[Preliminary Study on the Relationship between the Developmental Stage of Multiple Myeloma Disease and the Results of Bone Marrow Whole Exome Sequencing].

OBJECTIVE: To investigate the genetic results of whole exome sequencing of bone marrow from new onset multiple myeloma (MM) patients to analyze the process of genetic clonal evolution in MM patients. METHODS: Genomic DNA was extracted from bone marrow samples of 15 MM patients and the whole exomes sequencing was performed using next generation sequencing technology. Using own buccal cells as germline controls, combinated with clinical information, the mutation profile of genes from high-risk asymptomatic myeloma to symptomatic myeloma were analyzed, and genes that may be associated with the efficacy and side effects of bortezomib were screened. RESULTS: Except for two patients in whom no peripheral neuropathy was observed after a short treatment period, other patients peripheral neuropathy developed of various degrees during treatment with bortezomib containing chemotherapy, and the vast majority of patients achieved remission after receiving this bortezomib-related chemotherapy regimen. All patients had comparable levels of the inherited mutations number, but the somatic mutations was correlated with disease evolution. CONCLUSION: different gene "mutational spectra" exist in myeloma patients at different stages and are associated with progression through all stages of the disease.

[Expression and Significance of PD-1, PD-L1 and CTLA-4 in the Bone Marrow of Patients with Multiple Myeloma].

OBJECTIVE: To study the expression and significance of PD-1, PD-L1 and CTLA-4 tumor-associated antigens in multiple myeloma. METHODS: Bone marrow specimens from 122 patients with multiple myeloma were collected and divided into new-onset group (NDMM), complete remission group (CRMM) and relapsed and refractory group (RRMM) according to the disease progression stage. The proportion of CD4+ T lymphocytes, CD8+ T lymphocytes, Treg cells and plasma cells in the specimens and the expressions of PD-1, PD-L1 and CTLA-4 were detected by multi-parameter flow cytometry. RESULTS: There was no significant difference in the proportion of CD8+T and Treg cells among the three groups (P>0.05), while the proportions of CD4+T cells and PC in NDMM group were significantly higher than those in the CRMM group (P<0.05), the ratios of CD4+ to CD8+T in the NDMM and RRMM groups were significantly higher than those in the CRMM group (P<0.05). The expressions of PD-1, PD-L1 and CTLA-4 in CD8+ T cells was no significant difference among NDMM, CRMM and RRMM groups (P>0.05). While the expressions of PD-1, PD-L1 and CTLA-4 in CD4+ T cells and PC in the NDMM group were significantly lower than that in the CRMM group (P<0.05). There was significantly difference among the three groups in the expression of PD-1 in Treg cells, of which the NDMM group was significantly lower than that of the CRMM group (all P<0.05). The expressions of PD-1 and CTLA-4 in PC were significantly higher than those in CD8+ T, CD4+ T and Treg cells (P<0.05), the expression of PD-L1 in CD8+ T cells was significantly higher than that in CD4+ T and Treg cells (P<0.05). CONCLUSION: There is a correlation between the immune status of multiple myeloma and the expressions of PD-1, PD-L1 and CTLA-4 in plasma cells and lymphocyte subsets in vivo.

[Effects of Staphylococcus aureus supernatant on expression of various cytokines by neutrophils and retinal pigment epithelium cells].

OBJECTIVE: To evaluate the effects of Staphylococcus aureus supernatant on the levels of interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in co-cultures of neutrophils and retinal pigment epithelium (RPE) cells. METHODS: The co-culture system was established by co-culturing of human RPE cell line D407 and human peripheral blood neutrophils. Bacterium-free supernatant of Staphylococcus aureus ATCC29213 was added to the co-culture system for studying its effects. The volume of bacterium-free supernatant was divided into six groups: negative control, brain-heart infusion control, 50 µl, 100 µl, 250 µl and 500 µl group. The number of neutrophils was divided into four groups: negative control, 1 × 10(4), 5 × 10(4) and 5 × 10(5) group. The supernatants was collected at 6 h, 12 h, 24 h, 48 h and 72 h later and the levels of IL-1ß, TNF-α and IL-6 were measured by ELISA kits. RESULTS: When RPE cells were cultured with different doses of bacterium-free supernatant (0, 50, 100, 250 and 500 µl), the levels of IL-1ß was positively correlated with the volume of bacterium-free supernatant and the duration. The levels of IL-6 were significantly higher than that of the control group in the 500 µl group at 24 h [(23.17 ± 3.16) ng/L vs (7.61 ± 1.53) ng/L] and 48 h [(35.00 ± 4.37) ng/L vs (13.17 ± 3.27) ng/L] duration (P = 0.001 and P = 0.026, respectively). When RPE cells were co-cultured with the bacterium-free supernatant and the neutrophils (1 × 10(4), 5 × 10(4) and 5 × 10(5) cells) for 6 and 12 h, the levels of IL-1ß in the 5 × 10(5) group at both 6 h [(236.62 ± 8.20) ng/L] and 12 h [(447.42 ± 35.13) ng/L] was statistically higher than that in other groups (6 h: P = 0.000, P = 0.000, P = 0.002; 12 h: P = 0.000, P = 0.000, P = 0.000, respectively). The levels of IL-6 in the 5 × 10(5) group [(46.96 ± 2.72) ng/L] was significantly higher than that in the other groups at 12 h (P = 0.000, P = 0.000, P = 0.000, respectively). TNF-α could not be detected in the conditioned media from all cultures. CONCLUSIONS: Both IL-1ß and IL-6 are expressed in the co-cultures of neutrophils and RPE cells with Staphylococcus aureus supernatant. IL-1ß is upregulated at the early stage and the high level is maintained longer than that of IL-6. The virulence factor of bacteria and the neutrophil may play a role in the expression of IL-1ß and IL-6 in the RPE cells.

Sesquiterpenes from Laurencia similis.

One new sesquiterpene, (4E)-1-bromo-5-[(1'S*,3'R*)-3'-bromo-2',2'-dimethyl-6'-methylenecyclohexyl]-3-methylpent-4-ene-2,3-diol (1), and fifteen known sesquiterpenes, isopalisol (2), luzonensol (3), palisadin B (4), aplysistatin (5), palisadin A (6), 4-hydroxyl-palisudin C (7), 5-acetoxypalisadin B (8), 10-hydroxyaristolan-9-one (9), aristol-8-en-1-one (10), aristolan-9-en-1-one (11), aristolan-1(10)-en-9-one (12), aristolan-1(10)-en-9-ol (13), aristolan-1(10),8-diene (14), aristolan-1,9-diene (15) and aristofone (16), were isolated from a sample of marine red alga Laurencia similis. Their structures were established by detailed NMR spectroscopic analysis and comparison with literature data. Compounds 2-9, and 16 were isolated for the first time from this species. All these metabolites were submitted for a cytotoxicity assay against the tumor cell line BEL7402 (human liver adenocarcinoma), but all of them were found inactive (IC(50 )> 10 microg/mL).

[Comparative studies of wavefront aberration and contrast sensitivity after phacoemulsification and intraocular lens implantation].

OBJECTIVE: To study the difference of the contrast sensitivity and wavefront aberration of two eyes who undergone phacoemulsification and intraocular transplantation respectively. METHODS: Sixty-three patients with cataract were included in the study. With the consent of the patients, one eye was implanted SA60AT intraocular lens and another eye was implanted Tennis Z9001 intraocular lens after phacoemulsification. One and three months post-surgery, the best corrected visual acuity was recorded by computer and comprehensive optometry. Contrast sensitivity was analyzed by CSV-1000. The corneal aberration was measured by Option Keratron Scout. The whole ocular aberration was evaluated by Allegretto Waver Analyzer at pupillary diameter 4 mm and 6 mm. RESULTS: The best corrected visual acuity and the corneal spherical aberration, corneal coma aberration and RMS had no significant difference between the two groups. After one and three months, the contrast sensitivity of 3, 6, 12, 18c/d had statistic significance between the eye with and without glare through univariate split-plot analysis. The Z9001 contrast sensitivity value was much higher than the SA60AT groups. The results of Zernike polynomials C12, RMS4 and RMSh had statistic significance between the two groups through T-test. The Z9001 group value was much lower than the SA60AT groups. CONCLUSIONS: The Z9001 intraocular lens transplantation can reduce total ocular aberration, special the spherical aberration and improve visual acuity.

[Studies on chemical constitutes of Acantophora spicifera].

OBJECTIVE: To study the chemical constitutes of Acantophora spicifera. METHOD: Compounds were isolated by normal phase silica gel and Sephadex LH-20 gel column chromatography, and reverse-phase HPLC, as well as recrystallization. Their structures were elucidated by spectroscopic methods. RESULT: Seven compounds were isolated from A. spicifera and their structures were identified as aplysin (1), loloilide (2), (R)-(-)-dehydrovomifoliol (3), uracil (4), thymine (5), 1-methoxy-4-(1-propenyl) benzene (6). CONCLUSION: The compounds were obtained from this genus for the first time. Compound 6 was firstly obtained from marine organisms.

[Chemical constituents from red alga Corallina pilulifera].

OBJECTIVE: To investigate the chemical constituents of red alga Corallina pilulifera. METHOD: Compounds were isolated by normal phase silica gel and Sephadex LH - 20 gel column chromatography, reverse phase HPLC and recrystallization. Their structures were elucidated by spectroscopic methods including MS, 1H-NMR, 13C-NMR, HSQC, HMBC. Cytotoxicity of the compounds was screened by using standard MTT method. RESULT: Seven compounds were isolated from red alga C. pilulifera, their structures were identified as (E) -phytol epoxide (1), phytenal (2), phytol (3), dehydrovomifoliol (4), loliolide (5), 3beta-hydroxy-5alpha, 6alpha-epoxy-7-megastigmene-9-one (6), 4-hydroxybenzaldehyde (7). CONCLUSION: All of the compounds were obtained from this species for the first time. These compounds were inactive (IC50 > 10 microg x mL(-1)) in the MTT assay.

[Norisoprenoids from red alga Gymnogongrus flabelliformis].

OBJECTIVE: To investigate the chemical constituents of the red alga Gymnogongrus flabelliformis Harv. METHOD: Compounds were isolated by normal phase silica gel and Sephadex LH - 20 gel column chromatography and reverse phase HPLC. Their structures were elucidated by spectroscopic methods including MS, 1H-NMR, 13C-NMR. Cytotoxicity of the compounds was screened by using standard MIT method. RESULT: Five compounds were isolated from G. flabelliforrmis, their structures were identified as(3S, 6R, 7E)-( + )-3-hydroxyl-4, 7-mega-stigmadien-9-one (1), (3S, 5R, 6S, 7E)-(-)-3-hydroxy-5, 6-epoxy-7-megastigmene-9-one (2), (3S, 5S, 6R, 7E)-(+)3-hydroxy-5, 6-epoxy-7-megastigmene-9-one (3), dehydrovomifoliol (4), (3R)-(-)4-[(2R, 4S)-4-acetoxy-2-hydroxy-2, 6, 6-trimethylcyclohexylidene] -3-buten-2-one (5). CONCLUSION: All of the compounds were obtained from this species for the first time and compound 1 was a new natural product. These compounds were inactive (IC50 > 10 microg x mL(-1)) in the MTT assay against several human cancer cell lines.

[The effect of protein kinase C alpha on expression of developmental genes during differentiation of mouse embryonic stem cells into neuron-like cells in vitro].

OBJECTIVE: To study the effect of protein kinase C alpha on the expression of developmental genes pax-6, slit-2 and netrin-1 during differentiation of mouse embryonic stem cells into neuron-like cells in vitro, in an attempt to elucidate their roles in signaling. METHODS: ES-BALB/c cells were induced to form embryoid bodies in the ES conditioned medium for 4 days, and were plated separately on coated glass coverslip into 6-well culture dishes for immunohistochemical study and into 100 mm dishes for RT-PCR assay. These cultures were collected after 1, 3, 5, 7 and 14 days in the presence of 5 x 10(-7) mol/L retinoic acid (RA). The neuron-like cells were stained with antibody to NSE and NF-200. mRNA level of the development related genes (pax-6, slit-2 and netrin-1) in undifferentiated and differentiated ES cells was assessed by RT-PCR assay. Effects of PMA and D-sphingosine on the developmental genes were also observed. RESULTS: Most of the neuron-like cells stained with the antibodies to NSE and NF-200. RT-PCR assay showed that levels of PKC alpha, pax-6 and netrin-1, but not slit-2 transcripts decreased dramatically upon induction on the 1st day, but then raised slowly and resumed to normal level on 14th day. PMA upregulated the levels of PKC alpha, pax-6 and netrin-1; while D-sphingosine downregulated their levels. CONCLUSIONS: The results of the present experiments demonstrate that the developmental genes pax-6 and netrin-1 play a very important role during differentiation of mouse ES cells into neuron-like cells through PKC alpha signaling pathway.