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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.

X. Gong, X. Lu, X. Wu, R. Xu, Q. Tang, G. Xu, L. Wang, X. Zhang and X. Zhao Role of bone marrow imprints in haematological diagnosis: a detailed study of 3781 cases Objectives: To explore the role of imprints in routine bone marrow (BM) diagnosis. Methods: The cellularity and diagnostic accuracy of BM imprints, aspirate smears and trephine biopsy sections from 3781 patients were assessed using routine cytochemical staining. Seventy-nine cases of lymphoma and 114 cases of plasma cell myeloma (PCM) were selected for correlation analysis of tumour cell infiltration patterns. Another 21 cases of lymphoma were selected to detect t(14;18)(q32;q21) and t(11;14)(q13;q32) by fluorescent in situ hybridization (FISH) on BM imprints, and the G-banding technique was performed for comparison. Results: BM imprints were better than smears for evaluating cellularity. In the BM imprint group, diagnostic accuracy for metastatic carcinoma, myeloproliferative neoplasm, myelodysplastic/myeloproliferative neoplasm and PCM was better than in the smear group, while accuracy for megaloblastic anaemia, acute myeloid leukaemia, refractory cytopenia with unilineage or multilineage dysplasia, refractory anaemia with excess blasts and lymphoplasmacytic lymphoma was higher than in the section group, but not statistically different from the smear group. Good correlation of infiltration patterns of lymphoma and myeloma cells was found between BM imprints and sections (r = 0.90 and 0.78, respectively). Detection of t(11;14)(q13;q32) by FISH on imprints was higher than G-banding analysis. Conclusions: BM imprints show features of both smears and trephine sections. Imprints are superior to smears for evaluation of cellularity, and are also better than sections for analysis of cytological changes. In addition, FISH on BM imprints markedly improves the identification of chromosomal abnormalities.

The first set of 18 polymorphic microsatellite markers were developed from bluefin leatherjacket (Navodon septentrionalis Gunther, 1877). From a (GT)n-enriched genomic library, we got 121 microsatellites, of which 60 were randomly selected for designing microsatellite primers. Eighteen of these loci were polymorphic in a test population of 32 individuals with alleles ranging from 2 to 9, and expected and observed heterozygosities from 0.1463 to 0.8517 and from 0.1562 to 1.0000, respectively. No significant linkage disequilibrium between pairs of loci was found, but three loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction. These polymorphic microsatellite loci should provide sufficient level of genetic diversity to investigate population structure in bluefin leatherjacket.

In the present study, 10 polymorphic microsatellite DNA loci from Atlantic halibut (Hippoglossus hippoglossus) were isolated and characterized. The number of alleles for these loci ranged from 2 to 4 in tested 24 individuals. Observed and expected heterozygosities per locus varied from 0.21 to 0.70 and from 0.31 to 0.65, respectively. Most of these 10 microsatellite loci were successfully amplified and showed polymorphic in five related species. These loci will be useful for the assessment of genetic diversity and population structure of Atlantic halibut.

A microsatellite-enriched genomic DNA library of Navodon septentrionalis was generated and screened by sequencing. Ten dinucleotide microsatellite loci were characterized by genotyping 24 samples. The observed number of alleles ranged from two to seven with an average of 4.40, while the effective number of alleles ranged from 1.49 to 5.70 with an average of 3.31. The observed and expected heterozygosities ranged from 0.2917 to 0.9167 and from 0.3369 to 0.8422, respectively. No significant linkage disequilibrium between pairs of loci was found, but one loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction. These polymorphic microsatellite loci would be useful for investigating genetic population structure and molecule-assisted breeding for N. septentrionalis.

However,no reports are available on genetic diversity usingdifferent molecular marker techniques in China.Generally, the genetic diversity of introducedspecies will be reduced vastly due to the smaller ofeffective parent. In order to maintain sustaineddevelopment of the sole’s aquaculture in China,it is necessary to screen out molecular markersto analyze the genetic diversity in Senegal sole.MicrosatelliteDNAmarkershavebeenshowntobehighly polymorphic and useful as molecularmarkers for understanding population structure.

Small yellow croaker (Pseudosciaena polyactis) is an economically important marine fish species. About 43 microsatellite loci were isolated from two enriched genomic library of Pseudosciaena polyactis. Ten of these loci were polymorphic in a test population with alleles per locus ranging from two to six, and observed and expected heterozygosities per locus from 0.3750 to 0.8750 and from 0.3112 to 0.8121, respectively. No loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction and no significant linkage disequilibrium was found between pairs of loci. These polymorphic microsatellite loci would be useful for investigating genetic diversity of Pseudosciaena polyactis.

In the study, 34 microsatellite loci were isolated from a dinucleotide-enriched genomic library of Japanese Spanish mackerel (Scomberomorus niphonius). And 12 microsatellite loci were found to be polymorphic between 3 and 8 alleles. The number of observed and expected heterozygosity per locus in 23 individuals ranged from 0.6087 to 1.0000 and 0.8908 to 0.9773, respectively. Two loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction analysis and there was no significant linkage disequilibrium found between pairs of loci. As a result, 12 microsatellite loci probably located on different chromosome pairs and these polymorphic microsatellite loci should provide sufficient level of genetic diversity to investigate the fine-scale population structure and evaluate the breeding strategy in S. niphonius.

We describe the first isolation of 12 polymorphic microsatellite markers from Belenger’s jewfish (Johnius belengnerii Cuvier 1830). From a (GT)n-enriched genomic library, 54 microsatellites were selected for designing microsatellite primers, of which 36 gave working primer pairs. 12 of these loci were polymorphic in a test population of 21 individuals with alleles ranging from 3 to 18, and expected and observed heterozygosities from 0.5772 to 0.9449 and from 0.4286 to 0.9231, respectively. No significant linkage disequilibrium between pairs of loci was found, however, loci Jobe24 significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction. These polymorphic microsatellite loci should provide sufficient level of genetic diversity to investigate population structure in Belenger’s jewfish.

Given the ecological and commercial importance of the summer flounder (Paralichthys dentatus), there is a surprising paucity of information on the molecular genetics of this species. Some studies published to date are concentrated on the reproduction biology. To address this shortcoming, a microsatellite-enriched genomic DNA library of P. dentatus was generated and screened by sequencing. Twelve dinucleotide microsatellite loci were characterized by genotyping 24 samples. The observed number of alleles ranged from three to thirteen with an average of 8.25, while the effective number of alleles ranged from 2.21 to 8.28 with an average of 5.06. The observed and expected heterozygosities ranged from 0.0833 to 0.9583 and from 0.5594 to 0.8980, respectively. Significant deviations from Hardy–Weinberg expectations were detected at three loci and linkage disequilibrium between two loci was significant after applying Bonferroni correction. In cross-species amplification, three species showed at least two polymorphic loci. The 12 highly polymorphic microsatellite markers represent a powerful molecular tool, which will allow for detailed population genetic analyses on this important marine fish.

The first set of polymorphic microsatellite markers were developed from so-iuy mullet (Mugil soiuy Basilewsky 1855). From a (GT)n-enriched genomic library, 53 microsatellites were selected for designing microsatellite primers, of which 36 gave working primer pairs. Ten of these loci were polymorphic in a test population of 24 individuals with alleles ranging from 3 to 9, and observed and expected heterozygosities from 0.2083 to 0.9167 and from 0.2651 to 0.8812, respectively. No significant linkage disequilibrium between pairs of loci was found, but two loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction. These polymorphic microsatellite loci should provide sufficient level of genetic diversity to investigate the fine-scale population structure and evaluate the breeding strategy in Mullet.

Chinese sea perch (Lateolabrax maculates) is one of the most important commercial species of mariculture in China. In this study, we constructed a repeat-enriched genomic DNA library of L. maculates. Eighteen dinucleotide microsatellite markers were characterized by genotyping 32 samples. The number of alleles ranged from three to nine, and the observed and expected heterozygosities ranged from 0.4516 to 1.0000 and from 0.4045 to 0.8676, respectively. Significant deviations from Hardy–Weinberg expectations were detected at four loci and linkage disequilibrium between two loci was significant after applying Bonferroni correction. The 18 highly polymorphic microsatellite markers should provide sufficient level of genetic diversity to investigate the population structure and evaluate the breeding strategy in L. maculates.

Bcr-Abl tyrosine kinase inhibitors (TKIs) have been a remarkable success for the treatment of Ph+ chronic myeloid leukemia (CML). However, a significant proportion of patients treated with TKIs develop resistance because of leukemia stem cells (LSCs) and T315I mutant Bcr-Abl. Here we describe the unknown activity of the natural product berbamine that efficiently eradicates LSCs and T315I mutant Bcr-Abl clones. Unexpectedly, we identify CaMKII γ as a specific and critical target of berbamine for its antileukemia activity. Berbamine specifically binds to the ATP-binding pocket of CaMKII γ, inhibits its phosphorylation and triggers apoptosis of leukemia cells. More importantly, CaMKII γ is highly activated in LSCs but not in normal hematopoietic stem cells and coactivates LSC-related β-catenin and Stat3 signaling networks. The identification of CaMKII γ as a specific target of berbamine and as a critical molecular switch regulating multiple LSC-related signaling pathways can explain the unique antileukemia activity of berbamine. These findings also suggest that berbamine may be the first ATP-competitive inhibitor of CaMKII γ, and potentially, can serve as a new type of molecular targeted agent through inhibition of the CaMKII γ activity for treatment of leukemia.

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.

5 pages; International audience; This article documents the addition of 411 microsatellite marker loci and 15 pairs of Single Nucleotide Polymorphism (SNP) sequencing primers to the Molecular Ecology Resources Database. Loci were developed for the following species: Acanthopagrus schlegeli, Anopheles lesteri, Aspergillus clavatus, Aspergillus flavus, Aspergillus fumigatus, Aspergillus oryzae, Aspergillus terreus, Branchiostoma japonicum, Branchiostoma belcheri, Colias behrii, Coryphopterus personatus, Cynogolssus semilaevis, Cynoglossus semilaevis, Dendrobium officinale, Dendrobium officinale, Dysoxylum malabaricum, Metrioptera roeselii, Myrmeciza exsul, Ochotona thibetana, Neosartorya fischeri, Nothofagus pumilio, Onychodactylus fischeri, Phoenicopterus roseus, Salvia officinalis L., Scylla paramamosain, Silene latifo, Sula sula, and Vulpes vulpes. These loci were cross-tested on the following species: Aspergillus giganteus, Colias pelidne, Colias interior, Colias meadii, Colias eurytheme, Coryphopterus lipernes, Coryphopterus glaucofrenum, Coryphopterus eidolon, Gnatholepis thompsoni, Elacatinus evelynae, Dendrobium loddigesii Dendrobium devonianum, Dysoxylum binectariferum, Nothofagus antarctica, Nothofagus dombeyii, Nothofagus nervosa, Nothofagus obliqua, Sula nebouxii, and Sula variegata. This article also documents the addition of 39 sequencing primer pairs and 15 allele specific primers or probes for Paralithodes camtschaticus.

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.

Objective: To investigate the clinical value of plasma thrombmoclulin (PTM). Methold: PTM was determined in 979 patients and 60 healthy controls with ELISA method. Result: The PTM of the control group was 20.40±7.72 μg/L, there was no difference in genders and ages. The PTM of the patients with microangiopathy were higher than that of the patients with macroangiopathy (P Conclusion: PTM was a good criterion in evaluating the microangiopathy in clinical application. PTM was also a valuable index in prediction or assessment of the severity of diseases or in the observation of therapeutic effectiveness.

Objective: To study significance and character of fibrinolytic system in pleural and ascitic fluid with three disease. Methods: Urokinase plasminogen activator (uPA), urokinase plaminogen activator receptor (uPAR) were measured by ELISA, tissue plaminogen activator (tPA), plaminogen activator inhibitor-1(PAI-1), plasminogen (Plg), plasmin (Pl), and a2 plasmin inhibitor (a2PI) by colorimetric assay. Result: The levels of uPA and uPAR in pleural and ascitic fluid were marked higher in malignant tumor (2533.42±1369.89 ng/L and 7.32±1.59μg/L) and tuberculosis (2618.05±1542.50 ng/L, 8.75±3.48μg/L)than in cirrrhosis (961.25 ± 692.82ng/L and 2.73±1.59 μg/L, P Conclusions: There were significant difference in fibrinolytic charater in pleural and ascitic fluid with different diseases. To join test with fibrinolysis targets can not only help determine quality of pleural and ascitic fluid, but also can help deeply understand pathologic processes in desease, especially some biologic actions of malignant tumor.

Objective: To study the changes of thrombomodulin(TM) in both plasma and tissue extracts of cancer patients for evaluating its clinical significance. Methods: Plasma TM levels were measured by enzyme-linked immunosorbent assay(ELISA) in both plasma of 188 cancer patients and 24 cancer tissue extracts including their adjacent non-cancer tissues. Results: The plasma TM levels both in cancer patients and in metastasis patients were significantly higher than that in controls [(33.47±14.25)ug/L/(41.68±16.96)ug/L, vs (20.40±7.22)ug/L, P0.05). The TM levels in cancer tissue extracts were significantly lower than that in their adjacent non-cancer tissue extracts [(647.71±317.51)ug/L vs (1455.63±772.22)ug/L, P Conclusion: The rise of plasma TM levels in cancer patients was associated with metastasis and diffusion of cancers. The TM levels can be served as a sensitive index for judging progression and metastasis of cancers.

Objective: To study the expression character of fibrinolytic factors and its inhibitors in rabbits’ tissues extract. Methods: Tissue plasminogen activator ( tPA ), Plasminogen activator inhibitor-1 ( PAI-1 ), Plasminogen ( Plg ), Plasmin ( Pl ) and α2 Plasmin inhibitor ( α2PI ) were measured by colorimetric assay. Results: The tissues extract in renal, small intestine, lung, brain and spleen showed strong fibrinolytic function, and expressed markedly high activity levels of tPA, Plg and Pl. But striped muscle, tongue and stomach expressed markedly high activity levels of PAI-1 and α2PI, and showed low fibrinolytic character. There were linear correlation between levels of tPA and PAI-1 ( r=−0.8076, P Conclusion: The fibrinolytic character existed significant difference in tissues, it has profound significance in the function of heamostasis and thrombosis and physiologic function in tissues.