains a signal peptide suggestive of secretory function. The Dcpp mRNA level was maintained in the oviductal epithelium of pregnant females but decreased continuously in those of pseudopregnant mice. Exogenous estrogen stimulated the expression of Dcpp mRNA and protein in ovariectomized mice. The effect was abolished by an estrogen antagonist, ICI 182,780. Dcpp protein was present in mouse oviductal fluid but not in uterine fluid. More importantly, Dcpp immunoreactivity was found in embryos recovered from the oviduct but not in mature oocytes from the ovary. Supplementation of Dcpp to culture medium stimulated the development of mouse embryos to the blastocyst stage. Anti-Dcpp antibody decreased the beneficial effect of Dcpp on implantation of two-cell mouse embryos transferred to the oviducts of the foster mothers. In summary, our data demonstrated that Dcpp is highly expressed in the oviductal lumen in the presence of preimplantation embryos. It stimulates the growth of preimplantation embryos and may play an important role in embryo-maternal dialogue.

Differential growth, cell proliferation, and apoptosis of mouse embryo in various culture media and in coculture.

Sequential culture and coculture are two methods of improving the development of preimplantation embryos in vitro. Direct comparison of the efficiency of these methods is limited. Proliferation and apoptosis determine the total number of blastomere in preimplantation embryo, which is a sensitive parameter for evaluation of the development of embryo in vitro. In this study, we compared the proliferation and apoptosis of mouse embryo in different culture media, including CZB, KSOM, MTF, G1.2/G2.2 sequential culture media, and in human oviductal cell coculture. Sequential culture using G1.2/G2.2 was superior to KSOM, MTF, and CZB/CZB + G with respect to the formation of 3-4 cell embryos, morula, and blastocyst. G1.2/G2.2 cultured blastocyst had significantly more proliferating blastomeres and higher total cell number per blastocyst than those cultured in KSOM or CZB/CZB + G. Compared to embryos cultured in G1.2/G2.2, embryos cocultured in G1.2/G2.2 hatched more frequently. Cocultured blastocysts also had significantly higher percentage of proliferating cell and lower percentage of apoptotic cell per blastocyst than those cultured in G1.2/G2.2. It was concluded that G1.2/G2.2 facilitated the proliferation of blastomere whilst human oviductal cell coculture suppressed apoptosis in addition to stimulating proliferation of blastomere.

The embryotrophic activity of oviductal cell-derived complement C3b and iC3b, a novel function of complement protein in reproduction.

The oviduct-derived embryotrophic factor, ETF-3, enhances the development of trophectoderm and the hatching process of treated embryos. Monoclonal anti-ETF-3 antibody that abolishes the embryotrophic activity of ETF-3 recognized a 115-kDa protein from the conditioned medium of immortalized human oviductal cells. Mass spectrometry analysis showed that the protein was complement C3. Western blot analysis using an antibody against C3 confirmed the cross-reactivities between anti-C3 antibody with ETF-3 and anti-ETF-3 antibody with C3 and its derivatives, C3b and iC3b. Both derivatives, but not C3, were embryotrophic. iC3b was most efficient in enhancing the development of blastocysts with larger size and higher hatching rate, consistent with the previous reported embryotrophic activity of ETF-3. Embryos treated with iC3b contained iC3b immunoreactivity. The oviductal epithelium produced C3 as evidenced by the presence of C3 immunoreactivity and mRNA in the human oviduct and cultured oviductal cells. Cyclical changes in the expression of C3 immunoreactivity and mRNA were also found in the mouse oviduct with the highest expression at the estrus stage. Molecules involving in the conversion of C3b to iC3b and binding of iC3b were present in the human oviduct (factor I) and mouse preimplantation embryo (Crry and CR3), respectively. In conclusion, the present data showed that the oviduct produced C3/C3b, which was converted to iC3b to stimulate embryo development.

[Maxillary sinus lifting and simultaneously placement of implants from the top of alveoli].

OBJECTIVE: To study the effect of maxillary sinus floor elevation by the Frialit-2 Bone Condenser for implantation. METHODS: 11 patients underwent sinus floor lift by The Frialit-2 Bone Condenser and were inserted 14 implants. The time of following up was 10 - 21 months. RESULTS: There were no implant loose or lost, no clinical complaint of maxillary sinus area, and X-ray exam showed well osseointegration. CONCLUSIONS: The Frialit-2 bone condenser can be used for lifting sinus floor, and the sinus elevation without lateral access allows the insertion of implants with no additional surgical stress for the patients.

Coculture of human oviductal cells maintains mitochondrial function and decreases caspase activity of cleavage-stage mouse embryos.

OBJECTIVE: To investigate the mitochondrial function and caspase activity in mouse embryos after human oviductal cell coculture. DESIGN: Experimental laboratory study. SETTING: University gynecology unit. ANIMAL(S): MF-1 (female); BALB/c (male) mice. INTERVENTION(S): Mouse embryos were cocultured with human oviductal cells. MAIN OUTCOME MEASURE(S): Mitochondrial transmembrane potential (Delta psi(m)) and caspase activity. RESULT(S): Compared to embryos after coculture in Chatot-Ziomek-Bavister (CZB) medium supplemented with 0.5 mg/mL of BSA (CZB), Delta psi m of embryos cultured in CZB was significantly lower at the two-cell (CZB, 2.04 +/- 0.412; coculture, 4.34 +/- 0.563) and morula (CZB, 6.06 +/- 0.548; coculture, 7.12 +/- 0.568) stages. Cocultured embryos and in vivo developed embryos had comparable Delta psi m. Caspase activity was not detected in unfragmented cleavage-stage embryos and morula developed in vivo. In vitro cultured morula possessed caspase activity. The activity was significantly reduced in the cocultured morula. CONCLUSION(S): Human oviductal cells maintained the mitochondria function in terms of mitochondrial transmembrane potential and decreased the caspase activity to improve the development of mouse embryo.

Identification of mRNAs that are up-regulated after fertilization in the murine zygote by suppression subtractive hybridization.

Transcriptions occur in mouse preimplantation embryos as early as one-cell stage. However, our understanding on gene expression at this stage is lacking. The present study applied suppression subtractive hybridization (SSH) to compared gene expression profiles of mouse zygote and oocyte. Forty-four differentially expressed genes were selected and shown positive signals by reverse dot-blot hybridization. DNA sequences comparison of these putative clones with the GenBank/EMBL databases using BLAST search identified 38 clones with >90% identity to known genes and six novel clones with less than 70% homology to the databases. Eleven out of the 44 differentially expressed clones were either originally isolated from male embryo or testis-specific genes, suggesting that these genes may be derived from paternal genome. Five differentially expressed genes of interest, including bromodomain-containing protein BP75, spindlin, radixin, pituitary tumor-transforming gene (PTTG), and proteoglycan core protein (serglycin) were further studied by semi-quantitative RT-PCR. It is noted that spindlin which involves in cell division is highly expressed in zygote, suggesting that this protein may play an important role in zygotic gene activation (ZGA) and early stage development in 1-cell stage mouse embryos.

Early developing embryos affect the gene expression patterns in the mouse oviduct.

Fertilization and development of mouse embryos occur in the ampullae of oviduct. We hypothesize that fetal-maternal communication exists in the preimplantation period, allowing optimal development of embryos. It is known that embryotrophic factors from oviduct affect the development of embryos. Although embryos affect their own transport in the oviduct, the mechanism of action is unknown. As a step toward understanding the action of embryos on oviductal physiology, we adopted suppression subtractive hybridization (SSH) to compare the gene expression in the mouse oviduct containing early embryos with that of oviduct containing oocytes. Ten to twelve 1-cell mouse embryos were transferred to one oviduct of a foster mother and similar number of oocytes were transferred to the contralateral oviduct. The animals were sacrificed after 48 h and their oviducts were excised for mRNA study. Using SSH, we screened out 250 putative positive clones from the subtracted embryo-containing oviduct library and 97 of them were screened positive by reverse dot-blot analysis. DNA sequence analysis identified genes that shared high homology with sequences in GenBank/EMBL database with unknown functions. Overall, 13 of the 90 high-quality sequences (14%) were homologous to 6 different genes previously described. Reverse Northern analysis confirmed that the expression of these genes were higher in the embryo-containing oviduct than in the oocyte-containing oviduct. About 12% of these clones (11/90) were novel. This article is the first to report identification of genes in the oviduct that are upregulated in the presence of embryos during the preimplantation period.