The activation of Ca²⁺ entry through store-operated channels by agonists that deplete Ca²⁺ from the endoplasmic reticulum (ER) is a ubiquitous signaling mechanism, the molecular basis of which has remained elusive for the past two decades. Store-operated Ca²⁺-release-activated Ca²⁺ (CRAC) channels constitute the sole pathway for Ca²⁺ entry following antigen-receptor engagement. In a set of breakthrough studies over the past two years, stromal interaction molecule 1 (STIM1, the ER Ca²⁺ sensor) and Orai1 (a pore-forming subunit of the CRAC channel) have been identified. Here we review these recent studies and the insights they provide into the mechanism of store-operated Ca²⁺ channels (SOCCs).

Induction of tumor vasculature occlusion by targeting a thrombogen to newly formed blood vessels in tumor tissues represents an intriguing approach to the eradication of primary solid tumors. In the current study, we construct and express a fusion protein containing vascular endothelial growth factor (VEGF) and tissue factor (TF) to explore whether this fusion protein has the capability of inhibiting tumor growth in a colon carcinoma model. The murine cDNA of VEGF A and TF were amplified by reverse transcriptase polymerase chain reaction (RT-PCR), and then cloned into prokaryotic expression plasmid pQE30 with a linker. The expression product recombinant VEGF-TF (rVEGF-TF) was purified and proved to have comparable enzyme activity to a commercial TF and the capability of specific binding to tumor vessels. Significant decrease of tumor growth was found in the mice administered with rVEGF-TF on Day 6 after initiated rVEGF-TF treatment (P<0.05), and the tumor masses in 2 of 10 mice were almost disappeared on Day 14 after the first treatment. In addition, valid thrombogenesis and tumor necrosis were observed in the tumor tissues injected with rVEGF-TF. Our results demonstrate that occlusion of tumor vasculature with rVEGF-TF is potentially an effective approach for cancer therapy.

Objective: Leber’s hereditary optic neuropathy (LHON) is a maternally inherited degeneration of the optic nerve caused by point mutations of mitochondrial DNA (mtDNA). Many unsolved questions regarding the penetrance and pathophysiological mechanism of LHON demand efficient and reliable mutation testing. This study aims to develop a minor groove binder (MGB) probe assay for rapid detection of mtDNA11778 mutation and heteroplasmy in Chinese LHON patients by real-time polymerase chain reaction (PCR). Methods: Forty-eight patients suspected of having LHON and their maternal relatives underwent a molecular genetic evaluation, with 20 normal individuals as a control group at the same time. A real-time PCR involving two MGB probes was used to detect the mtDNA11778 mutation and heteroplasmy. A linear standard curve was obtained by pUCmLHONG and pUCmLHONA clones. Results: All 48 LHON patients and their maternal relatives were positive for mtDNA11778 mutation in our assay, 27 heteroplasmic and 21 homoplasmic. Eighteen cases did not show an occurrence of the disease, while 9 developed the disease among the 27 heteroplasmic mutation cases. Eleven did not show an occurrence of the disease, while 10 cases developed the disease among 21 homoplasmic mutation cases. There was a significant difference in the incidence between the heteroplasmic and the homoplasmic mutation types. The time needed for running a real-time PCR assay was only 80 min. Conclusion: This real-time PCR assay is a rapid, reliable method for mtDNA mutation detection as well as heteroplasmy quantification. Detecting this ratio is very important for predicting phenotypic expression of unaffected carriers.

Objective: To investigate the relationship between alanine aminotransferase (ALT) levels and metabolic syndrome (MS) in nonalcoholic fatty liver disease (NAFLD). Methods: A total of 26527 subjects who received medical health checkup in our hospital from January 2005 to July 2007 were enrolled in the study. The diagnosis of fatty liver was based on ultrasound imaging. MS was defined according to the criteria of the Adult Treatment Panel III. ALT, triglyceride (TG), high density lipoprotein cholesterol (HDL-c), fasting plasma glucose (FPG), height, weight, waist circumference (WC), systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured in each subject to analyze the relationship between MS and ALT activity. Results: (1) The prevalence of NAFLD in men (30.94%) was significantly higher than that in women (15.65%); (2) The incidence of MS in NAFLD (33.83%) was significantly greater than that in non-NAFLD (10.62%); (3) Of the 6470 subjects with NAFLD, in the age-adjusted partial correlation analysis, there were statistically significant correlations between the ALT levels and most metabolic risk factors in each sex (P<0.01), except that ALT levels had no correlation with HDL-c in women. Moreover, in the multiple stepwise regression analysis, SBP lost its significance, and WC, body mass index (BMI), age, DBP, TG and FPG were independently associated with ALT levels in both sexes (P<0.05). HDL-c remained significant and was independently related to ALT levels in men; (4) ALT levels were significantly higher in subjects with MS compared to those without MS (P<0.001). Mean ALT levels increased with the number of MS components in each sex (P<0.05 for trend). Conclusion: We found a strong relationship between ALT levels and MS in NAFLD and revealed that the cluster of MS components might be the predictor for ALT elevations.

Background: The adhesion of monocytes to the endothelium following accumulation of low-density lipoprotein (LDL) in subendothelial spaces is an important step in the development of intimal hyperplasia in arterially implanted vein grafts and atherosclerosis in both animals and humans. However, it is not well known how serum factors affect the adhesion of monocytes. Methods: We have studied the effect of fetal calf serum (FCS), which we considered a source of LDL, on the adhesion of monocytes to endothelial cells (ECs) by using human monocytic THP-1 cells and both a monolayer of cultured bovine aortic endothelial cells (EC monoculture) and a co-culture with bovine aortic smooth muscle cells (EC-SMC co-culture). Results: It was found that the addition of FCS to the medium greatly affected the adhesion of THP-1 cells, and the higher the concentration of FCS in the medium, the greater the adhesion of THP-1 cells to endothelial cells. Adhesion of THP-1 cells to an EC-SMC co-culture was approximately twofold greater than that to an EC monoculture, and after adhering to endothelial cells, many THP-1 cells transmigrated into the layer of smooth muscle cells. Conclusion: The results suggest that the elevation of the LDL (cholesterol) level in blood provides a favorable condition for the development of intimal hyperplasia and atherosclerosis by promoting the adhesion of monocytes to the endothelium and their subsequent migration into subendothelial spaces.

Background: Bone marrow mesenchymal stem cell (MSC) transplantation is a promising strategy in the treatment of myocardial infarction (MI). However, the time for transplanting cells remains controversial. The aim of this study was to find an optimal time point for cell transplantation. Methods: MSCs were isolated and cultured from Sprague-Dawley (SD) rats. MI model was set up in SD rats by permanent ligation of left anterior descending coronary artery. MSCs were directly injected into the infarct border zone at 1 h, 1 week and 2 weeks after MI, respectively. Sham-operated and MI control groups received equal volume of phosphate buffered saline (PBS). At 4 weeks after MI, cardiac function was assessed by echocardiography; vessel density was analyzed on hematoxylin-eosin stained slides by light microscopy; the apoptosis of cardiomyocytes was evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay; the expressions of proteins were analyzed by Western blot. Results: MSC transplantation improved cardiac function, reduced the apoptosis of cardiomyocytes and increased vessel density. These benefits were more obvious in 1-week group than in 1-h and 2-week groups. There are more obvious increases in the ratio of bcl-2/bax and the expression of vascular endothelial growth factor (VEGF) and more obvious decreases in the expression of cleaved-caspase-3 in 1-week group than those in other two groups. Conclusion: MSC transplantation was beneficial for the recovery of cardiac function. MSC transplantation at 1 week post-MI exerted the best effects on increases of cardiac function, anti-apoptosis and angiogenesis.

Objective: This study aimed to explore the effects of different types of palatal lateral excisions on the growth and development of the maxilla and dental arch, and to investigate the underlying mechanisms. Methods: A total of 112 3-week-old Sprague-Dawley (SD) male rats were randomly divided into a control and 3 experimental groups: the mucoperiosteal denudation group, the mucosal flap excision group, and the periosteum excision group. In the experimental groups, bilateral mucoperiosteal, mucosal flap and periosteum were excised respectively in the lateral one half of the palate. Four rats in each group were randomly chosen for sacrifice every two weeks. The maxilla was dissected following the excision. The widths of the maxilla and dental arch were measured and the histological phenomena were investigated at different phases. At the same time, 12 animals in each group were sequentially injected with calcein every two weeks. Three animals in each group, whose fluorescent labeling was used, were sacrificed for investigating bone formation at Week 8 following injection. Results: (1) Each experimental group presented the constriction of the maxilla and dental arch. The upper first molars in the experimental groups inclined medially. The mucoperiosteal denudation group showed the largest degree of effect followed by the periosteum excision group. The indices of the mucosal flap excision group, which retained the structures of the periosteum layer, had the most approximate values to the control group; (2) Different histological changes among the experimental groups were detected. The fibers penetrated into the palatal bone as Sharpey’s fibers in the mucoperiosteal denudation group. The pattern of bone deposition was the bundle type. Sharpey’s fibers were not found in the mucosal flap and periosteum excision groups and the depositions of palatal bone were the lamellar type as those in the control group; (3) The rates of bone deposition in the experimental groups decreased compared with the control group. The rates in different phases were the most approximate values to those of the control group in the mucosal flap excision group, which has the same structure of periosteum as the control group. Conclusion: There were different effects on the growth and development of the maxilla and dental arch in different types of palatal lateral excisions. Periosteum is important for bone formation and deposition pattern. The prevention of Sharpey’s fibers forming and attaching to the palatine can effectively avert the following malformation.

Objective: To evaluate the accuracy of a scoring system combining zygote and embryo morphology in predicting the outcome of in vitro fertilization (IVF) treatment. Methods: In a study group, 117 consecutive IVF or intracytoplasmic sperm injection (ICSI) cycles with embryo transfer were carried out and 312 embryos were scored using a combined scoring system (CSS) of zygote and embryo morphology before transplantation. In a control group, a total of 420 IVF or ICSI cycles were carried out and 1176 embryos were scored using a cumulative embryo score (CES). The effects of the combined scoring system on the embryo implantation rate and pregnancy rate per cycle were analyzed. Results: Using the combined scoring system, the embryo implantation rate (27.6%) and the clinical pregnancy rate (48.7%) were significantly higher than those in the control group (20.8% and 38.6%, respectively). Also, the implantation rate of embryos scoring ≥70 (38.5%: 82 sacs/213 embryos) was significantly higher (P<0.001) than that of embryos scoring <70 (4%: 4 sacs/99 embryos). The pregnancy rate of patients with embryos scoring ≥70 using the combined scoring system (66.7%) was significantly higher (P<0.001) than that of patients with embryos scoring ≥20 using the cumulative embryo score (59.0%). Conclusion: The results suggest that selecting embryos with a high score (≥70) using the combined scoring system could increase the implantation rate and pregnancy rate, and that using a scoring system combining assessments of human zygotes and pre-implantation embryos might predict IVF outcomes more accurately than using a cumulative embryo score.

Objective: To explore the effects of down-regulated tryptase expression in mast cells on the synthesis and release of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) of vascular endothelial cells. Methods: Tryptase-siRNA (small-interfering RNA) vector was constructed to inhibit tryptase expression in P815 cells. The medium of P815 cells treated by the tryptase-siRNA (RNAi-P815 group) or pure vector (P815 group) was collected and used to culture bEnd.3 cells. The messenger RNAs (mRNAs) of IL-6 and TNF-α in bEnd.3 cells and their protein levels in the medium were measured by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Results: IL-6 and TNF-α mRNAs in bEnd.3 cells cultured in RNAi-P815-conditioned medium decreased significantly compared to those in P815-conditioned medium. Consistently, IL-6 and TNF-α protein levels in the medium of bEnd.3 of RNAi-P815 group were lower than those of P815 group. Conclusion: Reduced tryptase expression significantly inhibited the synthesis and release of IL-6 and TNF-α in vascular endothelial cells. RNA interference targeting tryptase expression may be a new anti-inflammatory strategy for vascular diseases.

Objective: To examine the values of endoscopic ultrasonography (EUS) on diagnosis and treatment of esophageal hamartoma. Methods: We compared and analyzed various kinds of imaging examinations such as barium esophagram, contrast-enhanced computed tomography (CT) and conventional gastroscopy in retrospectively reviewing the clinical data of an esophageal hamartoma patient seen in our clinic in the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China. Having received various imaging examinations, this patient was finally diagnosed with esophageal hamartoma and underwent gastroscopic resection of hamartoma with the diagnostic information obtained from EUS. The patient had been regularly followed up for 13 months after treatment. Results: Barium esophagram, CT and conventional gastroscopy detected the lesion, but were unable to distinguish it from common esophagopolypus and other submucosal lesions, and unable to determine etiopathogenisis. EUS detected the hamartoma and identified its internal structure, echo, exact size, depth of invasion, origin and the relationship between adjacent tissues and organs, differentiating the lesion from other submucosal tumors and clearly defining the diagnosis. EUS-guided fine needle aspiration (FNA) also helped to identify the etiological diagnosis. Conclusion: EUS was superior to other imaging means in diagnosis and treatment of hamartoma.