Search

Objective: To study the effect of ABT-737 combined with cisplatin on apoptosis of breast cancer cell line T47D cells., Methods: T47D cells cultured in vitro was used for this experiment. Cell proliferation was measured by MTT assay. The expression of apoptosis-related protein was determined by Western blot. Morphological changes of apoptotic cells were observed by fluorescence microscopy. The apoptosis rate was examined by flow cytometry., Results: The MTT assay showed that ABT-737 significantly decreased the IC(50) of cisplatin in T47D cells [(26.00 ± 1.41) µmol/L of single cisplatin vs. (13.00 ± 1.11) µmol/L of combination (ABT-737 + cisplatin)]. As a single agent, ABT-737 did not inhibit the proliferation of T47D cells, but enhanced the inhibitory effect of cisplatin in a dose-dependent manner. The detection of the cleavage of PARP showed that ABT-737 lowered the doses of cisplatin to induce apoptosis and shortened the induction time of apoptosis in T47D cells. Compared with the single use of cisplatin, the combination of ABT-737 and cisplatin accelerated the cleavage of PARP and caspase3, but did not alter the expression levels of Bcl-2, Bcl-X(L), and Bax. Both flow cytometry and fluorescence microscopy showed that ABT-737 combined with cisplatin significantly increased the apoptosis induction in T47D cells (2.3% ± 0.1 % in the control, 30.0% ± 0.8% in the cisplatin alone, and 49.0% ± 0.5% in the cisplatin + ABT-737 groups, P < 0.05)., Conclusion: The Bcl-2 inhibitor ABT-737 can significantly enhance cisplatin-induced apoptosis in human breast cancer T47D cells in vitro.

Background: There is lack of consensus regarding the best option for the treatment of acute Achilles tendon rupture-operation or non-operation. The purpose of this meta-analysis was to identify and summarize the randomized controlled trials comparing the operative and non-operative lines of treatment of acute Achilles tendon ruptures., Methods: We searched multiple databases in English (including EMBASE, PubMed, and OVID) and in Chinese (including CNKI, WANFANG, and VIP), as well as reference lists of articles and main orthopaedic and sports medical journals. Two reviewers independently screened the studies for eligibility, evaluated the quality and extracted data from eligible studies, with confirmation by cross-checking. The major results and conclusions were concluded, and the different complication rates and functional outcomes were compared. Meta-analysis was processed by RevMan 5.0 software., Results: Eight randomized controlled trials (RCTs) involving 777 patients met the inclusion criteria. The rerupture rate in non-operative group was significantly higher (Z = 3.33, P < 0.01). However, the moderate (Z = 4.27, P < 0.01) and minor (Z = 5.59, P < 0.01) complication rate in the operative group were significantly higher. No significant difference in comparing the major and total complication rates. The return to work time in the operative group was shorter (Z = 2.65, P < 0.01). The inability to return to previous level sporting rate and ankle joint decreased range of motion (ROM) rate showed no significant difference in the two groups. Other functional outcomes were similar in the two groups., Conclusions: Operation could significantly reduce the risk of rerupture; however, it was associated with a higher risk of other complications. The functional outcomes were similar in two treatment methods except an earlier return to work in patients treated operatively. Thus operative treatment is preferable for patients with good physical condition. Non-operative treatment is an acceptable alternative especially for the older and patients with lower sporting requirements.

Objectives: A retrospective study was used to evaluate the short to intermediate outcomes of open reduction and internal fixation of acute Lisfranc joint injuries., Methods: From January 2003 to December 2009, 47 patients with Lisfranc injuries were treated in the study,41 patients were analyzed in current study with an average age of thirty-one years old. All of patients were treated with open reduction and internal fixation. According to the Myerson classification system, there were 9 type-A fractures, 7 type-B1, 11 type-B2, 10 type-C1, 5 type-C2. The AOFAS scale and VAS scale were used for the outcome measurement at the final follow-up., Results: There were 41 patients with 42 operations were followed up and with a mean time of 36 months (ranged, 12 to 71 months). The mean AOFAS score was (84.2 +/- 2.8) points (ranged, 65 to 100 points), with an excellent and good rate of 81 percent. The mean VAS score was (2.6 +/- 0.5) points (ranged, 0 to 6 points). Three patients developed superficial infection, and two developed skin necrosis. Nineteen patients had weight-bearing or walking pain. All of the patients had radiographic evidence of degenerative change, four with malunion of the second tarsometatarsal joint, and two of the patients with hallux valgus deformity change., Conclusion: According to the results of the study, anatomic reduction and internal fixation can get the satisfactory results for the treatment of acute Lisfranc injuries during a short to intermediate follow-up time. All of the patients would have different degree of tarsometatarsal joint degenerative changes, but these changes are non-progressive and associated with minimal loss of function.

Objective: To evaluate the therapeutic effect of hBNP on rats with chronic heart failure (CHF)., Methods: Thirty CHF rats defined by echocardiography at 12 weeks post abdominal aortic constriction were randomly divided into Ad-hBNP group (2.5 × 10(10) VP/ml NS Ad-hBNP 1 ml/week × 4, n = 14), Ad-Track group (n = 8), placebo group (NS, n = 8), 10 sham-operated rats served as control group. After 4 weeks treatment, cardiac function was evaluated by echocardiography and hemodynamic measurements. Heart weight (HW) and HW/body weight (BW) ratio were determined., Results: IVS, LVPW, LVEDD and LVESD were significantly reduced in the Ad-hBNP group [(2.34 ± 0.29) mm, (2.28 ± 0.18) mm, (6.50 ± 0.42) mm, (3.54 ± 0.59) mm] than those in the Ad-Track group [(2.71 ± 0.35) mm, (3.02 ± 0.85) mm, (7.71 ± 0.83) mm, (4.72 ± 0.80) mm] and in the NS group [(2.78 ± 0.23) mm, (2.83 ± 0.53) mm, (7.34 ± 0.97) mm, (4.55 ± 0.77) mm, all P < 0.05]. The LVEF and LVFS of the Ad-hBNP group [(79.27 ± 7.01)%, (43.38 ± 6.73)%] were significantly higher than in the Ad-Track group [(70.85 ± 4.81)%, (35.72 ± 3.68)%] and in the NS group [(69.67 ± 6.90)%, (34.91 ± 5.10)%, all P < 0.01]. HR [(417.48 ± 32.57) beats/min, (446.85 ± 61.49) beats/min, P < 0.05; (440.83 ± 32.18) beats/min, P < 0.05], LVEDP [(-4.24 ± 4.00) mm Hg (1 mm Hg = 0.133 kPa); (21.99 ± 6.80) mm Hg, P < 0.01; (18.00 ± 12.25) mm Hg, P < 0.01] were significantly decreased and while LVSP [(131.79 ± 15.76) mm Hg; (112.99 ± 32.35) mm Hg, P < 0.05; (117.13 ± 15.26) mm Hg], +dP/dt(max) [(5037.20 ± 430.41) mm Hg/s; (4217.40 ± 1354.15) mm Hg/s, P < 0.05; (4310.50 ± 1293.97) mm Hg/s, P < 0.05] and -dP/dt(max) [(-4382.00 ± 1304.79) mm Hg/s; (-3725.00 ± 791.34) mm Hg/s, P < 0.05; (-3890.00 ± 1043.73) mm Hg/s, P < 0.05]were significantly increased in Ad-hBNP group than in Ad-Track group and NS group (all P < 0.05). HW and HW/BW were also decreased in Ad-hBNP group than in the Ad-Track group and the NS group., Conclusion: Exogenous hBNP improved the cardiac function and attenuated remodeling in CHF rats.

Objectives: To provide a cumulative data about the complications of second or third generation ankle prostheses in the literature, and to provide a summary high-grade complications associated with implant failure., Methods: A comprehensive search for all relevant articles published in English from January 1995 to December 2010 was conducted. Two reviewers evaluated each study to determine whether it was eligible for inclusion and collected the data of interest. Meta-analytic pooling of results across studies was performed for the complications and failure rate., Results: Thirty-five primary studies with 4395 implants were identified. The three highest complications of total ankle arthroplasty were aseptic loosening (12.51%), intra-operative bone fracture (11.97%) and bony impingement (11.27%). The three high-grade complications associated with implant failure were aseptic loosening (45.00%), infection (33.00%) and malalignment (29.00%). The pooled mean failure rate was 10.98% (95%CI: 8.80% - 13.16%), and the pooled mean failure rate of STAR implant was 14.20% (95%CI: 10.64% - 17.76%)., Conclusions: It is found that aseptic loosening, infection and malalignment are high-grade complications associated with implant failure in total ankle arthroplasty. The orthopaedic surgeons should be more careful in the operation, and the patients should coordinate with the post-operative rehabilitation plan.

Objective: To establish a colon cancer cell line with stable expression of carcinoembryonic antigen(CEA)., Methods: Recombinant lentivirus conjugated with CEACAM5 cDNA were used to transfect wild CT26 cells. Antibiotics were given for 2 weeks to select CEA positive cells. A single transfected clone was obtained using limiting dilution. The 7th and 14th passages of cells cultured in vitro were detected for CEACAM5 mRNA by RT-PCR, and protein by western blot. The location of CEACAM5 expression was examined using fluorescent microscope and immunocytochemistry. The 14th passage cells were injected subcutaneously into mice to create BALB/c model and CEACAM5 protein was detected by in vivo fluorescence image analysis system and immunohistochemistry., Results: CEACAM5 mRNA and protein were found in the 7th and 14th passages of CT26CEA cells, which were proved to locate in the cytoplasm by fluorescence microscope and immunohistochemistry. Abundant CEACAM5 protein was found in subcutaneous tumors by in vivo fluorescence image analysis system and immunohistochemistry., Conclusion: Colon cancer cell line CT26 with stable expression of CEA in vitro and in mice can be used as a suitable tool to facilitate research on the impact and mechanism of CEA on colon cancer under normal immune environment.

Distraction osteogenesis is a valuable treatment method that allows limb lengthening or reconstruction of large bone defects. However, its major disadvantage is the long period required for the consolidation of a distraction callus. Osteogenic growth peptide (OGP) stimulates endochondral bone formation in fracture callus, but its capacity to promote regenerate ossification during distraction osteogenesis has not been evaluated. This study investigated whether intravenously administered OGP accelerated bone healing during distraction osteogenesis in 36 male New Zealand White rabbits, randomized into two groups. The treatment group received OGP (200 ng/kg body weight) in phosphate-buffered saline (PBS), intravenously, each day; the control group received PBS alone. A 15-mm lengthening of the right lower leg was performed using the method of Ilizarov. Evidence from biomechanical, histological and radiographic evaluations demonstrated that systemic OGP treatment promoted optimal new bone formation during distraction osteogenesis in this rabbit model.

Objective: Review the series cases of open calcaneal fractures, to discuss the clinical outcome and evaluate the effective factors associated with the prognosis., Methods: Between 2004 and October 2009, 427 calcaneal fractures were treated, 22 of these fractures were open, 15 feet of 15 patients were available for the follow-up. There were 9 males and 6 females with a mean age of 34.7 years (range 18 to 66). According to the Sanders classification, there were 2 type I fractures, 3 type II, 6 type III and 4 type IV. According to the Gustilo classification, there were 1 of type 1, 4 of type II, 5 of type III A, and 5 of type III B open fracture. According to the open calcaneal injury subtypes (OCIS), there were 1 of type IA, 2 of type II A,7 of type II B, 2 of type IIIA and 3 of type III B. All patients were treated with intravenous antibiotics, immediate and repeat irrigation and debridement, temporary wound coverage, and initial stabilization of the limb. Clinical examination, radiographs and AOFAS ankle-hindfoot scores were used for outcome measurement., Results: The average follow-up time was 16.4 months (8 to 31 months). The mean AOFAS score was (74.6 +/- 10.4) (range 58 to 94). There were 4 feet required tissue transfer for wound coverage. An infection developed at the sites of 5 fractures,4 of witch were Gustilo type III, 2 deep infections with osteomyelitis developing at the site of 1 patient, no patient required amputation., Conclusion: These findings do not reflect as high a deep infection and osteomyelitis rate for open calcaneal fractures as previously reported, if early and satisfying debridement, evaluate the soft tissue injury carefully, and choose the right time and indications for internal fixation. In addition, early internal fixation should be avoided for Gustilo type III and OCIS type B calcaneal fractures.

Objective: To explore the significance of the expression of carcinoembryonic antigen receptors (CEAR) in digestive organs., Methods: Specimens were procured from 20 male BALB/c mice including esophagus, small intestine, stomach, colon, pancreas, and liver. Kupffer cells were obtained by density gradient centrifugation following enzymatic digestion of the fresh liver specimen. Immunohistochemistry and immunocytochemistry methods were used to detect CEAR in those organs or Kupffer cells., Results: CEAR was found both in cytoplasm and nuclei of the digestive tract mucosal epithelial cells and pancreas islet cells, but only in the cytoplasm of liver cells, Kupffer cells, and smooth muscle cells of the whole digestive tract. The mean ranks of CEAR expression were 174.33 in the mucosal epithelial cells of colon, 160.70 in epithelial cells of small intestine, 139.18 in Kupffer cells, 137.43 in pancreas islet cells, 131.70 in liver cells, 124.23 in gastric epithelial cells, 77.15 in esophageal epithelial cells and 57.80-71.00 in smooth muscle cells of the entire digestive tract examined. There were significantly differences in the CEAR expression intensity among those positive cells (chi2=99.58, P<0.01) while CEAR was not present in submucosal connective tissue cells, pancreatic exocrine cells, or hepatic sinusoid endothelial cells., Conclusion: There are significantly differences in the expression of CEAR in the main digestive organs according to the different tissue and cells, which may play an important role in the carcinogenesis and hepatic metastasis from tumors of the digestive system.

Chlamydia trachomatis, an obligate intracellular bacterial pathogen, is the major cause of sexually transmitted diseases worldwide. Although a variety of strategies have been taken to promote the development of a protective vaccine, no ideal vaccine has been generated so far. In this study, we transfected dendritic cells (DCs) with recombinant adenovirus carrying C. trachomatis serovar E major outer membrane protein gene (Ad-MOMP), and investigated their ability to induce specific protection against genital tract chlamydial challenge infection. The results showed that when DCs were transfected with Ad-MOMP in vitro, the DCs exhibited increased expression of CD80 and MHC-II molecules as well as enhanced IL-12 secretion and were able to stimulate T-cell proliferation. The level of IFN-gamma secreted by stimulated T cells was also up-regulated significantly. When the Ad-MOMP transfected DCs were adoptively transferred intravenously to naive mice, they generated Th1-biased cytokine production and mucosal IgA responses specific for C. trachomatis. More importantly, the mice immunized with Ad-MOMP-DC mounted protection against genital tract challenge infection, shown by lower body mass loss, lower chlamydial loads, and less severe pathological changes. In conclusion, Ad-MOMP transfected DCs are capable of inducing effective protective immune responses against C. trachomatis genital infection.

Xe lamp was employed to simulate the sunlight as light source to study the direct photolysis of three kinds of phenolic pollutants under different pH values. The effects of the dissociation on their photolysis were investigated, and a calculation method and a forecasting model for the photolysis rate constants under the situation of dissociation and non-dissociation of phenolic pollutants were established. The experiment results showed that the photolysis rate constant of pentachlorophenol (PCP) increased from 25.30 x 10(-4) min(-1) to 82.90 x 10(-4) min(-1) when pH value varied from 4.0 to 9.0, and that of nitrophenol (PNP) decreased from 11.90 x 10(-4) min(-1) to 3.18 x 10(-4) min(-1) Similar to the PNP, the photolysis rate constant of phenol decreased from 32.50 x 10(-4) min(-1) to 13.40 x 10(-4) min(-1) with the pH value increased from 4.0 to 11.0. From analysis of the results, a power function relationships between the total photolysis rate constants (K) and dissociation degrees (alpha) of these three phenolic compounds could be established. If alpha had been determined, the dissociation and non-dissociation species photolysis rate constants could be calculated by the tangent equation of the power function. The effect of dissociation on the photolysis should be mainly attributed to the formation of the negative oxygen ions, which led to the changes of the activities of benzene ring and substituent groups. These results will provide theoretical references to further understand environmental behaviors of phenolic compounds in natural waters.

Background: Human epidermal growth factor 2 (HER2) is one of the most important prediction factors, but only 25% - 30% of breast cancer patients HER2 are positive. It is unknown whether there are other molecular markers that could be used to predict prognosis and recurrence in HER2 negative patients. This study investigated correlations of cyclin A2 and HER2 levels with clinical outcomes in 281 patients with invasive breast cancer in order to identify whether cyclin A2 can serve as a prognostic factor in HER2 negative patients., Methods: Immunohistochemical staining was used to detect cyclin A2 and HER2 expression in 281 patients. Cyclin A2 and HER2 gene amplifications were analyzed using gene analysis and RT-PCR in 12 patients. Risk and survival estimates were analyzed using Log-rank, Kaplan-Meier, and Cox regression analysis; cyclin A2 and HER2 consistency with survival were analyzed using Kappa analysis., Results: Patients with higher cyclin A2 and HER2 expressions had significantly shorter disease-free survival periods (P = 0.047 and P = 0.05, respectively). Kappa analysis performed that cyclin A2 and HER2 showed a low Kappa index (kappa = 0.37), allowing us to conclude that cyclin A2 and HER2 detect different pathologies. Gene analysis and RT-PCR showed that cyclin A2 was upregulated in patients with early relapse; the average increase was 3.69 - 2.74 fold., Conclusions: Cyclin A2 and HER2 are associated with proliferation and high recurrence, particularly when combined. Cyclin A2 is easily detected by nuclear staining and might be a useful biomarker for recurrence risk in HER2 negative patients.

To elucidate the roles of humic acids (HAs) and iron on the environmental fate and transport of organic pollutants in natural water, the interactions of HAs with Fe(III) were characterized by Fourier transform infrared spectroscopy (FTIR) spectra, Ultraviolet-visible (UV-vis) spectra and fluorescence spectra, indicating the formation of HAs-Fe(III) complex. Electron paramagnetic resonance (EPR) spectra show *OH radicals are generated and can participate in the photoreaction in solutions containing HAs, Fe(III) and HAs-Fe(III) complex. Under Xe lamp irradiation (lamda >290 nm), the photodegradation of 2,4-dichlorophenoxyacetic acid (2,4-D), as a kind of herbicide, followed the pseudo-first-order reaction kinetics. The pseudo-first-order rate constant of 2,4-D photodegradation with the presence of only 2,4-D (2 mg x L(-1)) was 0.007 h(-1). In the presence of HAs (5 mg x L(-1)), Fe(III) (0.2 mmol x L(-1)) and HAs-Fe(III) complex, the rate constants of 2,4-D degradation were 0.004, 0.034 and 0.046 h(-1), respectively. It was interesting to note that in the existence of HAs, 2,4-D photodegradation was inhibited. While in the presence of Fe(III), 2,4-D photodegradation was enhanced. Furthermore, in the coexistence of HAs and Fe(III), HAs-Fe(III) complex showed better increased effect on the photodegradation of 2,4-D than Fe(III) alone.

Quantum chemical protocols explaining the crystal structures and the visible light absorption properties of 3,4:9,10-perylene tetracarboxylic acid bisimide (PBI) derivates are proposed. Dispersion-corrected density functional theory has provided an intermolecular potential energy of PBI dimers showing several energetically low-lying minima, which corresponds well with the packing of different PBI dyes in the solid state. While the dispersion interaction is found to be crucial for the binding strength, the minimum structures of the PESs are best explained by electrostatic interactions. Furthermore, a method is introduced, which reproduces the photon energies at the absorption maxima of PBI pigments within 0.1 eV. It is based on time-dependent Hartree-Fock (TD-HF) excitation energies calculated for PBI dimers with the next-neighbor arrangement in the pigment and incorporates crystal packing effects. This success provides clear evidence that the electronically excited states, which determine the color of these pigments, have no significant charge-transfer character. The developed protocols can be applied in a routine manner to understand and to predict the properties of such pigments, which are important materials for organic solar cells and (opto-)electronic devices.

Objective: To study the clinical significance of extracapsular extension (ECE) of axillary lymph node metastases in breast cancer., Methods: The clinicopathological data of 1230 cases of nodal positive breast cancer treated in our department from 1989 to 1995 were analyzed retrospectively., Results: 486 (39.5%) from the 1230 cases were ECE positive. There was a higher incidence of ECE in postmenopausal women than premenopausal ones (47.5% versus 35.5%, respectively, P < 0.001). The patients in ECE positive group had a larger tumor size (5.11 +/- 2.53 cm versus 3.90 +/- 1.80 cm, P < 0.001). 18.3% of patients with stage T1 were ECE positive, stage T2 were 36.4%, and stage T3 were 54.4%, and the difference was significant (P < 0.001). ECE was correlated with the number of positive axillary lymph nodes. The ECE positive group had more positive nodes than ECE negative group (16.96 +/- 12.16 versus 5.24 +/- 6.60, P < 0.001). 6.1% of patients with 1 positive node were ECE positive, 13.5% with 2 - 3, 35.8% with 4 - 9, 62.3% with 10 - 19, and 84.0% with more than 20 positive axillary nodes, and there was a significant difference among those groups (P < 0.001). ECE had no association with ER/PR status (P = 0.706). ECE was a risk factor of local-regional recurrence, but the relapse time had no significant difference (P = 0.559). ECE was also a risk factor of distant metastasis, and the relapse time had a significant difference (P < 0.001). The median metastasis free time was 30.0 (2 approximately 172) months in ECE positive group, while 37.5 (2 approximately 170) months in ECE negative group (P = 0.006). CE occurred in 60.4% of the patients with firstly diagnosed bone, skin and distant lymph node metastasis, but in 42.0% of the patients with firstly diagnosed visceral metastasis (P = 0.001). The metastasis-free survival rate, locoregional recurrence-free survival rate and overall survival rate of the ECE positive group were much shorter than that of the ECE negative group. COX proportional hazard regression single factor analysis and multi-factor analysis suggested that ECE is an independent factor of metastasis-free survival, locoregional free recurrence and overall survival., Conclusion: The presence of ECE in breast cancer is positively related with tumor size and the number of positive lymph nodes. It is also a risk factor of locoregional recurrence and distant metastasis. ECE positive group has a much shorter metastasis-free survival, locoregional recurrence-free survival and overall survival. ECE is a risk factor of those three indexes.

Class I of phosphoinositide 3-kinases (PI3Ks) is characterized as a group of intracellular signal proteins possessing both protein and lipid kinase activities. Recent studies implicate class I of PI3Ks acts as indispensable mediators in early development of mouse embryos, but the molecular mechanisms are poorly defined. In this paper, mouse one-cell embryos were used to investigate a possible contribution of the catalytic subunit of PI3K, p110 alpha, to cell cycle progression. The expression level of p110 alpha was determined in four phases of one-cell embryos. Silencing of p110 alpha by microinjection of p110 alpha shRNA into one-cell embryos resulted in a G2/M arrest and prevented the activation of Akt and M-phase promoting factor (MPF). Further, microinjection of the synthesized mRNA coding for a constitutively active p110 alpha into one-cell embryos induced cell cleavage more effectively than microinjection of wild-type p110 alpha mRNA, whereas microinjection of mRNA of kinase-deficient p110 alpha delayed the first mitotic cleavage. Taken together, this study demonstrates that p110 alpha is significant for G2/M transition of mouse one-cell embryos and further emphasizes the importance of Akt in PI3K pathway., ((c) 2008 Wiley-Liss, Inc.)

Access to excited-state structures and dynamics of pi-chromophor aggregates is needed to understand their fluorescence behavior and the properties of related materials. A quantum-chemistry-based protocol that provides quantitative and qualitative insight into fluorescence spectra has been applied to perylene bisimide dimers and provides excellent agreement with measured fluorescence spectra. Both dispersion and dipol-dipole interactions determine the preferred relative arrangements of the chromophores in ground and excited states of the dimer. An exciton trapping mechanism is identified, which may limit the energy transfer properties of perylene bisimide and other dye materials.

The denitrification and excessive P removal can be realized by denitrifying phosphate-accumulating organisms (DNPAOs) under low carbon condition. DNPAOs use poly-3-hydroxy- butyrate (PHB) as electron donor, and use nitrate and nitrite as electron acceptor during the denitrifying phosphate uptake. In this study the biological phosphorus removal granular sludge was induced into the denitrifying and phosphate uptake granular sludge under an anaerobic/anoxic/aerobic alternating operation (referred to as an A/A/O) in a sequencing batch reactor (SBR). When the system is stable, the P and N removal ratio is over 90% and 93% respectively; the release phosphorus is 25-33 mg/L at anaerobic stage; 1g NOx-N approximately takes up P 1.3 g at anoxic stage. In the typical cycle the anaerobic maximum specific release phosphorus rate (max. SRPR) is 18.39 mg/(g x h); the anoxic max. specific uptake phosphorus rate (SUPR) and the max. specific denitrification rate (SDNR) is 23.72 mg/(g x h) and 18.19 mg/(g x h) respectively; the aerobic max. SUPR is 17.15 mg/(g x h). The fraction of DNPAOs rises from 14.9% to 80.7%. Compared with the physical-chemical property of the biological phosphorus removal granular sludge, the settling velocity and the specific gravity of the denitrifying phosphate uptake granular sludge is elevated 0.16-0.7 times and 0.0031 respectively.

Wheat cultivar Jing 411 which is susceptible to powdery mildew, wheat cultivar Brock and near isogenic lines (NILs) of Jing411, which are resistant to powdery mildew were analysized for polymorphisms using 225 pairs of AFLP primers Only two pairs of primers Pst+GAC/Mse+ TCT (P1) and Pst+AGC/Mse+ACC (P2) stably produced polymorphic bands between the resistant and susceptible plants. Two specific fragments were obtained. By cloning and sequencing these two specific fragments, it was showed that the specific fragment amplified by primer P1 had 268bp, and the fragment amplified by P2 had 227bp. They were named AFLP marker P1(268) and P2(227) respectively. Linkage analysis of these two markers revealed that the polymorphism existed in a 106 F2 segregating population. These two markers closely linked to a powdery mildew resistance gene in wheat cultivar Brock, linkage distance were 3.6 and 1.9 cM respectively. These two markers will be useful for marker-assisted selection and gene pyramiding in wheat resistance breeding.

Objective: To observe the effect of Bawei Xilei powder on CD3, CD4, CD8 T-lymphocytes in peripheral blood and colonic mucosa of rat with ulcerative colitis., Method: Sixty SD rats were randomly divided into 6 groups, normal group, model group, low, middle and high dosage Bawei Xilei powder group, Sulfasalazine group. Ulcerative colitis was induced by immunization with rabbit 's colonic mucous emulsified with completely Freund's adjuvant in all rats. Rats in low, middle and high dosage Bawei Xilei powder group were administered with 0.05, 0.1, 0.2 mg Bawei Xilei powder for 18 days by enema respectively. While rats in Sulfasalazine group were enema administered with 100 mg Sulfasalazine, and the rats in other group were administered with equal volume of saline enema as control. We analyzed expression of CD3, CD4, CD8 T-lymphocytes in peripheral blood by flow cytometry and in colonic mucous by immunohistochemistry., Result: In peripheral blood, compared with normal group, in model group, the increased of CD4 T-lymphocytes and CD4 /CD8 ratio, the reduced of CD8 T-lymphocytes, these results were significant discrepancy (P < 0.01). Compared with model group, after treatment with Bawei Xilei powder, CD8 T-lymphocytes increased, but only high dosage Bawei Xilei powder group had discrepancy (P < 0.05). But low dosage Bawei Xilei powder group, other treatment groups' rats showed CD4/CD8 ratio were reduced significantly (P < 0.05). In colonic mucous, compared with normal group, in model group, Rats showed that expression of CD3, CD4 T-lymphocytes reduced and CD8 T-lymphocytes increased obviously (P < 0.05, P < 0.01). Compared with model group, expression of CD8 T-lymphocytes reduced significantly in all treatment groups (P < 0.05, P < 0.01)., Conclusion: Bawei Xilei powder may regulate their balance between T-lymphocytes subgroup, consequently relieve inflammatory injury in favor of ulcer reparation and tissue regeneration.

Phytoremediation, use of plants for remediation, is an emerging technology for treating heavy metals or a final polishing step for the high-level organic contamination, and may be suitable for remediation of heavy metal and organic co-contaminated soil. The aim of this study was to investigate the influence of co-contamination on the growth of Zea mays L. and the fate of both heavy metal and organic pollutants, using Cu and pyrene as the model pollutants. Results showed that shoot and root biomass were affected by the copper-pyrene co-contamination, although maize grown in spiked soils showed no outward signs of phytotoxicity. With the initial concentration of 50,100 and 500 mg/kg, pyrene tended to alleviate the inhibition of Cu to Z. mays L. Pyrene in both planted and non-planted soil was greatly decreased at the end of the 4-week culture, accounting for 16-18% of initial extractable concentrations in non-planted soil and 9-14% in planted soil, which indicated that the dissipation of soil pyrene was enhanced in the presence of vegetation probably due to the biodegradation and association with the soil matrix. With the increment of Cu level, residual pyrene in the planted soil tended to increase. The pyrene residual in the presence of high concentration of Cu was even higher in the planted soil than that in the non-planted soil, which suggested that the change of the microbial composition and microbial activity or the modified root physiology under Cu stress was probably unbeneficial to the dissipation of pyrene. A more thorough understanding of the mechanisms by which metals affect the dissipation of organic pollutants in the rhizosphere could provide a much better framework on which to base manipulation. Unlike pyrene, heavy metal copper cannot be degraded. Decontamination of Cu from contaminated soils in this system required the removal of Cu by plants. It was observed that the ability of Cu phytoextraction would be inhibited under co-contamination of high level of pyrene in highly Cu-polluted soil. In the treatment of 400 mg Cu/kg and 500 mg pyrene/kg, the accumulation of Cu was less than half of that in 400 mg Cu/kg treatment.

The aim of this study was to evaluate the soil microbial characteristics in historically heavy-metal polluted soil, which was also affected by organic co-contaminants, 2,4-dichlorophenol or pentachlorophenol, which often occur due to the conventional use of pesticides. It was observed that the normalized microbial biomass (microbial biomass per unit soil organic C) of the contaminated soil was very low, less than 1% in both non-planted and ryegrass planted soil, and showed a decreasing trend with the treatment of organic co-contaminants. The microbial biomass and substrate-induced respiration (SIR) in the ryegrass planted soil were much larger, as compared with the non-planted soil with or without organic pollutants. The different resistant bacterial community and its physiological diversity in the rhizosphere further suggested that the effect of vegetation on microbial activity was not just a general increase in the mass or activity of pre-existing microorganisms, but rather acted selectively on microbial growth so that the relative abundance of different microbial groups in soil was changed. In sum, high concentrations of organic co-contaminants, especially pentachlorophenol (PCP), could strengthen the deterioration of microbial ecology. The adverse effect of heavy metal-organic pollutants on the soil microbial biomass and activity might be the reason for the slow degradation of PCP that has high chlorinated and high toxicity. Vegetation might be the efficient way to assist in improving and restoring the utilization of agricultural ecosystems. The beneficial microbial effect of vegetation could cause the rapid dissipation of 2,4-dichlorophenol (2,4-DCP) that has less chlorinated and less toxicity in the planted soils.

Objective: To assess the antitumor activity and safety of aromatase inhibitors in advanced breast cancer., Methods: Fifty-two advanced and female breast cancer patients with measurable and /or bone valuable tumor lesions were observed from June 2003 to September 2006. They were treated by aromatase inhibitors for at least 24 weeks, of whom 11 were treated less than 24 weeks because of disease progress; their age range was from 37 to 75 years (median 58); 8 patients were pre-menopausal, 6 with ovarian ablation and 2 with Goserelin to suppress ovarian function. Thirty-six patients were treated with exemestane, 13 with anastrozole and 3 with letrozole. Major items were observed including objective response rate (ORR=CR+PR), clinical benefit rate (CBR=CR+PR+SD>or=24 weeks), time to progress (TTP), time to failure (TTF), safety and toxicity., Results: CR were 6 cases (11.5%) , with 1 case going on for 152 weeks, 1 case for 96 weeks, and other 4 cases for longer than 60 weeks; PR were 19 cases (36.5%), lasting 32-96 weeks; 16 cases obtained SD>or=24 weeks(30.8%); and 11 cases PD (progress of disease)+SD<24 weeks (21.2%). ORR were 48%, CBR 78.8%, and TTP 78.87 weeks (95% CI 61.13%-96.61%); although the patients who did not achieve objective response (group B) were treated with chemotherapy, radiotherapy or another kind of aromatase inhibitor, but their survival time was significantly different from that of the patients who achieved objective response (group A) when defined by Kaplan-Meier survival estimate. The over survival was 92% in group A, and 81.5% in group B when patients follow up more than 24 weeks [Log Rank (Mantel-Cox) analysis, chi2=3.85, P=0.047]; side effects were observed such as arthralgia, sweating, hot flash, and 2 patients developed heart failure with uncertain related drug before recovery., Conclusion: The single agent effective rate of aromatase inhibitor was 48%. The patients had long term survival if they obtained CR or PR, and the side effects were well tolerated.

TiO2 and montmorillonite composite photocatalysts were prepared and applied in degrading gamma-hexachlorocyclohexane (gamma-HCH) in soils. After being spiked with gamma-HCH, soil samples loaded with the composite photocatalysts were exposed to UV-light irradiation. The results indicated that the photocatalytic activities of the composite photocatalysts varied with the content of TiO2 in the order of 10% < 70% < 50% < 30%. Moreover, the photocatalytic activity of the composite photocatalysts with TiO2 content 30% was higher than that of the pure P25 with the same mass of TiO2. The strong adsorption capacity of the composite photocatalysts and quantum size effect may contribute to its increased photocatalytic activities. In addition, effect of dosage of composite photocatalysts and soil pH on gamma-HCH photodegradation was investigated. Pentachlorocyclohexene, trichlorocyclohexene, and dichlorobenzene were detected as photodegradation intermediates, which were gradually degraded with the photodegradation evolution.

Objective: To identify the FTIR spectroscopic characterization of breast cancer and explore the possibility of application of FTIR in differentiation of malignant and benign breast lesions., Methods: FTIR spectra of surgically removed fresh breast tissues were measured by spectrometer equipped with mid-infrared fiber optics and an ATR probe. Peaks in the spectra were measured and relative intensity ratios were calculated and analyzed if there are significant differences between the spectra of malignant and benign breast lesions., Results: There were significant differences (P < 0.05) between the spectra of malignant breast cancers and benign breast tissues in the relative intensity ratios of different peaks (I1640/ I1550 and I1160/I1120 for protein structures; I1640/I1460 and I1550/I1460 for relative content of protein and lipid; I1460/I1400 for lipid structures; I1310/I1240 for nucleic acid)., Conclusion: FTIR spectroscopy could be a useful tool in clinical diagnosis of breast cancer.

The adsorption kinetics for model pollutants on the activated carbon fiber (ACF) by polarization was investigated. Kinetics data obtained for the adsorption of these model pollutants at open-circuit and 400mV, -400mV polarization was applied to the Lagergren equation, and adsorption rate constants (Ka) were determined. With the anodic polarization of 400mV, the capacity of sodium phenoxide increases from 0.0083mmol x g(-1) at open-circuit to 0.18mmol x g(-1), and a seventeen-fold enhancement is achievable; however, the capacity of p-nitrophenol decreases from 2.93 mmol x g(-1) at open-circuit to 2.65 mmol x g(-1). With the cathodal polarization of -400mV, the capacity of aniline improves from 3.60 mmol x g(-1) at open-circuit to 3.88 mmol x g(-1); however, the capacity of sodium dodecylbenzene sulfonate reduces from 2.20 mmol x g(-1) at open-circuit to 1.59 mmol x g(-1). The enhancement for electrosorption changes with different groups substituting. Anodic polarization enhances the adsorption of benzene with electron-donating group. But whether anodic or not cathodal polarization has less effect on the adsorption of electron-accepting aromatic compounds, and decreases the adsorption of benzene bearing donor-conjugate bridge-acceptor but increases the adsorption rate. Electrostatic interaction plays a very important role in electrosorption of ion-pollutants.

The C-terminal region of the merozoite surface protein 1 (MSP119) is one of the most promising vaccine candidates against the erythrocytic forms of malaria. In the present study, a gene encoding Plasmodium falciparum MSP119 was expressed in yeast Pichia pastoris. A non-glycosylated form of the recombinant protein MSP119 was purified from culture medium. This recombinant protein maintains its antigenicity. Significant immune responses were seen in C57BL/6 mice after the second immunization. Moreover, the specific antibodies recognized the native antigens of P. falciparum. The prevailing isotypes of immunoglobulin (Ig) G associated with immunization were IgG1, IgG2a and IgG2b. The antibodies isolated from mouse sera immunized with MSP119 can inhibit parasite growth in vitro. Based on these immunological studies, we concluded that MSP119 deserves further evaluation in pre-clinical immunizations against P. falciparum.

Objective: To study the expression of cerbB-2 and its clinical biology value in Chinese breast cancer patients by evidence based medical analysis., Methods: All the published studies about cerbB-2 and breast cancer for the last 10 years were reviewed and standard techniques of meta-analysis to combined with the results of these studies were used to produce a more precise estimate of the prognostic significance expression of cerbB-2., Results: The mean of cerbB-2 positive expression was 50% (95% confidence interval 48%-54%), cerbB-2 positive expression was related with node metastasis, recurrence after surgery and survival time; the RR values were 1.71, 2.14 and 2.32 respectively; tumor size, nuclear grade and pathology type were also the important factors that were related with the expression of cerbB-2, while the expression was not related with age; cerbB-2 was a special and sensitive prognostic factor for breast cancer., Conclusion: cerbB-2 can be used as an independent molecular marker for definitive prognosis of breast cancer, as well as a reliable marker for choice of standard and individual therapy.

Aim: To investigate the effects of peripheral orexins on myoelectric activity of the sphincter of Oddi (SO) in fasted rabbits, and carry out a preliminary investigation into the mechanisms underlying these effects., Methods: Myoelectric activity of SO in fasted rabbits was recorded before and after intravenous or local application of orexins. The effects of intravenous atropine on orexin-increased myoelectric activity of SO were tested., Results: Myoelectric activity of SO was activated by both intravenous and local injection of orexin-A or orexin-B. Intravenous application of atropine completely inhibited the excitatory effect of orexins on SO., Conclusion: Peripheral application of orexins can increase myoelectric activity of SO in fasted rabbits, which is partially associated with the activation of the cholinergic pathway.

Ba-wei-xi-lei powder is a classical herbal mixture, and is widely used for the treatment of oral ulcer and ulcerative colitis. This study aimed to explore the effect of Ba-wei-xi-lei powder with enema application on ulcerative colitis in rats. Ulcerative colitis was induced by immunization with rabbit's colonic mucosal protein emulsified with Completely Freund's Adjuvant. The mucosal inflammatory reaction and ulcer have been observed in the model rats. Characteristic changes of ulceractive colitis include that CD4 lymphocyte increased in peripheral blood while CD8 lymphocyte decreased; CD8 lymphocyte and TNF-alpha expression area increased in colonic mucosa, while CD4 lymphocyte decreased. Ba-wei-xi-lei powder and sulfasalazine with enema application could alleviate the pathological changes in the model rats. The results suggest that the pharmacological effects of Ba-wei-xi-lei powder on ulcerative colitis in rats are similar to the effect of sulfasalazine.

Photodegradation of pentachlorophenol (PCP) and p-nitrophenol (PNP) in soil was carried out in a designed rotary reactor, which can provide the soil particles with continually uniform irradiation, and on a series of thin soil layers. TiO2, as a kind of environmental friendly photocatalyst, was introduced to the soil to enhance the processes. Compared with that on the soil layers, photodegradation of PCP at initial concentration of 60 mg/kg was improved dramatically in the rotary reactor no matter whether TiO2 was added, with an increase of 3.0 times in the apparent first-order rate constants. The addition of 1 wt% TiO2 furthered the improvement by 1.4 times. Without addition of TiO2, PNP (initial concentration of 60 mg/kg) photodegradation rate in the rotary reactor was similar to that on the soil layers. When 1 wt% additional TiO2 was added, PNP photodegradation was enhanced obviously, and the enhancement in the rotary reactor was 2 times of that on the soil layers, which may be attributed to the higher frequency of the contact between PNP on soil particles and the photocatalyst. The effect of soil pH and initial concentrations of the target compounds on the photodegradation in the rotary reactor was investigated. The order of the degradation rate at different soil pH was relative to the aggregation of soil particles during mixing in the rotary reactor. Photodegradation of PCP and PNP at different initial concentrations showed that addition of TiO2 to enhance the photodegradation was more suitable for contaminated soil with higher concentration of PCP, while was effective for contaminated soil at each PNP concentration tested in our study.

Since the first publication of the Chinese Journal of Ophthalmology on October 1st, 1950, following the establishment of the People's Republic of China, more than fifty years have already elapsed. The editors and publishers of the elder generation of this magazine have witnessed the hardships in starting publication, the disappointments of the discontinuation of publication during certain periods, as well as the great delight of restarting publication. Having entered the 21st century, editors and publishers of the new generation have made dramatic achievements by keeping up with the times and continuously seeking innovations. However all of these achievements have only turned out to be the past. There are even more challenges for moving ahead in the future. The Chinese Journal of Ophthalmology has noted its achievements on the one hand; while on the other hand, has analyzed the main existing problems of this magazine. Some feasible approaches to improve the magazine have been proposed and the direction in the following few years was ascertained. We will collaborate with Chinese ophthalmologists to work towards the continuous development of this magazine.

The authors detected ten normal breast tissue samples and eight breast cancer samples by FTIR spectroscopy with an ATR probe. Nineteen variables of thirteen bands in the spectra were compared using standard statistic methods. The results demonstrated that bands of protein, lipid, carbohydrate, and nucleic acid from cancerous samples were significantly different from those from normal ones: (1) The relative intensity of N-H band increased and amide I band shifted to lower wave number significantly; (2) Symmetric and antisymmetric vibrations of -CH2 group, C=O vibration, and relative intensity of (-CH2)n decreased; (3) The intensity of 1160 cm(-1) band was much weaker than that of 1120 cm(-1); (4) The band of P=O or P-O-C shifted toward lower wave number. The authors believe that FTIR spectroscopy has a promising future in breast cancer diagnosis.

A novel process, microwave assisted catalytic wet air oxidation (MW-CWO), was applied for the degradation of H-acid (1-amino-8-naphthol-3, 6-disulfonic acid) in aqueous solution. Ni-loaded granular activated carbon (GAC), prepared by immersion-calcination method, was used as catalyst. The results showed that the MW-CWO process was very effective for the degradation of H-acid in aqueous solution under atmospheric pressure with 87.4% TOC (total organic carbon) reduction in 20 min. Ni on GAC existed in the form of NiO as specified by XRD. Loss of Ni was significant in the initial stage, and then remained almost constant after 20 min reaction. BET surface area results showed that the surface property of GAC after MW-CWO process was superior to that of blank GAC.

We report here the discovery of Malassezia furfur from a groin abscess of a renal transplant patient. A 33-year-old male patient was admitted to our hospital because of a high fever and a persistent inflammatory nodule on his right groin for one week. He had received a renal transplant 3 years before and remained on immunosuppressive agents. He was treated with broad-spectrum antibiotics after hospitalization but the nodule formed a large abscess and then a deep ulcer instead of resolving. Examination of the culture by light microscopy revealed ovoid budding yeasts displaying collar-shaped structure. Subculture of the primary colonies onto Sabouraud's dextrose agar and medium containing rapeseed oil resulted in growth only on the medium containing rapeseed oil. All of the isolates was identified as Malassezia furfur. The pathogenicity of the isolates was tested in mice by intravenous injection of (3-5) x 10(8) cfu per mouse after immunosuppression with 500 mg/kg of prednisone intraporitoneally on day-2. In the mouse model, micro-abscess and inflammatory reaction and oval yeasts with budding were noted in histopathologic section of the viscera of the mice. A rib-like or serrate-like structure of the inner side of cell wall, characteristic for Malassezia spp., was observed by transmission electron microscopy. The patient received oral fluconazole and topical amphotericin B. The isolate before antifungal therapy was sensitive to both fluconazole and amphotericin B, while the isolate after antifungal treatment was only sensitive to amphotericin B. Proteinase activity of the isolates increased 1.43 times after antifungal treatment. This case indicated the invasive power of M. furfur in deep infection. Renal transplantation and reception of long-term immunosuppressive treatment are risk factors for the invasive infection of this fungus.

The TiO2 nanotube sample was prepared via a NaOH solution in a Teflon vessel at 150 degrees C. The as-prepared nanotubes were then treated with H2 SO4 solutions. The TiO2 nanotube has a crystalline structure with open-ended and multiwall morphologies. The TiO2 nanotubes before and after surface acid treatment were characterized by X-ray diffraction (XRD), scanning electron microscopy(SEM), transmission electron microscopy (TEM) and UV-VIS dispersive energy spectrophotometry (DRS). The photocatalytic activity of the samples was evaluated by photocatalytic degradation of acid orange II in aqueous solutions. It was found that the order of photocatalytic activity was as follows: TiO2 nanotubes treated with 1.0 mol/L H2SO4 solution (TiO2 (1.0M H2SO4) snanotubes) > TiO2 nanotubes treated with 0.2 mol/L H2SO4 solution (TiO2 (0.2M H2O4) nanotubes) > TiO2 nanotubes > TiO2 powder. This was attributed to the fact that TiO2 nanotubes treated with H2SO4 was composed of smaller particles and had higher specific surface areas. Furthermore, the smaller TiO2 particles were beneficial to the transfer and separation of photo-generated electrons and holes in the inner of and on the surface of TiO2 particles and reduced the recombination of photo-generated electrons and holes. Acid treatment was particularly effective for TiO2 nanotubes, this increase in activity was correlated with the concentration of H2 SO4 solution.

The present work extended the knowledge on the binding and complexation of humic substances( humic acid or fulvic acid) and hematite by Fourier transform infrared spectroscopy( FTIR) . The FTIR data gained gave the consist evidences by two different sampling preparation methods that the interaction mechanism between humic substances and hematite was mainly conform to the ligand-exchange involving carboxylic functional groups of humic substances and the surfaces sites of hematite. The present method, although associated with some uncertainties, provided an opportunity to increase the knowledge in this field.

Objective: To confirmed reliability and feasibility of intranasal nerve growth factor (NGF) bypassing the blood-brain barrier and its potential neuroprotective effects on acute cerebral ischemia., Methods: (1) To assay NGF concentrations in different brain regions after middle cerebral artery occlusion (MCAO). Rats were randomly divided into intranasal (i.n.) NGF, intravenous (i.v.) NGF, and untreated group (n = 4). The concentrations of NGF of different brain regions in the three groups after MCAO were measured by ELISA. (2) To observe neuroprotective action of NGF on focal cerebral ischemic damage. Rats were randomly assigned to 4 groups: i.n. vehicle, i.n. NGF, i.v. vehicle, i.v. NGF (n = 8). Treatment was initiated 30 minutes after onset of MCAO and given again 24 hours later. Three neurologic behavioral tests were performed 24 and 48 hours following onset of MCAO. Corrected infarct volumes were determined 48 hours after onset of MCAO., Results: The olfactory bulb in i.n. NGF group obtained the highest concentration (3252 pg/g) of NGF among all regions, followed by the hippocampus. The NGF concentrations in the olfactory bulb and hippocampus in i.n. NGF group were markedly higher than that in i.v. NGF and control groups. The infarct volume in i.n. NGF group was markedly reduced by 38.8% compared with i.n. vehicle group. I.n. NGF group vestibulum function markedly improved compared with i.n. vehicle group at 24 and 48 hours after onset of MCAO (P24h = 0.02 and P48h = 0.04, respectively)., Conclusion: Intranasal NGF could pass through the blood-brain barrier, reach the central nervous system, reduce infarct volume, and improve neurologic function in rats following MCAO. Intranasal delivery of NGF may be a promising treatment for stroke.

Objective: To study the material basis of the tonifying effect of Radix Changii by observing its contents of nutrient elements., Methods: The contents of vitamins in Radix Changii and inorganic elements in the water extracts of it were tested by high performance liquid chromatography, fluorescence spectrophotography and scanning electron microanalysis., Results: Radix Changii contains fat-soluble vitamins E and K 259.2 and 64.1 microg/kg respectively, and water-soluble vitamins C, B1 and B2 78.5, 0.6 and 0.7 mg/kg respectively. Its decoction contains abundant potassium and calcium ions., Conclusion: Radix Changii is rich in elements necessary for human body. It has the material basis for tonification.

In this study, L929 cell adhesion, proliferation and apoptosis were investigated on chitosan (CS) and chitosan-gelatin (CS-Gel) membranes having different degrees of deacetylation (DD). The mitochondrial activity [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay results demonstrated that the DD played a key role in the L929 cell adhesion. Indeed, the higher the DD of a CS membrane, the stronger the cell adhesion. The cell cycle analysis showed that the CS surface inhibits most of the cells entering the cell cycle and the DD of CS had little effect on the cell cycle progression. On the other hand, this study also confirmed that CS, blended with Gel, induced L929 cells to enter the cell cycle, encouraged proliferation, decreased the degree of apoptosis on the CS membranes, and are comparable to the results from L929 cells on tissue culture plates. In brief, CS modified with Gel improves cytocompatibility by shielding the positively charged CS with a high charge density.