[The long-term efficacy of left cardiac sympathetic denervation in long QT syndrome].

Objective: To investigate the long-term efficacy and safety of left cardiac sympathetic denervation(LCSD) for long QT syndrome(LQTS) patients with either recurrence on drug therapy intolerance/refusal. Methods: This study was a retrospective cohort study. The cases selected from 193 patients with LQTS who were enrolled in the Chinese Channelopathy Registry Study from November 1999 to November 2012. This study selected 28 LQTS patients with either recurrence on drug therapy intolerance/refusal and underwent LCSD surgery in the Peking University People's Hospital or Beijing Tongren Hospital. The patients were allocated into 3 groups: high-risk group(n=13, baseline QTc ≥550 ms or symptomatic in the first year of life or highly malignant genetics); intermediate-risk group(n=10, 500 ms≤baseline QTc<550 ms, symptomatic after the first year and without highly malignant genetics); low-risk group(n=5, baseline QTc<500 ms, symptomatic after the first year and without highly malignant genetics). LCSD was performed with the traditional supraclavicular approach or video assisted thoracoscopic surgery (VATS). Patients were regularly followed up until 20 years after the surgery. Data were collected before and 1 year after surgery and at the last follow-up. Patients' electrocardiograph(ECG), cardiac events and surgery-related complications were recorded. Kaplan-Meier survival analysis was used to determine the cardiac event-free survival based on different risk stratification and genotypes. Results: A total of 28 LQTS patients, aged 20.5 (15.0, 37.5) and underwent LCSD surgery, were enrolled in this study, including 23(82.1%) women. There were 11(39.3%) patients treated with traditional approach while 17(60.7%) with VATS-LCSD. There were 19(67.9%) patients had positive genetic test results, including 4 LQT1, 12 LQT2, 1 LQT1/LQT2 mixed type, and 2 Jervell-Lange-Nielsen (JLN) syndrome. The median follow-up period was 189.3(138.7, 204.9) months. The dropout rate was 10.7%(3/28) while 3 patients in the intermediate-risk group were lost to follow-up. Horner syndrome occurred in 1 patient (in the high-risk group). Sudden cardiac deaths were observed in 3 (12.0%) patients (all in the high-risk group), and 12 patients (48.0%) had syncope recurrences (2 in low-risk group, 3 in intermediate-risk group and 7 in high-risk group). A significant reduction in the mean yearly episodes of cardiac events was observed, from (3.5±3.3) before LCSD to(0.2±0.1) at one year after LCSD and (0.5±0.8) at last follow up(P<0.001). The mean QTc was shortened from (545.7±51.2)ms before the surgery to (489.0±40.1)ms at the last follow-up (P<0.001). Among the 20 patients with basic QTc ≥500 ms and completing the follow-up, the QTc intervals of 11(55.0%) patients were shortened to below 500 ms. The event free survival rates for any cardiac events after LCSD decreased sequentially in the low-, intermediate- and high-risk groups, and the difference was statistically significant (χ²=7.24, log-rank P=0.026). No difference was found in the event free survival rates among LQT1, LQT2 and undefined gene patients (χ²=5.20, log-rank P>0.05). Conclusions: LCSD surgery can reduce the incidence of cardiac events and shorten the QTc interval in patients with LQTS after the long-term follow-up. LCSD surgery is effective and safe for patients with LQTS ineffective or intolerant to drug therapy. However, high-risk patients are still at a high risk of sudden death after surgery and should be actively monitored and protected by combined therapies.

[The IL-6/STAT3 signaling pathway mediated by radiotherapy regulates the expression of PD-L1 in esophageal cancer cells].

Objective: To explore the possible mechanism of radiotherapy regulating the expression of PD-L1 in esophageal carcinoma. Methods: Three esophageal cancer cell lines (Eca109, Kyse150, TE1) were irradiated with different doses of X-rays, and 6 Gy+ AG490 group was set. The mRNA expression of PD-L1 was detected by real-time quantitative polymerase chain reaction (RT-qPCR). The protein expressions of PD-L1, STAT3, p-STAT3 were detected by western blotting and the protein level of IL-6 was detected by ELISA. Results: The mRNA expressions of PD-L1 in Eca109, Kyse150 and TE1 were 2.86±0.30, 960.01±21.27 and 106.78±6.67, higher than 1.07±0.15 in normal esophageal cell line HET-1A (P<0.01). The protein expressions of PD-L1 in Eca109, Kyse150 and TE1 were 0.091±0.036, 1.533±0.079 and 0.914±0.035, higher than 0.063±0.01 in normal esophageal cell line HET-1A (P<0.01). After 48 hours of 6 Gy irradiation, the protein expression levels of PD-L1 in Eca109, Kyse150 and TE1 were 0.135±0.007, 1.66±0.06 and 1.32±0.06, higher than 0.09±0.01, 1.21±0.05 and 0.93±0.03 of the 0 Gy group (P<0.01), while the protein expression levels of p-STAT3 in Eca109, Kyse150 and TE1 were 1.44±0.26, 0.75±0.04 and 1.92±0.17, higher than 0.18±0.05, 0.48±0.02 and 0.36±0.06 of the 0 Gy group (P<0.01). IL-6 protein expression increased significantly after different doses of irradiation (P<0.01). After the IL-6/STAT3 signaling pathway was blocked by the specific inhibitor AG490, the expressions of PD-L1 of Eca109, Kyse150 and TE1 in the 6 Gy+ AG490 groups were 0.11±0.03, 1.07±0.08 and 0.96±0.11, without significant differences of 0.09±0.01, 0.96±0.05 and 0.85±0.09 of the 0 Gy group (P>0.05), while the protein expressions of p-STAT3 were 0.76±0.11, 0.59±0.06 and 0.96±0.12, without significant differences of 0.67±0.08, 0.54±0.06 and 0.84±0.11 of the 0 Gy group (P>0.05). Conclusion: Radiotherapy may regulate the expression of PD-L1 in esophageal cancer cells through IL-6 / STAT3 signaling pathway.

[Analysis of factors influencing the false-negative diagnosis of cervical/vaginal liquid based cytology].

Objective: To investigate the possible influencing factors of false-negative diagnosis of cervical/vaginal liquid based cytology, and further improve the sensitivity of cervical/vaginal cytology. Methods: The results of cervical/vaginal cytology of outpatients and inpatients in Department of Obstetrics and Gynecology, Peking University First Hospital from July 2015 to December 2018 were analyzed retrospectively. Cytological false-negative cases were defined as the patients whose cytological results showed no intraepithelial neoplasia and malignant (NILM), but whose biopsy was diagnosed as cervical intraepithelial neoplasia (CIN) 2 or vaginal intraepithelial neoplasia (VAIN) 2 or above within 6 months of the diagnosis. The review of false-negative cytology smear was completed by two senior cytologists. Two-class logistic regression was used to evaluate the influence of age, location or number of lesion, and degree of lesion on the false-negative diagnosis of cytology. The reasons for the inconsistency of false-negative diagnosis were analyzed with the review results. Results: Among 1 009 cases of CIN2+ and VAIN2+ lesions, 180 cases (17.8%) showed NILM. After reviewing the smear, 123 cases (68.3%) were identified as NILM and 57 cases(31.7%) as abnormal. The false-negative rate was the highest (20.8%) in the patients with age≤30 years, and the risk was 8.85 times higher than the patients aged 31 to 60 years (P<0.001), 9.26 times than the patients aged≥60 years (P<0.001). The highest cytological false-negative rate was 50.0% for cervical polyps or intraductal lesions. The false-negative rate of vaginal wall or vaginal pedicle rupture was 13.0%; that of single cervical lesion was 22.3%; that of high-grade squamous intraepithelial lesion(HSIL) and adenocarcinoma in situ of cervix(AIS) was 13.7% and that of malignant lesions was 3.9%. The most common cell types in the reviewed abnormal cases were squamous cells in the middle surface layer (38.6%) and squamous cells in the outer bottom layer (24.6%). The abnormal cells in all smears was the most common distribution (59.7%), the number of abnormal cells in the smear was less than 10 (31.6%), nuclear enlargement and light staining were common (42.2%), and inflammatory lesions or keratotic changes in the background were most common (59.7%). Conclusions: Age of the patient, location or number of lesion, and degree of lesion are associated with false-negative diagnosis of cytology. Summarizing sampling experience and improving sampling skills will help reduce the occurrence of false-negative cases. Cytopathologists should examine slightly abnormal changes more carefully and learn how to further reduce the false-negative rate procactively.

[Paediatric nodal marginal zone lymphoma: a clinicopathological study of seven cases].

Objective: To investigate the pathological features and clinical manifestation of pediatric nodal marginal zone lymphoma(NMZL). Methods: Histological morphology and immunophenotype of 7 cases of pediatric NMZL were retrospectively reviewed at Beijing Friendship Hospital Affiliated to Medical University between January 2008 to October 2018. Clonal rearrangement analysis was performed. Clinical information including patient follow-up data were analyzed. Results: All 7 patients were male with a median age of 15 years aged from 10 to 26 years. All patients presented with only lymph node enlargement without B symptoms, including cervical lymph node (5 cases), preauricular lymph node (1 case) and retroauricular lymph node (1 case). Histologically, all cases showed irregular large follicles on the edges with widened marginal areas and intervesicular areas, and lesional cells were uniform with progressive transformation of germinal center centers along with a small amount of intrinsic lymphoid tissue. All 7 cases showed diffuse CD20 positivity both follicle and interfollicular region along with 30%-40% positivity in the interfollicular region (pathological region). Markers of other B-cell lymphomas werenot expressed. All 7 cases were positive for immunoglobulin(Ig) gene rearrangement. None of the patients showed no recurrence up on after follow-up for an average of 13 months. Conclusions: Pediatric NMZL is a rare type of lymphoma that has a unique morphology and occurs almost exclusively in male children and young adults and often in head and neck lymph nodes. It has an excellent prognosis. Therefore, awareness of the disease with accurate diagnosis is important.

[Application of intraoperative CT in oral and maxillofacial surgery].

With the introduction of the concept of precision surgery and the development of modern compound operating rooms, intraoperative computerized tomography (ICT) came into being. Due to the multidisciplinary nature of ICT, its use in oral and maxillofacial surgery has also been increasing in recent years. This article describes the application of ICT in oral and maxillofacial trauma, orthognathic surgery, and craniofacial tumor treatment, and analyzes its advantages and limitations.

General solvothermal approach to synthesize telluride nanotubes for thermoelectric applications.

One-dimensional tellurides are good candidates for thermoelectric applications, but the fabrication of telluride nanotubes is still challenging. To this end, the solvothermal approach is proposed to synthesize Bi2Te3, PbTe, CuxTe and Ag2Te nanotubes. In this scheme, single-crystal Te nanotubes are produced first and then used as the sacrificed template for epitaxial growth of metal telluride. It was demonstrated that polycrystalline telluride nanotubes are produced. Considering Bi2Te3 nanotubes as an example, the pellets are prepared by spark plasma sintering, and the thermoelectric properties are measured. Compared to the nanowire counterpart, the higher-energy barrier to electrons at the grain boundaries (GBs) leads to an optimized power factor of 1.04 mW m-1 K-2 at 373 K in the nanotube samples. Furthermore, the thermal conductivity of nanotubes is in the range of 0.503-0.617 W m-1 K-1, which is much smaller than that of the nanowires. The ultralow thermal conductivity could be attributed to both the higher potential barrier of GBs and the additional scattering of phonons at the side walls of the nanotubes. In all, a ZT value of 0.74 was obtained at 373 K, which is much higher than that of nanowires. This synthesis route is ready to be extended to other telluride nanotubes.

[Acute suppurative otitis media caused by pasteurella multocida :a case report].

The pasteurella multocida (PM) is widely gastrointestinal parasitic on dogs, cats and other animals. The PM human infections is caused by animal bites orits close contact. Clinically foundin focal wound infection after bite, and acute suppurative otitis media reportshave not been caused by PM. A 45 years old nasopharyngeal cancer who sudden stabbing painof the left ear for 5h. Physical examination found that the left earexisted a lot of yellowish white purulent secretion. Distribution and drug sensitivity test of bacteria showed PM and it was sensitive to many antibiotics. nasopharygo fiberscope revealed that eustachian tube and theleft ear had a large number of purulentsecretion. The main diagnosis was: ①Recurrent nasopharyngealcarcinoma; ② Acute suppurative otitis media. TREATMENT: according to the results of drug sensitive test and skin test inpatients with selection of levofloxacin (0.2 g/12 h), clindamycin (0.6 g/12 h) anti-infection treatment, the patient get better in the end.

[Clinical application of intraoperative CT in management of maxillofacial fracture].

Nine patients with maxillofacial fracture that received intraoperative CT examination in Lanzhou General Hospital of Lanzhou Military Command from January 2017 to March 2017 were retrospectively studied. The procedure of intraoperative CT was introduced. The value of this technique was preliminarily discussed in order to provide a new method for the accurate implementation of maxillofacial fracture surgery.

ERK1/2/p53 and NF-κB dependent-PUMA activation involves in doxorubicin-induced cardiomyocyte apoptosis.

OBJECTIVE: Numerous studies have demonstrated that Doxorubicin (DOX) induces cardiomyocyte apoptosis, which is associated with DOX-induced acute and chronic cardiotoxicity. DOX activated ERP1/2 and NF-KB signals has been linked to DOX-induced apoptosis and cardiotoxicity. However, the underlying mechanisms responsible for DOX-induced apoptosis have not been completely elucidated. In this study, we determine whether both ERK1/2/p53-dependent and NF-κB dependent-PUMA activation was related to DOX-induced apoptosis in H9c2 cells. MATERIALS AND METHODS: H9c2 cells were treated with DOX (1 µM) for 2-48 hours. To explore the effect of ERK1/2, NF-KB, P53 and PUMA on DOX-induced apoptosis in H9c2 cells, H9c2 cells were transfected with PUMA siRNA or p65 siRNA, or treated with PFT-α (a chemical inhibitor of p53), or PD98059 (ERK inhibitor) before DOX treatment. MTT, Flow cytometry, TUNEL, Western blot and EMSA assay was used to detect cell survival, apoptosis, protein expression and NF-KB activity. RESULTS: DOX induced apoptosis and inhibited growth of H9c2 cells in a time-dependent manner. DOX activated ERK1/2, NF-KB, p53 and PUMA. Knockdown of PUMA completely blocked DOX-induced cell apoptosis and survival inhibition. Knockdown of NF-KB or ERK1/2 alone could partly block DOX-induced PUMA upregulation and cell apoptosis. However, knockdown of NF-KB and ERK1/2 together completely blocked DOX-induced cell apoptosis and PUMA upregulation. In addition, knockdown of ERK1/2 blocked p53-dependent PUMA upregulation. CONCLUSIONS: DOX induced apoptosis and inhibited growth of H9c2 cells by activation of ERK1/2/p53 and NF-κB dependent-PUMA signaling pathway.

Upregulation of salivary α2 macroglobulin in patients with type 2 diabetes mellitus.

We investigated the expression of salivary α2-macroglobulin (α2-MG) in patients with type 2 diabetes mellitus (T2DM) to investigate its value for predicting damage to the salivary glands. A total of 116 patients with T2DM and 60 patients with impaired fasting glucose (IFG) were included in this study. Sixty health volunteers were enrolled as a control group. Unstimulated saliva was collected at 8 a.m. prior to breakfast. Expression of α2-MG was determined using an enzyme-linked immunosorbent assay. The correlation between salivary α2-MG, serum α2-MG, and concentration of fasting glucose was analyzed using Pearson correlation analysis. No significant difference was observed in the expression of serum α2-MG in the T2DM group, IFG group, and control group (P > 0.05). Compared with the control group and IFG group, a statistical difference was observed in the salivary α2-MG in the T2DM group (P < 0.01). No statistical difference was observed in the salivary α2-MG in the IFG group compared with the control group (P > 0.05). In the patients with T2DM, a close correlation was identified in the expression of serum α2-MG and salivary α2-MG (r = 0.52, P < 0.01). A poor correlation was identified between salivary α2-MG and blood sugar level (r = -0.12, P = 0.199). The expression of salivary α2-MG showed a remarkable increase in T2DM patients, which may be associated with functional disorders of the salivary gland.

High-quality, single-layered epitaxial graphene fabricated on 6H-SiC (0001) by flash annealing in Pb atmosphere and mechanism.

High-quality epitaxial graphene is produced on silicon carbide by flash annealing of 6H-SiC in a lead (Pb) atmosphere at ∼1400 °C for 30 s. Nearly three top bilayers of SiC are decomposed due to fast heating and cooling, and sublimation of Si atoms from SiC is retarded by the Pb atmosphere. The synergetic effects promote the growth of continuous single-layered graphene sheets on the SiC terraces, and a model is established to elucidate the effects and growth mechanism.

Summer flounder (Paralichthys dentatus) sperm cryopreservation and application in interspecific hybridization with olive flounder (P olivaceus).

The present study established an efficient technology for summer flounder (Paralichthys dentatus) sperm cryopreservation, and successfully applied the cryopreserved sperm into interspecific hybridization with olive flounder (P olivaceus). The best motility of postthaw sperm (78.00 ± 4.70% and 76.60 ± 7.90%), fertilization rates (95.70 ± 3.60% and 79.40 ± 5.20%), and hatching rates (93.10 ± 4.00% and 77.20 ± 2.90%) were achieved when using propylene glycol or DMSO as cryoprotectant. Furthermore, we have successfully improved the cryopreservation method from using 2-mL cryotube to 5-mL cryotube, and the dilution ratio has been increased to 4:1. By this method, the cryopreservation efficiency has been improved to 30 times of that with routine method. Computer-assisted sperm motion analysis showed the freezing-thawing process decreased the sperm speed but did not significantly change the sperm movement pattern, and the progressive linear motion still was the dominant movement pattern. The ultrastructural analysis showed 50% to 60% of the spermatozoa had normal morphology, 20% to 30% were slightly damaged, such as swelling or rupture of head, midpiece, and tail region, and 10% to 20% were severely damaged. In the artificial hybridization experiment, high fertilization rates and hatching rates were achieved when using 15% DMSO (95.7 ± 3.6% and 79.4 ± 5.2%, respectively) and 15% propylene glycol (93.1 ± 4.0% and 77.2 ± 2.9%, respectively), with no significant difference in comparison with control (94.7 ± 2.6% and 72.5 ± 6.5%, respectively). In addition, we found the embryos and larvae from postthaw sperm of P dentatus developed and grew normally. The results of the present study further validated the safety of the cryopreserved sperm in breeding production by assessing the fertilization capacity, embryo development, and larval growth.

Screening miRNAs related to different subtypes of breast cancer with miRNAs microarray.

AIM: The aim of this study was to screen miRNAs related to different subtypes of breast cancer and their target genes to identify new markers of tumor subtype. MATERIALS AND METHODS: The miRNA expression profiles of breast cancer GSE38867 including 7 ductal carcinoma in situ breast (DCIS) cancer samples, 7 invasive breast cancer samples, 7 metastatic breast cancer samples, and 7 normal breast samples) were downloaded from Gene Expression Omnibus (GEO) database. Limma package in R software was applied to identify specific differentially expressed miRNAs of different subtypes of breast cancer. MicroRNA.org database source was used to predict the target genes of the identified differentially expressed miRNAs. We integrated the target genes and their interacted genes (predicted by STRING) into DAVID to perform the GO function and KEGG pathway analyses. RESULTS: Compared to the normal control, a total of 21, 47, and 107 differentially expressed miRNAs were screened in DCIS, invasive and metastatic breast cancer, respectively. Specific differentially expressed miRNAs of the three subtypes were identified, including hsa-miR-99a and hsa-miR-151-3p for DCIS breast cancer, hsa-miR-145 and hsa-miR-210 for invasive breast cancer, and has-miR-205 and has-miR-361-5p metastatic breast cancer. Furthermore, 220, 43, 446, 307, 587 and 328 interaction pairs of the specific miRNA targets were predicted. Multiple GO functions and KEGG pathways were enriched with the miRNA targets and their interacted genes. CONCLUSIONS: We screened the most representative miRNAs of the three different subtypes of breast cancer, which may act as the putative markers in the diagnosis of different subtypes of breast cancer.

CpG methylation of dmrt1 and cyp19a promoters in relation to their sexual dimorphic expression in the Japanese flounder Paralichthys olivaceus.

To better understand the effects of DNA methylation on the expression patterns of dmrt1 (Doublesex and Mab-3-related transcription factor 1) and cyp19a (Cytochrome P450 19a) in the Japanese flounder Paralichthys olivaceus, quantitative expressions, cellular distributions and cytosine-p-guanine (CpG) methylation patterns of these two genes in the gonads were analysed. The results showed that P. olivaceus dmrt1 expression was 70 times higher in the testis than in the ovary (P < 0·05). Its mRNA was detected clearly in spermatocytes and Sertoli cells of the testis, but weakly in the ovary. Paralichthys olivaceus cyp19a expression was 40 times higher in the ovary than in the testis (P < 0·01). Its mRNA was detected clearly in follicular cells of the ovary, but weakly in spermatocytes of the testis. The dmrt1 promoter CpGs were not methylated in the testis, whereas 57·69% were methylated in the ovary. For the cyp19a promoter CpGs, 97·5% were methylated in the testis and 73·33% were methylated in the ovary. These findings demonstrate that P. olivaceus dmrt1 and cyp19a are sex-related genes with sexual dimorphic expression, CpG methylation levels of the two genes are consistent with their expression quantities, and this epigenetic modification can influence the differential expression of genes in the gonads of P. olivaceus.

Germline-specific and sexually dimorphic expression of a dead end gene homologue in turbot (Scophthalmus maximus).

Germ cells are indispensable for gonadal development and fertility. However, the physiological mechanisms regulating germ cell development in marine fish are poorly understood due to a lack of germ cell markers. The dead end (dnd) gene is a vertebrate-specific component of germplasm crucial for primordial germ cells (PGCs) migration and development in teleosts. In this study, we identified a dnd homologue (Smdnd) in turbot (Scophthalmus maximus) and investigated its expression pattern during embryogenesis and gonadal development. The deduced amino acid sequence of Smdnd shared several conserved motifs of Dnd homologues as well as high identity to other Dnd proteins. Phylogenetic analysis revealed that the SmDnd was closely related to its teleost counterparts. Reverse transcription polymerase chain reaction (RT-PCR) and in situ hybridization revealed that Smdnd transcripts could be exclusively detected in germ cells, including presumptive PGC and adult male and female germ cells. In addition, an interesting sexually dimorphic expression of Smdnd during gonadal development was observed by real-time PCR. Female turbot showed greater (P < 0.05) Smdnd expression than male before sex maturation. This difference reduced gradually due to the upregulation of Smdnd in the male during the period corresponding to spermatogonia proliferation and meiosis. These results indicate that Smdnd can be used as a germ cell marker in turbot. In addition, the temporal and sex differences in Smdnd expression indicate that this gene may play different roles in gonadal development in both sexes.

Germ cells and fertilization differences among Japanese flounder Paralichthys olivaceus, summer flounder Paralichthys dentatus and their first and second generations.

The morphology of gametes and the fertilization biology of Japanese flounder Paralichthys olivaceus (Po), summer flounder Paralichthys dentatus (Pd) and their hybrids were examined. Multiple generations (two hybrids: Poâ™€× Pd♂ (F1) and Pdâ™€× Po♂; two backcrosses: F1â™€× Po♂ and F1â™€× Pd♂) were obtained by artificial insemination. Comparison of egg ultrastructure among Po, Pd and F1 showed the morphology of micropyle region and the distribution density of pores were species specific. There were c. 100-200 accessory openings around the micropyle in Po, but not in Pd and F1. The zona radiata thickness and number of parallel bands were similar between F1 and Po, which were different from Pd. Comparison of spermatozoa ultrastructure revealed a close relationship between Po and Pd. Cytologically, the six crosses obeyed normal fertilization and cleavage processes, and only one male pronucleus was observed in a fertilized egg, indicating a monospermic fertilization pattern. Analysis of the time distribution from fertilization to first cleavage revealed an obvious delay at pronucleus fusion in the Pd × Po cross. The delay might indicate some cytoplasmic-nuclear incompatibility during the process of fertilization.

Skeletal development and abnormalities of the vertebral column and of the fins in hatchery-reared turbot Scophthalmus maximus.

To describe the skeletal development and abnormalities in turbot Scophthalmus maximus, samples were collected every day from hatching to 60 days after hatching (DAH). A whole-mount cartilage and bone-staining technique was used. Vertebral ontogeny started with the formation of anterior haemal arches at 5·1 mm standard length (L(S) ) c. 11 DAH, and was completed by the full attainment of parapophyses at 16·9 mm L(S) c. 31 DAH. Vertebral centra started to develop at 6·3 mm L(S) c. 16 DAH and ossification in all centra was visible at 11·0 mm L(S) c. 25 DAH. The caudal fin appeared at 5·1 mm L(S) c. 11 DAH and ossification was visible at 20·6 mm L(S) c. 37 DAH. The onset of dorsal and anal fin elements appeared at 5·8 mm L(S) c. 15 DAH and 6·3 mm L(S) c. 16 DAH, respectively. Ossifications of both dorsal fin and anal fin were visible at 20·6 mm L(S) c. 37 DAH. The pectorals were the only fins present before first feeding, their ossifications were completed at 23·5 mm L(S) c. 48 DAH. Pelvic fins began forming at 7·2 mm L(S) c. 19 DAH and calcification of the whole structure was visible at 19·8 mm L(S) c. 36 DAH. In the present study, 24 types of skeletal abnormalities were observed. About 51% of individuals presented skeletal abnormalities, and the highest occurrence was found in the haemal region of the vertebral column. As for each developmental stage, the most common abnormalities were in the dorsal fin during early metamorphic period (stage 2), vertebral fusion during climax metamorphosis (stage 3) and caudal fin abnormality during both late-metamorphic period (stage 4) and post-metamorphic period (stage 5). Such research will be useful for early detection of skeletal malformations during different growth periods of reared S. maximus.

Digestive enzyme activities of turbot (Scophthalmus maximus L.) during early developmental stages under culture condition.

Digestive enzyme activities were analysed in turbot (Scophthalmus maximus) from hatching until 60 days after hatching (DAH). Trypsin sharply increased to the climax at 17 DAH and decreased until 31 DAH followed by a stable level thereafter. Amylase was determined at 4 DAH, reached the maximum value at 19 DAH and declined sharply to 39 DAH and remained at a low level thereafter, suggesting the carbohydrate component should remain at a low level in formulated diets. Pepsin was detected at 9 DAH and increased to 34 DAH and then remained at a stable level. The above results revealed pancreatic enzymes are no longer main enzymes for food digestion after the formation of functional stomach. Leucine-alanine peptidase (Leu-ala) and alkaline phosphatase (AP) and leucine aminopeptidase N (LAP) were found in newly hatched larvae. Both AP and LAP activities markedly increased to 23 DAH, decreased abruptly to 50 DAH and increased gradually to 60 DAH. Leu-ala reached the plateau from 23 to 39 DAH, followed by a decline to 46 DAH and an increase until 60 DAH. The brush border membrane (BBM)-bound enzyme activities increased from 30% at 31 DAH to 81% at 38 DAH of the total activities, indicating the maturation of intestinal tract.