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Half-smooth tongue sole (Cynoglossus semilaevis) is one of the most important flatfish species for aquaculture in China. To produce a monosex population, we attempted to develop a marker-assisted sex control technique in this sexually size dimorphic fish. In this study, we identified a co-dominant sex-linked marker (i.e., CyseSLM) by screening genomic microsatellites and further developed a novel molecular method for sex identification in the tongue sole. CyseSLM has a sequence similarity of 73%–75% with stickleback, medaka, Fugu and Tetraodon. At this locus, two alleles (i.e., A244 and A234) were amplified from 119 tongue sole individuals with primer pairs CyseSLM-F1 and CyseSLM-R. Allele A244 was present in all individuals, while allele A234 (female-associated allele, FAA) was mostly present in females with exceptions in four male individuals. Compared with the sequence of A244, A234 has a 10-bp deletion and 28 SNPs. A specific primer (CyseSLM-F2) was then designed based on the A234 sequence, which amplified a 204 bp fragment in all females and four males with primer CyseSLM-R. A time-efficient multiplex PCR program was developed using primers CyseSLM-F2, CyseSLM-R and the newly designed primer CyseSLM-F3. The multiplex PCR products with co-dominant pattern could be detected by agarose gel electrophoresis, which accurately identified the genetic sex of the tongue sole. Therefore, we have developed a rapid and reliable method for sex identification in tongue sole with a newly identified sex-linked microsatellite marker.

Objective: To evaluate the effects of tetrandrine citrate, a novel tetrandrine salt with high water solubility, on the growth of imatinib (IM)-resistant chronic myeloid leukemia (CML) in vitro and in vivo, and reveal action molecular mechanisms. Methods: Cell viability in vitro was measured using methyl thiazolyl tetrazolium (MTT) assay. CML cell growth in vivo was assessed using a xenograft model in nude mice. Bcr-Abl and β-catenin protein levels were determined using Western blotting. Bcr-Abl messenger RNA (mRNA) was measured by reverse transcription polymerase chain reaction (RT-PCR). Flow cytometry (FCM) was used to determine cell cycle status. Results: Tetrandrine citrate inhibited the growth of IM-resistant K562 cells, primary leukemia cells, and primitive CD34+ leukemia cells, and their inhibition concentration that inhibited 50% of target cells (IC50) ranged from 1.20 to 2.97 μg/ml. In contrast, tetrandrine citrate did not affect normal blood cells under the same conditions, and IC50 values were about 10.12–13.11 μg/ml. Oral administration of tetrandrine citrate caused complete regression of IM-resistant K562 xenografts in nude mice without overt toxicity. Western blot results revealed that treatment of IM-resistant K562 cells with tetrandrine citrate resulted in a significant decrease of both p210Bcr-Abl and β-catenin proteins, but IM did not affect the Bcr-Abl protein levels. Proteasome inhibitor, MG132, did not prevent tetrandrine-mediated decrease of the p210Bcr-Abl protein. RT-PCR results showed that tetrandrine treatment caused a decrease of Bcr-Abl mRNA. FCM analysis indicated that tetrandrine induced gap 1 (G1) arrest in CML cells. Conclusions: Tetrandrine citrate is a novel orally active tetrandrine salt with potent anti-tumor activity against IM-resistant K562 cells and CML cells. Tetrandrine citrate-induced growth inhibition of leukemia cells may be involved in the depletion of p210Bcr-Abl mRNA and β-catenin protein.

The half-smooth tongue sole (Cynoglossus semilaevis) is an important cultured marine fish as well as a promising model fish for the study of sex determination mechanisms. In the present study, a protocol for artificial induction of mitogynogenesis by hydrostatic pressure using heterologous sperm was developed in half-smooth tongue sole in order to assess homozygosity of gynogens and to identify WW super-female. The optimal initiation time for pressure shock of mitogynogenetic embryos was determined to be 21.5 min after insemination when water temperature is at 22-23°C, while the optimal pressure and treatment duration were determined to be 70 MPa for 4 min. About 1,500 mitogynogenetic diploid larvae were obtained. Ten tongue sole microsatellite markers were used for homozygosity analysis of 24 mitogynogenetic larvae. Among the 24 larvae, the percentage of homozygosity ranged from 73.91% to 87.50% with an average homozygosity of 80.54%. Sex-specific simple sequence repeat (SSR) markers, CseF-SSR1, were isolated and used for identifying WW super-female mitogynogens in the tongue sole. The amplification of genomic DNA using the sex-specific SSR marker produced one DNA band of 206 bp in ZZ males, two DNA bands of 206 and 218 bp in ZW females, and one DNA band of 218 bp in WW super-females. Four WW "super-female" gynogens were observed in 39 mitogynogenetic diploids, indicating a ZW sex determination mechanism in the tongue sole. Thus, a protocol for the induction of artificial mitogynogenesis has been developed for the first time in half-smooth tongue sole, and the WW super-female diploids were identified in the mitogynogens by sex-specific SSR markers. These findings lay the foundation and provide important tool for the elaboration of sex determination mechanism, generation of WW super-females, and development of clone line and breeding of all-female stock in the half-smooth tongue sole.

To observe the inhibitory effect of 4-chlorobenzoyl berbamine (BBD9) on imatinib-resistant cell line K562 (K562/IR) in vitro and in vivo and explore the mechanisms.The IC50 of BBD9 and berbamine (BBM) was determined by MTT assay. The expressions of p210(Bcr-Abl), IKKa, cytoplasmic and nuclear NF-κBp65 were determined using Western blotting in K562/IR cells following a 48-h exposure to 0.5 µg/ml BBD9 or 8 µg/ml BBM. Flow cytometry was used to analyze the cell viability, apoptosis and necrosis; Western blotting was employed to determine the expressions of PARP, caspase-3, caspase-9 and LC3II in K562/IR cells exposed to different concentrations of BBD9 for 48 h. In nude mouse models bearing K562/IR cell xenograft, the tumor weight, tumor regression, and body weight changes of the mice were measured after treatments with 15 mg/kg and 30 mg/kg BBD9 and 100 mg/kg imatinib.The IC50 of BBD9 and BBM was 0.73 µg/ml and 5.43 µg/ml, respectively. In K562/IR cell cultures, the expressions of p210(Bcr-Abl), IKKa and nuclear NF-κB p65 were all decreased following BBD9 and BBM treatments, but BBD9 produced more potent effect; cytoplasmic NF-κB p65 showed no obvious changes after the treatments. The cell apoptosis and necrosis increased with the concentrations of BBD9, which also dose-dependently increased the levels of cleaved caspase-3, csapase-9, PARP, and LC3II expression. In the tumor-bearing mouse model, BBD9 showed stronger effects than imatinib in reducing the tumor weight, promoting tumor regression, and increasing the body weight.BBD9 can effectively inhibit the growth of K562/IR cells in vitro and in vivo by activating cell apoptosis, necrosis and autophage pathways, down-regulating expressions of p210(Bcr-Abl) and IKKa and suppressing the cytoplasm-to- nucleus translocation of NF-κBp65.

To explore the role of bone marrow (BM) imprint in the diagnosis of hematological diseases.Between January 2002 and June 2008, a total of 3024 cases with BM smears, imprints and sections conducted simultaneously were recruited. There were 1667 males and 1357 females with a median age of 55 years old (range: 7 to 92). The cellularity on imprint and smear was evaluated with the standard cellularity on BM section. With the integrative diagnosis (including all examinations and clinical outcomes) as the standard, the diagnostic accuracy of hematological diseases were compared between BM imprint, smear and section groups. Another 79 cases of lymphoma and 114 cases of plasma cell myeloma (PCM) were selected for a correlation analysis of tumor cell infiltration patterns.BM imprint contained hematopoietic and non-hematopoietic regions and cells retained integrated structure. The cellularity evaluation by imprint was superior to smear overall. In BM imprint group, the diagnostic accuracy for hypersplenism (n = 130), metastatic carcinoma (n = 67), refractory anemia with excess blasts, myeloproliferative neoplasm (n = 174), and PCM (n = 94) were better than smear group (96.9% vs 80.7%, 91.0% vs 76.1%, 92.6% vs 81.5%, 92.5% vs 76.4%, and 97.8% vs 92.6% respectively, all P0.05); And the diagnostic accuracy for megaloblastic anemia (n = 69), acute myeloid leukemia (n = 104), refractory cytopenia with unilineage dysplasia (n = 15), refractory cytopenia with multilineage dysplasia (n = 22), and lymphoplasmacytic lymphoma (n = 12) were higher than biopsy section group (100% vs 84.0%, 91.3% vs 74.0%, 86.7% vs 60.0%, 90. 9% vs 72.7%, and 66.6% vs 50.0% respectively, all P0.05); And the diagnostic accuracy for myelodysplastic/myeloproliferative neoplasm (n = 26) was higher than smear group (76.3%, P0.05) and biopsy section group (78.2%, P0.05). Excellent correlations existed between BM imprint and section of the patients with lymphoma or with PCM (r = 0.90, r = 0.78, both P0.05).BM imprint contains the characteristics of both smear and section. BM imprint is superior to smear for an evaluation of cellularity. And it is also better than section for an analysis of cytological changes.

The aim of this study was to improve the dyeing method of hydroperoxidase (HPO), to analyze the morphologic features of Phi bodies and to evaluate the clinical application of this method. 128 bone marrow or peripheral blood smears from patients with myeloid and lymphoid malignancies were stained by improved HPO staining. The Phi bodies were observed with detection rate of Phi bodies in different leukemias. 69 acute myeloid leukemia (AML) specimens were chosen randomly, the positive rate and the number of Phi bodies between the improved HPO and POX stain based on the same substrate of 3, 3'diaminobenzidine were compared. The results showed that the shape of bundle-like Phi bodies was variable, long or short. while the nubbly Phi bodies often presented oval and smooth. Club-like Phi bodies were found in M(3). The detection rates of bundle-like Phi bodies in AML M(1)-M(5) were 42.9% (6/14), 83.3% (15/18), 92.0% (23/25), 52.3% (11/21), 33.3% (5/15) respectively, and those of nubbly Phi bodies were 28.6% (4/14), 66.7% (12/18), 11.1% (3/25), 33.3% (7/21), 20.0% (3/15) respectively. The detection rate of bundle-like Phi bodies in M(3) was significantly higher than that in (M(1) + M(2)) or (M(4) + M(5)) groups. The detection rate of nubbly Phi bodies in (M(1) + M(2)) group was higher than that in M(3) group. In conclusion, after improvement of staining method, the HPO stain becomes simple, the detection rate of Phi bodies is higher than that by the previous method, the positive granules are more obvious, and the results become stable. This improved method plays an important role in differentiating AML from ALL, subtyping AML, and evaluating the therapeutic results.

The half-smooth tongue sole (Cynoglossus semilaevis, hereafter, “tongue sole”) is a marine flatfish with great commercial importance for fisheries and aquaculture in China. It has also been a promising model for the study of sex determination mechanisms in fish. Here, we report the construction of a genetic linkage map for the tongue sole, based on 137 markers including 103 AFLP markers, 33 microsatellite markers, and one female-specific DNA marker. Twenty-six linkage groups (LGs) were found. The total map length was 934.6 cM (Kosambi), with an average spacing of 8.4 cM, covering 64.4% of the estimated genome size. Furthermore, a female-specific SCAR marker, CseF-382, was mapped on LG5. This study represents the first genetic linkage map in the tongue sole. This map has great potential in the identification of quantitative traits loci and sex-related genes and marker-assisted selection in the tongue sole. Meanwhile, the new set of polymorphic microsatellite markers developed in this study is not only useful for genetic mapping but also of critical importance for studies on genetic diversity and broodstock management in tongue sole.

To observe and investigate the risk factors and pathogen diversification of nosocomial lower respiratory infections in patients with hematological malignancy after chemotherapy.Respiratory tract microbial population of fifty patients with different kinds of hematological malignancy and para-prepared to chemotherapy was quantitatively analyzed before and after chemotherapy at an arranged time from April, 2004 to December, 2005. Susceptibility test was determined for bacterium of nosocomial infection, and the homology of the same species of the bacteria was analyzed by a pulsed field gel electrophoresis (PFGE).Incidence rate of lower respiratory infections in patients with the hematological malignant after chemotherapy was 16%. The major nosocomial infectious pathogens were Acinetobacter spp; Escherichia coil and Fungus. Among them, Acinetobacter spp, were highly resistant to cephalosporins, quinolones, aminoglycosides, carbapenems and antibiotic with enzyme inhibitor, respectively but susceptible to Cefoperazone/Sulbactam belonging to antibiotic with enzyme inhibitor. And it was shown that there were two clones by the pulsed field gel electrophoresis (PFGE).Following-up of nosocomial lower respiratory infection in patients with hematological malignancy after chemotherapy might offer theoretical evidence for the rational use of antibiotics and the control of nosocomial infections.

The purpose of this study was to investigate the clinical value of plasma thrombomodulin (PTM) in different diseases or in different severity or complications of diseases, PTM in 979 patients and 60 healthy controls was determined by ELISA method. The results showed that the PTM level in the control group was 20.40 +/- 7.72 microg/L, there was no difference in sex and ages. In chronic primary glomerular disease, the PTM level in chronic renal failure (CRF) group was higher than that in non-CRF group (P0.01). PTM level70 microg/L was defined as its positive criterion. The sensitivity, specificity and positive predictive value in PTM were 85.7%, 82.4% and 77.8% respectively. The PTM level in septemia group was higher than that in non-septemia group (P0.01), the sensitivity, specificity and positive predictive value were 86.6%, 89.5% and 76.5% respectively (50 microg/L as its positive criterion). With respect of multiple trauma, the PTM level in multiple organ failare (MOF) group was higher than that in non-MOF group (P0.01), while the sensitivity, specificity and positive predictive value were 77.8%, 77.3% and 73.7% respectively (40 microg/L as its positive criterion). For systemic lupus erythematosus (SLE), the PTM level in the patients with albuminuria was higher than that in the patients without albuminuria (P0.01), and the sensitivity, specificity and positive predictive value were 77.8%, 92.3% and 93.3% respectively (35.54 microg/L as its positive criterion). For diabetes, the PTM level in complication group was higher than that in group without complications, the sensitivity, specificity and positive predictive value were 53.4%, 97.1% and 98.6% respectively (35.54 microg/L as its positive criterion). The PTM level in microangiopathy group was higher than that in macroangiopathy group (P0.01). The sensitivity, specificity and positive predictive value were 71.2%, 97.1% and 97.9% respectively. Acute leukemia (AL) and multiple myeloma (MM) had higher PTM level and PTM level was extremely high when renal failure developed (P0.01). As compared the acute stage with the restoration stage in stroke, pre-chemotherapeutics with post-chemotherapeutics in AL and MM, and pre-operation with post-operation in cancer, the PTM level was connected with clinical development. The PTM level in the patients with microangiopathy was higher than that in the patients with macroangiopathy (P0.01). The defined PTM level was higher than its normal upper limit as PTM positive criterion in microangiopathy diseases, the sensitivity, specificity and positive predictive value were 77.7%, 71.2% and 75.6% respectively. It is concluded that PTM level is a good criterion in evaluating the microangiopathy, and PTM is also a valuable indicator in prediction or assessment of the severity of diseases, or evaluation of therapeutic effectiveness.

To study the value of bone marrow biopsy imprint in evaluating cellularity.The bone marrow tissues were obtained by trephine biopsy from 272 patients, and then put on the slides to make the imprints. The imprints was stained by Wright-Giemsa method, and the bone marrow smears and imprints were examined simultaneously according to the bone marrow cellularity criteria.In bone marrow cellularity, four grades (distinct decrease, extreme decrease, distinct increase, and extreme increase) were significantly higher in bone marrow imprints than those in bone marrow smears (P0.05), but there was no significantly differences between bone marrow imprints and sections (P0.05). Using bone marrow sections as standard, in cellularily decreasing samples, the consistent rate of bone marrow imprints and smears were both high (84.4% and 97.9%), in the group of the normal and increased cellularity, the consistent rate of the bone marrow imprints (84.4% and 97.7%) was significantly higher than that in smears (60% and 64%, P0.01). The sensitivity, specificity, Youden index, positive predictive value and positive likelihood rate of bone marrow imprints were all higher than those of the smears. Using the bone marrow sections as gold standard, in 124 cases with decreased cellularity in smears, the positive diagnosis rate for aplastic anemia and dyshaematopoiesis based on bone marrow imprints was 37.1% with a false positive rate of 7.3% which was lower than that of the bone marrow smears (false positive rate of 29.8%, P0.01).To evaluate bone marrow cellularity, bone marrow imprint is better than bone marrow smear. The combination of the two examinations can make the diagnosis more convenient and quicker.

To investigate the morphological changes of megakaryocytes with nuclear extrusion and nucleocytoplasmic separation, the morphological characteristics of megakaryocytes in peripheral blood films, bone marrow smears, and bone marrow biopsies from 4 newly diagnosed patients with primary myelofibrosis (PMF), myelodysplastic syndrome (MDS), myeloblastic leukemia with maturation (M(2)) and erythroleukemia (M(6)) were studied by using light microscope. The results showed that many kinds of dysmegakaryocytes were observed in bone marrow smears of 4 cases, while in case A (PMF) and case D (M(6)) micromegakaryocytes were ripped apart; in case B (MDS) and case C (M(2)) megakaryocytes were accompanied by nuclear extrusion or nucleocytoplasmic separation, and their bodies were large or giant, the part of nucleus separated from their body and little cytoplasm remained as micromegakaryocytes. The nucleocytoplasmic separation could be displayed by immunocytochemistry stain. It is concluded that the phenomenon of nuclear extrusion and nucleocytoplasmic separation in megakaryocytes suggested the process that dispersed multinuclear releasing towards surround or even totally left the cell body during the megakaryocyte maturation. It also showed that the micromegakaryocytes may be the result of nucleocytoplasmic separation or splittings from multi-separated nucleus.

To study the plasma levels of urokinase-type plasminogen activator(u-PA) and its soluble receptor(suPAR )in patients with multiple myeloma(MM) and to evaluate their clinical significance.Plasma u-PA and suPAR levels in 34 MM cases were measured with enzyme- linked immunosorbent assay (ELISA). The changes of plasma u-PA and suPAR levels in 6 MM cases were observed in succession before and after chemotherapy.The plasma u-PA and suPAR levels of MM patients were significantly higher than those of controls. The plasma u-PA and suPAR levels in the progress period was significantly higher than those in the stable period of MM patients as well as in controls (P0.01), whereas there were no significant difference between the stable period of MM patients and controls (P0.05). Among 6 cases,the plasma u-PA and suPAR levels after chemotherapy were significantly lower than those before chemotherapy (P0.05). MM patients with tumor cells20% in bone marrow smear had higher levels of plasma u-PA and suPAR than those with tumor cells19% (P0.05 P0.01). The plasma u-PA and suPAR levels were positively correlated with the levels of serum globulin and the percentage of tumor cells in bone marrow,but negatively correlated with the levels of serum albumin.Plasma u-PA and suPAR levels can serve as an index for clinical staging and assessing the therapeutic effect in MM patients.

To study the changes of soluble Apo-1/Fas levels in plasma, pleural and ascites fluid of malignant tumor patients and to evaluate their clinical significance.The soluble Apo-1/Fas levels were measured by enzyme-linked immunosorbent assay (ELISA) in the plasma of 157 malignant tumor patients and 25 normal controls as well as in the pleural and ascite fluids of 129 patients with various diseases.The plasma soluble Apo-1/Fas levels in acute and chronic leukemia and multiple myeloma were significantly higher than those in normal controls (P0.05). The plasma soluble Apo-1/Fas levels in chronic myeloid leukemia and chronic lymphocytic leukemia were significantly higher than those in acute myeloid leukemia and acute lymphocytic leukemia, respectively (P0.05). After chemotherapy, the plasma soluble Apo-1/Fas levels in complete remission group were distinctly decreased(P0.05),whereas the levels in no remission and recurrence groups remained high. Compared with normal controls, the plasma soluble Apo-1/Fas levels in solid tumors were significantly increased (P0.01), and the levels in metastasis cancers were significantly higher than those in non-metastasis cancer (P0.0 1). Simultaneously the levels in remission cancer patients after operation and radiotherapy were distinctly lower than those before treatment(P0.01), but were significantly increased in recurrence cancer patients (P0.01). The soluble Apo-1/Fas levels in pleural and ascites fluid of malignant tumors were significantly higher than those in tuberculous effusions and transudates.The soluble Apo-1/Fas levels in plasma, pleural and ascites fluid of malignant tumor patients are markedly increased, which might be associated with the progress of cancers. The changes of soluble Apo-1/Fas levels may be useful for understanding the pathologic process of cancers and to differential diagnosis of various pleural and ascites fluids.

To study the changes of thrombomodulin(TM) in both plasma and tissue extracts of cancer patients for evaluating its clinical significance.Plasma TM levels were measured by enzyme-linked immunosorbent assay(ELISA) in plasma of 188 cancer patients and in 24 cancer tissue extracts including their adjacent normal tissue.The plasma TM levels both in cancer patients and in metastasis patients were significantly higher than that in controls [(33.47+/-14.25) microg/L/ (41.68 +/-16.96) micro/L, compared with (20.40+/-7.22) microg/L, P0.01]. The plasma TM levels in cancer patients after operation decreased obviously [(18.45+/-9.96) microg/L, compared with (28.29+/-11.74) microg/L,P0.01]. Whereas, the plasma TM levels in patients with recurrence and metastasis after operation increased obviously [(34.50+/-12.57 micro/L]. The plasma TM levels in metastasis of lung cancers, gastric cancers and pancreatic cancers were significantly higher than that in non-metastasis (P0.05 approximate, equals 0.01) respectively, but no significant differences were found between controls and non-metastasis cancers including gastric cancers, pancreatic cancers, nasopharyngeal cancers, large intestine cancers and laryngeal cancers (P0.05). The TM levels in cancer tissue extracts were significantly lower than that in their adjacent normal tissue extracts [(647.71+/-317.51)) microg/L,compared with (1455.63+/-772.22) microg/L,P0.01]. On the contrary, the plasma TM levels in these cancers were higher than that in controls.The rise of plasma TM levels in cancer patients is associated with metastasis and diffusion of cancers. The TM levels can be used as an sensitive index for judging progression and metastasis of cancers.

To study the relationship between abnormal reactions of free radicals in bodies of patients with acute organophosphorus pesticide poisoning (AOPP) and damages induced by free radicals.58 AOPP patients and 58 healthy adult volunteers (HAV) were enrolled in an independent samples control design, in which spectrophotometric methods were used to determine the concentrations of nitric oxide (NO) and lipoperoxides (LPO) in plasma, and LPO in erythrocytes, vitamin C (VC), vitamin E (VE) and beta-carotene (beta-CAR) in plasma as well as activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and acetylcholinesterase (AChE) in erythrocytes.Compared with the average values of every biochemical parameter in the HAV group, the average values of LPO in plasma and in erythrocytes, and NO in plasma in the AOPP group were significantly increased (P = 0.000001), while the average values of VC, VE, beta-CAR in plasma as well as SOD, CAT, GSH-Px and AChE in erythrocytes in the AOPP group were significantly decreased (P = 0.000001). The findings of Pearson product-moment correlation analysis between the value of AChE in erythrocytes and the values of above biochemical parameters for 58 AOPP patients showed that there was a significant linear negative correlation between AChE in erythrocytes and LPO, NO in plasma, and LPO in erythrocytes (P = 0.000001-0.001319), while there was a significant linear positive correlation between AChE in erythrocytes and VC, VE, beta-CAR in plasma as well as SOD, CAT, GSH-Px in erythrocytes (P = 0.000013-0.000824). The results of discriminant analysis of above chemical parameters for 58 AOPP patients and 58 HAV suggested that the correct rates of discriminant analysis were increased to 100% when the values of AChE and LPO in plasma and in erythrocytes, or AChE and others, were jointly used for the discriminant analysis.The findings of the present study suggest that a series of free radical reactions in AOPP patients' bodies are pathologically aggravated, and the discriminant analysis used the above biochemical parameters could markedly increase its correct rates for AOPP patients.