[Study on artificial compound feed for Buthus martensii].

OBJECTIVE: To solve the problem that the single feed causing malnutrition, extension of the life cycle and low survival rates of Buthus martensii. METHODS: By using Minitab (R) 15.1.1.0.0., 7 different kinds of compound feed were designed, including minced meat mud (pork,chicken and rabbit), bran (fried yellow), sugar, milk, vegetable paste and multivitamin as raw material. RESULTS: Different proportions of compound feed that taking the yellow mealworm as main component had a significant effect on the growth, mortality and birth number of Buthus martensii. Compound feed 5 significantly reduced mortality of youth scorpion. The compound feed 4 was the best in improving the weight of scorpion larvae and youth scorpion, and the farrowing number of mother scorpion. Other indicators were also good. Proportions in meatmud (pork, chicken and rabbit), bran (fried yellow), sugar, milk, vegetable paste and multivitamin was 30.00%, 25.00%, 20.08%, 15.58%, 8.08% and 1.25%, respectively. CONCLUSION: The growth of Buthus martensii is significantly influenced by the type of feed. In the production of compound feed, the yellow mealworm with compound feed 4 can be popularized.

[Study on spatial distribution pattern and seasonal distribution of Buthus martensii in yan'an].

OBJECTIVE: To study population dynamics of Buthus martensii and its wild spatial distribution pattern in Yan'an. METHODS: Every month, the 1st, 10th and 20th day were designated to collect Buthus martensii samples and count on the collection by using expansion pattern target, Taylor power law and Lwao m-x regression analysis. RESULTS: Taylor power law regression equation was Ig S2 = 1.6671 lg x + 0.1554; Lwao m-x regression equation was m = 1.3179 + 1.5133x. The spatial distribution pattern of Buthus martensii was negative binomial distribution and its population fluctuation curve in the wild presented a single peak. CONCLUSION: Spatial pattern of Buthus martensii in Yan'an is aggregated distribution which based on individual group, and the higher density,the greater aggregation degree.

The association between unilateral high-riding vertebral artery and atlantoaxial joint morphology: a multi-slice spiral computed tomography study of 396 patients and a finite element analysis.

BACKGROUND CONTEXT: A high-riding vertebral artery (HRVA) can deviate too medially, too posteriorly, or too superiorly to allow the safe insertion of screws. However, it is unknown whether the presence of a HRVA is associated with morphological changes of the atlantoaxial joint. PURPOSE: To investigate the association between HRVA and atlantoaxial joint morphology in patients with and without HRVA. STUDY DESIGN: A retrospective case-control study and finite element (FE) analysis. PATIENT SAMPLE: A total of 396 patients with cervical spondylosis underwent multi-slice spiral computed tomography (MSCT) of cervical spine at our institutions from 2020 to 2022. OUTCOME MEASURES: A series of atlantoaxial joint morphological parameters, including C2 lateral mass settlement (C2 LMS), C1-2 sagittal joint inclination (C1-2 SI), C1-2 coronal joint inclination (C1-2 CI), atlanto-dental interval (ADI), lateral atlanto-dental interval (LADI), and C1-2 relative rotation angle (C1-2 RRA) were measured, and lateral atlantoaxial joints osteoarthritis (LAJs-OA) was recorded. The stress distribution on the C2 facet surface under different torques of flexion-extension, lateral bending, and axial rotation was analyzed by FE models. A 2-Nm moment was applied to all models to determine the range of motion (ROM). METHODS: A total of 132 consecutive cervical spondylosis patients with unilateral HRVA were enrolled in the HRVA group, and 264 patients without HRVA matched for age and sex were enrolled in the normal (NL) group. Atlantoaxial joint morphological parameters were compared between two sides of C2 lateral mass within HRVA or NL group, and between HRVA and NL groups. A 48-year-old woman with cervical spondylosis without HRVA was selected for cervical MSCT. A three-dimensional (3D) FE intact model of the normal upper cervical spine (C0-C2) was created. We established the HRVA model by simulating atlantoaxial morphological changes of unilateral HRVA with FE method. RESULTS: The C2 LMS was significantly smaller on the HRVA side than that on the non-HRVA side in the HRVA group, but C1-2 SI, C1-2 CI, and LADI on HRVA side were significantly larger than those on non-HRVA side. There was no significant difference between left and right sides in the NL group. The difference in C2 LMS (d-C2 LMS) between HRVA side and non-HRVA side in the HRVA group was larger than that in the NL group (P < 0.05). Meanwhile, the differences in C1-2 SI (d-C1/2 SI), C1-2 CI (d-C1/2 CI), and LADI (d-LADI) in the HRVA group were significantly larger than those in the NL group. The C1-2 RRA in the HRVA group was significantly larger than that in the NL group. Pearson correlations showed that d-C1/2 SI, d-C1/2 CI, and d-LADI were positively associated with d-C2 LMS (r=0.428, 0.649, 0.498, respectively, p<.05 for all). The incidence of LAJs-OA in the HRVA group (27.3%) was significantly larger than that in the NL group (11.7%). Compared with the normal model, the ROM of C1-2 segment declined in all postures of the HRVA FE model. We found a larger distribution of stress on the C2 lateral mass surface of the HRVA side under different moment conditions. CONCLUSIONS: We suggest that HRVA affects the integrity of the C2 lateral mass. This change in patients with unilateral HRVA is associated with the nonuniform settlement of the lateral mass and an increase in the lateral mass inclination, which may further affect the degeneration of the atlantoaxial joint because of the stress concentration on the C2 lateral mass surface.

High-performance SERS substrate based on hybrid structure of graphene oxide/AgNPs/Cu film@pyramid Si.

We present a novel surface-enhanced Raman scattering (SERS) substrate based on graphene oxide/silver nanoparticles/copper film covered silicon pyramid arrays (GO/AgNPs/PCu@Si) by a low-cost and simple method. The GO/AgNPs/PCu@Si substrate presents high sensitivity, good homogeneity and well stability with R6G molecules as a probe. The detected concentration of Rhodamine 6 G (R6G) is as low as 10-15 M. These sensitive SERS behaviors are also confirmed in theory via a commercial COMSOL software, the electric field enhancement is not only formed between the AgNPs, but also formed between the AgNPs and Cu film. And the GO/AgNPs/PCu@Si substrates also present good property on practical application for the detection of methylene blue (MB) and crystal violet (CV). This work may offer a novel and practical method to facilitate the SERS applications in areas of medicine, food safety and biotechnology.

[In vitro expansion of cord blood megakaryocyte progenitor].

OBJECTIVE: To expand cord blood megakaryocyte progenitor cells in vitro. METHODS: Cord blood CD34+ cells were selected by magnetic cell sorting (MACS), and thrombopoietin (TPO), interleukin-11 (IL-11), and heparin were used in the expansion system of megakaryocyte progenitor. The expansion efficiency was measured by fluorescence-activated cell sorting (FACS) using the megakaryocytic specific monoclonal antibodies (CD34+, CD41a+, CD61+, CD34+CD41a+, CD41a+CD61+) and colony-forming units-megakaryocyte (CFU-MK) analysis. The expanded megakaryocyte progenitor were determined by histochemistry staining using CD41a and the observation of the ultrastructure of megakaryocyte (MK) by electron microscopy. The megakaryocyte function were examined by the platelet activation in vitro and nonobese diabetic/severe combined immunodifficiency (NOD/SCID) mice transplantation in vivo. RESULTS: CD34+CD41a+ cells was expanded (4.0 +/- 1.7) folds on day 7 in TPO (50 ng/ml) group and (10.5 +/- 4.8) fold in TPO combined with IL-11 group; after heparin was joined in on day 0, a more significantly elevated expansion was found in the heparin, TPO, and IL-11 group [(29.9 +/- 6.4) folds than the above two groups; P < 0.05]. Meanwhile, the large CFU-MK colony (> 50 cells/colony) was (106.8 +/- 26.9) folds on day 7 (P < 0.05). The megakaryocyte expanding with TPO, IL-11 and heparin for 7 days in vitro transplanted the NOD/SCID mice fasten the recovery of platelet and white blood cell account and improved the survival. Megakaryocyte under culture displayed certain development of territories membrane. Platelet activation test comfirmed that the expanding megakaryocyte progenitor had the normal function. CONCLUSION: TPO, IL-11, and heparin combination system for ex vivo expansion is an effective expansion system of megakaryocyte progenitor.

[Activation of auto-mesenchymal stem cells of skeletal muscle by bone morphogenetic protein for rescuing bone marrow failure].

OBJECTIVE: To explore the effect of bone morphogenetic protein (BMP) to activate mesenchymal stem cells of skeletal muscle for rescuing bone marrow failure. METHODS: The study was performed on lethal rat acute aplastic anemia model induced by combined 5-fluorouracil (5-FU) and busulfan. The rh-BMP-2 was implanted into the thigh muscle of the rats at 3 days before aplastic anemia was induced. In the control group the rats were implanted with agar into the thigh muscle. The blood picture, pathologic changes and the mortality in two groups were observed. At the same time, rh-BMP-2 were implanted into the thigh muscle of normal Kun-min mice for dynamic control observation of the implantation local morphological changes, colony forming units-spleen (CFU-S) and stem cell growth factor (SCF) expression of the stroma cells of ectopic ossicles induced by BMP. RESULTS: At 7 days after BMP implantation in the mice the mesenchymal cells around BMP in muscle proliferated, and appeared in bone marrow to form an ectopic ossicles. The SCF expression of stroma cells in ectopic ossicles were higher than that of self-bone marrow. 56.3% of BMP-treated aplastic rats were survived over 3 months and its hematopoiesis was completely reconstituted and the histo-morphological picture of the spleen and bone marrow were recovered to normal. But in the control group only one of 23 rats was survived, the remainder died of hematopoietic failure. CONCLUSIONS: BMP-implantation into the skeletal muscle could rescue the bone marrow hematopoietic failure. The mechanism might be related to the BMP activated auto-mesenchymal cells of skeletal muscles to direct hematopoietic cell differentiation. In our hands it might create a new pathway for utilization of auto-muscle derived mesenchymal cells to reconstitute hematopoiesis.

[Interferon-alpha-2b induces molecular responses of patients with polycythemia vera and its post-polycythemic myelofibrosis].

To evaluate the efficacy and safety of interferon-alpha-2b (IFN-α-2b) in polycythemia vera patients(PV patient) with or without post-polycythemic myelofibrosis (post-PV MF), 30 patients with mutated JAK2V617F were enrolled in this study, from which 29 patients were evaluable. The percentage of mutated JAK2V617F allele (V617F%) was evaluated by real-time polymerase chain reaction (RT-PCR) before and after treatment with IFN-α-2b. The correlation of V617F allele burden with the major clinical outcomes was studied. Adverse effects appeared in patients was observed. The results showed that the median follow-up was 24 (12 - 42) months for 29 evaluable patients. Complete hematologic response was achieved in 10%, 48%, 72% and 78% of patients after treatment for 6, 12, 24 and 36 months respectively. The detection of V617F allele burden revealed that the molecular remission of patients (V617F%) was achieved in 41%, 76%, 89% and 89% after treatment for 6, 12, 24 and 36 months respectively. Molecular complete remission (JAK2V617F undetectable) was achieved in 4 patients, lasted from 6 to 12 months after IFN-α-2b discontinuation. The decrease of V617F% in patients with post-PV MF was significantly higher than that in patients without post-PV MF (53 ± 18% vs 32 ± 22%, respectively; p = 0.031) after treatment for 12 months. PV patients had a good tolerance to IFN-α-2b. It is concluded that IFN-α-2b can decrease the mutated V617F allele burden. Patients with PV, especially with post-PV MF, can achieve molecular remission after treatment with IFN-α-2b.

[Relation of HLA-DRB1*15 with pathogenesis in 162 childhood cases of acute lymphoblastic leukemia].

To unravel the relation of HLA-DRB1*15 with childhood acute lymphoblastic leukemia (ALL), 162 childhood patients with ALL were selected for this investigation. 1 000 normal umbilical cord blood samples were used as control.HLA-DRB1*15 and HLA-DRB5* were typed by polymerase chain reaction (PCR) analysis. The relation of HLA-DRB1*15 with childhood ALL was studied by calculating the chi-square test and relative risk. The results showed that the antigen frequencies and allele frequencies of HLA-DRB1*15 in childhood patients with ALL were 40.12% and 22.62% respectively, while the antigen frequencies and allele frequencies of HLA-DRB1*15 in control were 30.80% and 16.81% respectively, there were significant difference between them (chi(2) = 5.560, p = 0.018, RR = 1.506). In conclusion, the antigen frequencies and allele frequencies of HLA-DRB1*15 in childhood patients with ALL were higher than those in control, so the HLA-DRB1*15 gene is one of the genetic risk factors for childhood ALL. These preliminary data may be useful for further study on the pathogenesis of childhood ALL.

[Studying of clinical and laboratory features of chronic eosinophilic leukemias /hypereosinophilic syndrome].

OBJECTIVE: To investigate the clinical and laboratory features of chronic eosinophilic leukemias (CEL) and hypereosinophilic syndrome (HES). METHODS: The clinical manifestations, laboratory parameters were retrospectively analyzed in 20 patients with HES/CEL. Detection of the FIP1L1-PDGFRA fusion gene was performed by nested RT-PCR. JAK2 V617F mutation screening was processed through allele-specific PCR combined with sequence analysis. PCR-RFLP was used to discriminate homozygous from heterozygous mutation patterns. TCR gamma rearrangement was detected by PCR. RESULTS: Of the 20 patients, 19 were males and one female, with a median age of 33 (20 to 57) years. The FIP1L1-PDGFRA fusion gene positivity in bone marrow mononuclear cells in 12 cases was identified. All the breakpoints were identified by direct sequencing of cloned RT-PCR products in FIP1L1 intron 10 - 12 and in PDGFRA exon 12. In CEL the most common involved organs were lungs, heart and nervous system. Splenomegaly was significantly more frequent in CEL than in HES (92.5% vs 42.5%, P = 0.031). Anemia and myelofibrosis were common in CEL. There was no significant difference in circulating absolute eosinophil, leukocyte, platelet counts, hemoglobin level and percentages of eosinophil and blast cell in bone marrow between CEL and HES. The morphological abnormalities of eosinophils on bone marrow smear were easily found in CEL, including hypogranularity, and cytoplasmic vacuolization, increased basophilic granule. One patient with HES was found to have heterozygous JAK2 V617F mutation. Six patients had TCR gamma rearrangement, including 4 CEL and 2 HES. CONCLUSIONS: (1) There is a male predominance in HES/CEL, and the median age was in the thirties. (2) The most common involved organs in CEL were lung, heart and nervous system. Bone marrow morphology might be of a little help in diagnosis of CEL. (3) JAK2 V617F may be involved in the pathogenesis of HES. (4) Patients with CEL carried the FIP1L1-PDGFRA fusion gene and TCR gamma rearrangement concurrently, their relationship warrants further study.

[Clinical study on relationship between JAK2 V617F mutation and chronic myeloproliferative disorders].

OBJECTIVE: To investigate JAK2V617F mutation and its clinical significance in patients with chronic myeloproliferative disorders (cMPD). METHODS: A retrospective study was performed on 523 cMPD patients diagnosed according to the current World Health Organization (WHO) criteria. Allele-specific PCR (ASP) was used to identify JAK2V617F mutation, the mutation status was analyzed by PCR-RFLP, and the results were confirmed by sequence analysis. The mutation burden was calculated by the ratio of T/G. The correlation between the allele burden and the clinical and hematologic features was analysed. For those without JAK2 V617F, MPL W515L mutation was analyzed. RESULTS: JAK2 V617F was detected in 66% of all patients (94% in PV, 80% in ET, 78% in CIMF, 75% in CMPD-U and 14% in HES). The majority of patients carried JAK2 V617F mutation were heterozygous , homozygote was found in only 5 cases (4 in PV and 1 in ET). The mutation burden in most patients (71.5%) was low with PV>ET>CIMF (P =0.003). Hemoglobin level was significantly related to high mutation burden in PV (r = 0. 203, P =0.033). Bone marrow megakaryocyte counts were found to be marked increased in ET with high JAK2 V617F loads (P = 0.024), and hepatomegaly in CIMF was significantly associated with high JAK2 V617F mutation burden (r = 0.315, P = 0.001). CONCLUSIONS: 1) Most cMPD patients, especially those with PV, carry JAK2 V617F mutation, except for CML. 2) .98% of JAK2 V617F mutation occurs of heterozygous status. 3) The mutation burden is PV>FT>CIMF. High JAK2 V617F loads are significantly associated with higher hemoglobin level in PV and higher bone marrow megakaryocyte counts in ET. 4) The positive correlation between hepatomegaly and JAK2 V617F mutation burden is found in CIMF.

[Monitoring IgH levels in patients with B-cell malignancy by real-time quantitative PCR after hematopoietic stem cell transplantation and its significance].

The study was purpose to evaluate the value of real time quantitative-PCR for monitoring IgH level in patients with B-cell malignancy after hematopoietic stem cell transplantation (HSCT). Quantification of IgH levels was performed on bone marrow mononuclear cells from 9 patients with B-cell malignancy before and after HSCT by PCR using the consensus JH TaqMan probe in combination with an allele-specific oligonucleotide (ASO) upstream primer. The IgH levels was normalized by control gene GAPDH. The results indicated that the reproducible sensitivity of RQ-PCR was 1 copy, the significant reduction of IgH copies was observed in bone marrow samples of 9 patients at one month post HSCT (6.67x10(3)/10(6) GAPDH vs 29/10(6) GAPDH, p<0.01). 3 out of 9 patients who achieved complete clinical and molecular cytogenetic remission (CCyR) contained persistently measurable low IgH level of 10(2)/10(6) GAPDH within 15 months and no detectable IgH at 18 months post HSCT. Whereas 5 out of 9 patients whose IgH copies were less than 10(2)/10(6) GAPDH within 3 months and less than 10(3)/10(6) GAPDH 3 months post HSCT achieved a sustained complete remission (CR). IgH copies in one patient were 4.5x10(3)/10(6) GAPDH at 3 months post HSCT, who relapsed at 4 months post HSCT. The median levels of tumor contamination in the stem cell harvests from 8 patients measured by RQ PCR were 3.68x10(2) (0-1720)/10(6) GAPDH. RQ PCR showed that PBPC harvests were less contaminated than BM harvests [75 (0-890)/10(6) GAPDH vs 1.1x10(3) (527-1720)/10(6) GAPDH, p<0.05]. 8 patients whose stem cell harvest were avaiable for RQ PCR were still in CR despite of the tumor contamination. The level of tumor contamination in stem cell harvest well correlated with IgH levels at diagnosis and one month after HSCT (r=0.810, r=0.708, p<0.05). It is concluded that RQ PCR can effectively monitor the IgH levels in patients with B-cell malignancy after auto-HSCT. 10(3)/10(6) GAPDH within 3 months post HSCT may be a cut-off level of IgH copies, which may be used to evaluate different prognoses of patients.

[Long-term therapeutic outcome of patients with acute promyelocytic leukemia].

OBJECTIVE: To analyze the long-term therapeutic outcome of patients with acute promyelocytic leukemia(APL). METHODS: Newly diagnosed APL patients were treated with ATRA as induction therapy followed by 3-4 courses of combined consolidation chemotherapy and 2 year maintenance therapy with ATRA and 6-MP + methrotrexate, alternatively. Patients were regularly monitored with nested RT-PCR for PML-RARalpha fusion transcript at the end of consolidation chemotherapy and in the following 4 to 5 years. RESULTS: A total of 81 patients with APL were entered the trial, 75 (92.6%) patients achieved CR. Early death (ED) rate was 6.6%. ED patients had significantly higher WBC count and higher percentage of peripheral promyelocyte than those achieved CR. Of 65 patients received consolidation, 60 (92.3%) were proved PML-RARalpha fusion gene negative at the end of the 3rd courses and 3 (4.6%) the end of the 4th courses of consolidation. The mean follow-up was 21.2 (8-64) months, 6 patients relapsed (relapse rate 9.2%). The 5-year Kaplan-Meier estimates of overall survival (OS) rate was (86.6 +/- 4.6)%. For 65 patients received consolidation therapy, the 5-year relapse-free survival (RFS) rate was 82.7%. COX-regression analyses showed only high WBC count (>10 x 10(9)/L) had an adverse prognostic influence on OS. CONCLUSION: More than 80% of APL patients treated with systemic therapy could experience long-term relapse-free survival.

[Detection of CBFbeta/MYH11 fusion transcripts and study of the mechanism of leukemogenesis of CBFbeta/SMHHC fusion protein].

OBJECTIVE: To explore CBFbeta/MYH11 fusion transcripts and its expressing product CBFbeta/SMHHC fusion protein in mechanism of leukemogenesis. METHODS: CBFbeta/MYH11 fusion transcripts were detected by combined RT-PCR with sequencing. Transcription assays were examined using pM-CSFR-Luc as reporting plasmid, and subcellular localization of encoding proteins were assayed by double immunofluorescent staining and Western blot. RESULTS: Two types of CBFbeta/MYH11 fusion transcripts were found in 26 patients with acute leukemia, most being of type A (23/26 cases, 92%) and a few of type D (2/26 cases, 8%). The inhibition of CBF-mediated M-CSFR promotor transactivation by CBFbeta/SMHHC fusion protein was increasing with the increase in amount of the fusion protein. CBFalpha subunit (AML1) located in nucleus, both CBFbeta subunit (CBFbeta) and CBFbeta/SMHHC located in cytoplasm. When AML1 and CBFbeta were coexpressed, CBFbeta still located mainly in cytoplasm, but when AML1 and CBFbeta/SMHHC were coexpressed, CBFbeta/SMHHC located mainly in nucleus. CONCLUSIONS: (1) The types of CBFbeta/MYH11 fusion transcripts of Chinese leukemia patients are almost the same as that reported in western literature. (2) CBFbeta/SMHHC inhibits CBF-mediated transactivation through competing with CBFbeta for binding to AML1.