Osimertinib Covalently Binds to CD34 and Eliminates Myeloid Leukemia Stem/Progenitor Cells.

Osimertinib is a third-generation covalent EGFR inhibitor that is used in treating non-small cell lung cancer. First-generation EGFR inhibitors were found to elicit pro-differentiation effect on acute myeloid leukemia (AML) cells in preclinical studies, but clinical trials yielded mostly negative results. Here, we report that osimertinib selectively induced apoptosis of CD34+ leukemia stem/progenitor cells but not CD34- cells in EGFR-negative AML and chronic myeloid leukemia (CML). Covalent binding of osimertinib to CD34 at cysteines 199 and 177 and suppression of Src family kinases (SFK) and downstream STAT3 activation contributed to osimertinib-induced cell death. SFK and STAT3 inhibition induced synthetic lethality with osimertinib in primary CD34+ cells. CD34 expression was elevated in AML cells compared with their normal counterparts. Genomic, transcriptomic, and proteomic profiling identified mutation and gene expression signatures of patients with AML with high CD34 expression, and univariate and multivariate analyses indicated the adverse prognostic significance of high expression of CD34. Osimertinib treatment induced responses in AML patient-derived xenograft models that correlated with CD34 expression while sparing normal CD34+ cells. Clinical responses were observed in two patients with CD34high AML who were treated with osimertinib on a compassionate-use basis. These findings reveal the therapeutic potential of osimertinib for treating CD34high AML and CML and describe an EGFR-independent mechanism of osimertinib-induced cell death in myeloid leukemia. SIGNIFICANCE: Osimertinib binds CD34 and selectively kills CD34+ leukemia cells to induce remission in preclinical models and patients with AML with a high percentage of CD34+ blasts, providing therapeutic options for myeloid leukemia patients.

N6-methyladenosine demethylase FTO related to hyperandrogenism in PCOS via AKT pathway.

BACKGROUND: Polycystic ovary syndrome (PCOS) was known as the common endocrine disease in women, featured as hyperandrogenism, ovulation disorders, etc. Fat mass and obesity-associated protein (FTO), a m6A demethylase, is abnormal in the occurrence of ovarian diseases. However, the mechanism of FTO in the pathogenesis of PCOS is still unclear. METHODS: The level of FTO in clinical samples, PCOS rat with hyperandrogenism and granulosa cells (GCs) lines effected by DHT were investigated by ELISA, qRT-PCR, WB, and IHC, while m6A RNA methylation level was studied by m6A Colorimetric and androgen level was tested through ELISA. Changes in steroid hormone synthetase and androgen receptor (AR)/prostate-specific antigen (PSA) levels in vitro were visualized by WB after transient transfection silenced FTO. The effect of DHT combined with FTO inhibitor meclofenamic acid (MA) on FTO, AR/PSA, and AKT phosphorylation were also demonstrated by WB. The co-localization of FTO and AR in KGN cells was analyzed by confocal microscopy, and the physiological interaction between FTO and AR was studied by Co-IP assay. The effect of FTO-specific inhibitor MA, AKT phosphorylation inhibitor LY294002, and the combined them on GCs proliferation and cell cycle were evaluated by drug combination index, EDU assay, and flow cytometry analysis. RESULTS: FTO expression was upregulated in follicular fluid and GCs in PCOS patients clinically. The high FTO expression in patients was negative with the level of m6A, but positive with the level of androgen. The upregulation of FTO was accompanied with a decrease in the level of m6A in PCOS rat with hyperandrogenism. Dihydrotestosterone (DHT) promoted the FTO expression and inhibited m6A content as a dose-dependent way in vitro. In contrast, suppression of FTO with siRNA attenuated the expression of steroid hormone synthetase such as CYP11A1, CYP17A1, HSD11B1, HSD3B2 except CYP19A1 synthetase, ultimately inducing the decrease of androgen level. Suppression of FTO also decreased the biological activity of androgen through downregulation AR/PSA. MA treatment as the specific FTO antagonist decreased cell survival in time- and dose-dependent way in GCs lines. Correspondingly, MA treatment decreased the expression of FTO, AR/PSA expression, and AKT phosphorylation in the presence of DHT stimulation. Additionally, we also speculate there is a potential relation between FTO and AR according to FTO was co-localized and interacted with AR in KGN cells. Compared with AKT phosphorylation inhibitor LY294002 or MA alone, LY294002 combined with MA synergistically inhibited cell survival and increased G2/M phase arrest in GC line. CONCLUSIONS: We first evaluated the correlation of FTO and m6A in PCOS clinically, and further explored the mechanism between FTO and hyperandrogenism in PCOS animal and cell models. These findings contributed the potential therapy by targeting the FTO for hyperandrogenism in PCOS.

Economical Production of Phenazine-1-carboxylic Acid from Glycerol by Pseudomonas chlororaphis Using Cost-Effective Minimal Medium.

Phenazine compounds are widely used in agricultural control and the medicine industry due to their high inhibitory activity against pathogens and antitumor activity. The green and sustainable method of synthesizing phenazine compounds through microbial fermentation often requires a complex culture medium containing tryptone and yeast extract, and its cost is relatively high, which greatly limits the large-scale industrial production of phenazine compounds by fermentation. The aim of this study was to develop a cost-effective minimal medium for the efficient synthesis of phenazine compounds by Pseudomonas chlororaphis. Through testing the minimum medium commonly used by Pseudomonas, an ME medium for P. chlororaphis with a high production of phenazine compounds was obtained. Then, the components of the ME medium and the other medium were compared and replaced to verify the beneficial promoting effect of Fe2+ and NH4+ on phenazine compounds. A cost-effective general defined medium (GDM) using glycerol as the sole carbon source was obtained by optimizing the composition of the ME medium. Using the GDM, the production of phenazine compounds by P. chlororaphis reached 1073.5 mg/L, which was 1.3 times that achieved using a complex medium, while the cost of the GDM was only 10% that of a complex medium (e.g., the KB medium). Finally, by engineering the glycerol metabolic pathway, the titer of phenazine-1-carboxylic acid reached the highest level achieved using a minimum medium so far. This work demonstrates how we systematically analyzed and optimized the composition of the medium and integrated a metabolic engineering method to obtain the most cost-effective fermentation strategy.

Mechanism of human chorionic gonadotropin in endometrial receptivity via the miR-126-3p/PI3K/Akt/eNOS axis.

Human chorionic gonadotropin (hCG) might affect endometrial receptivity, exerting integral roles in embryo implantation. This study explored the action of hCG in endometrial receptivity via the miR-126-3p/PIK3R2/PI3K/Akt/eNOS axis. The embryo implantation dysfunction (EID) mouse models were established by administrating mifepristone and human endometrial epithelial cells (EECs) were used for in vivo experiments, both followed by hCG treatment. Expression level of CD105 and protein levels of cadherin CD144 and CD146 in mice were determined by immunohistochemistry and Western blot. The levels of miR-126-3p and PIK3R2 mRNA and PIK3R2, p-PI3K p85 α, PI3K p110 α, p-Akt, Akt, p-eNOS, and eNOS protein levels were measured. Cell proliferation was evaluated by CCK-8 and EdU assays. The binding sites of miR-126-3p and PIK3R2 were predicted and verified. hCG-treated EECs were further transfected with miR-126-inhibitor for functional rescue experiments. hCG ameliorated endometrial receptivity in EID mice. Moreover, hCG promoted miR-126-3p and suppressed PIK3R2 in EID mice and EECs. miR-126-3p targeted PIK3R2. EEC proliferation was enhanced after hCG treatment but inhibited by miR-126-3p downregulation. Both in vivo and in vitro experiments validated that hCG activated the PI3K/Akt/eNOS pathway through the miR-126-3p/PIK3R2 axis. Collectively, hCG improves endometrial receptivity by activating the PI3K/Akt/eNOS pathway via regulating miR-126-3p/PIK3R2.

C1Q labels a highly aggressive macrophage-like leukemia population indicating extramedullary infiltration and relapse.

Extramedullary infiltration (EMI) is a concomitant manifestation that may indicate poor outcome of acute myeloid leukemia (AML). The underlying mechanism remains poorly understood and therapeutic options are limited. Here, we employed single-cell RNA sequencing on bone marrow (BM) and EMI samples from a patient with AML presenting pervasive leukemia cutis. A complement C1Q+ macrophage-like leukemia subset, which was enriched within cutis and existed in BM before EMI manifestations, was identified and further verified in multiple patients with AML. Genomic and transcriptional profiling disclosed mutation and gene expression signatures of patients with EMI that expressed high levels of C1Q. RNA sequencing and quantitative proteomic analysis revealed expression dynamics of C1Q from primary to relapse. Univariate and multivariate analysis demonstrated adverse prognosis significance of C1Q expression. Mechanistically, C1Q expression, which was modulated by transcription factor MAF BZIP transcription factor B, endowed leukemia cells with tissue infiltration ability, which could establish prominent cutaneous or gastrointestinal EMI nodules in patient-derived xenograft and cell line-derived xenograft models. Fibroblasts attracted migration of the C1Q+ leukemia cells through C1Q-globular C1Q receptor recognition and subsequent stimulation of transforming growth factor ß1. This cell-to-cell communication also contributed to survival of C1Q+ leukemia cells under chemotherapy stress. Thus, C1Q served as a marker for AML with adverse prognosis, orchestrating cancer infiltration pathways through communicating with fibroblasts and represents a compelling therapeutic target for EMI.

An additional medium renewal of D4 embryo culture improves the concordance of noninvasive chromosome screening with trophectoderm biopsy.

Our research question was to evaluate the chromosome concordance of trophectoderm (TE) biopsy with noninvasive chromosome screening (NICS) using embryo culture medium renewed twice on Day 3 (D3) and Day 4 (D4). In this study, we evaluated 64 cycles with 223 biopsied blastocysts. These were categorized into two groups based on replacing embryo culture medium on D3 (control group) or on D3 and D4 (experimental group). The fundamental characteristics and main outcomes were compared. The concordance rates of NICS results with TE biopsy were determined according to next generation sequencing results. In total, 103 experimental and 120 control embryo cultures were collected, and the euploid status was analyzed using NICS technology. The overall concordance rates with TE biopsy of the experimental and control groups were 0.86 and 0.75, respectively. Statistically significant difference was found between the two groups. An additional medium renewal of the D4 embryo culture can improve the concordance of NICS with TE biopsy.

Mitochondrial oxidative phosphorylation is dispensable for survival of CD34+ chronic myeloid leukemia stem and progenitor cells.

Chronic myeloid leukemia (CML) are initiated and sustained by self-renewing malignant CD34+ stem cells. Extensive efforts have been made to reveal the metabolic signature of the leukemia stem/progenitor cells in genomic, transcriptomic, and metabolomic studies. However, very little proteomic investigation has been conducted and the mechanism regarding at what level the metabolic program was rewired remains poorly understood. Here, using label-free quantitative proteomic profiling, we compared the signature of CD34+ stem/progenitor cells collected from CML individuals with that of healthy donors and observed significant changes in the abundance of enzymes associated with aerobic central carbonate metabolic pathways. Specifically, CML stem/progenitor cells expressed increased tricarboxylic acid cycle (TCA) with decreased glycolytic proteins, accompanying by increased oxidative phosphorylation (OXPHOS) and decreased glycolysis activity. Administration of the well-known OXPHOS inhibitor metformin eradicated CML stem/progenitor cells and re-sensitized CD34+ CML cells to imatinib in vitro and in patient-derived tumor xenograft murine model. However, different from normal CD34+ cells, the abundance and activity of OXPHOS protein were both unexpectedly elevated with endoplasmic reticulum stress induced by metformin in CML CD34+ cells. The four major aberrantly expressed protein sets, in contrast, were downregulated by metformin in CML CD34+ cells. These data challenged the dependency of OXPHOS for CML CD34+ cell survival and underlined the novel mechanism of metformin. More importantly, it suggested a strong rationale for the use of tyrosine kinase inhibitors in combination with metformin in treating CML.

[Diagnosis value of MRI in acute rotator interval injury].

OBJECTIVE: To compare clinical application of 1.5 T MRI in acute rotator interval injury. METHODS: Totally 160 patients with acute rotator cuff tear by clinical diagnosis were retrospectively analyzed by MRI examination and arthroscopy from March 2016 to February 2019, including 122 males and 38 females, aged from 22 to 71 years old with an average of (42.35±3.48) years old. Based on the results of arthroscopy as the gold standard, the shape and signal changes of rotator cuff, rotator interval, peripheral bursa, bone and soft tissue were observed by MRI on axial, oblique coronal and oblique sagittal imagese. RESULTS: The direct MRI signs of acute rotator interval injury displayed thickening, diminution, distortion, interruption of the coracohumeral ligament and superior glenohumeral ligament complex with highsignal intensity on fat-suppression by proton weighted sequence. The indirect MRI signs displayed rotator cuff, peripheral bone and soft tissue injury. The consistency of the results between the two methods was quite satisfactory (Kappa=0.85), and the concordance rate of the two methods has statistically significant (Z=10.75, P<0.00). There was no statistical difference in the inconsistent parts between two methods (χ2= 0.400, P>0.05). The true positive rate, true negative rate, false positive rate and false negative rate of MRI diagnosis of rotator interval injury were 93.75%, 91.30%, 94.74%, 5.26%, 8.70% respectively. CONCLUSION: MRI could clearly display acute rotator interval, and could accurately diagnose acute rotator interval injury, which provide more accurate imaging basis for clinical diagnosis and treatment.

SUMOylation disassembles the tetrameric pyruvate kinase M2 to block myeloid differentiation of leukemia cells.

Leukemia arises from blockage of the differentiation/maturation of hematopoietic progenitor cells at different stages with uncontrolled proliferation of leukemic cells. However, the signal pathways that block cell differentiation remain unclear. Herein we found that SUMOylation of the M2 isoform of pyruvate kinase (PKM2), a rate-limiting glycolytic enzyme catalyzing the dephosphorylation of phosphoenolpyruvate to pyruvate, is prevalent in a variety of leukemic cell lines as well as primary samples from patients with leukemia through multiple-reaction monitoring based targeted mass spectrometry analysis. SUMOylation of PKM2 lysine 270 (K270) triggered conformation change from tetrameric to dimeric of PKM2, reduced PK activity, and led to nuclear translocation of PKM2. SUMO1 modification of PKM2 recruits and promotes degradation of RUNX1 via a SUMO-interacting motif, resulting in blockage of myeloid differentiation of NB4 and U937 leukemia cells. Replacement of wild type PKM2 with a SUMOylation-deficient mutant (K270R) abrogated the interaction with RUNX1, and the blockage of myeloid differentiation in vitro and in xenograft model. Our results establish PKM2 as an essential modulator of leukemia cell differentiation and a potential therapeutic target, which may offer synergistic effect with differentiation therapy in the treatment of leukemia.

LncRNA NEAT1 promotes endometrial cancer cell proliferation, migration and invasion by regulating the miR-144-3p/EZH2 axis.

Background Endometrial cancer (EC) is one of the most common gynaecological tumours in the worldwide. Long non-coding RNA (lncRNA) nuclear enriched abundant transcript 1 (NEAT1) promotes cell proliferation, migration and invasion in EC cells. However, the molecular mechanisms of NEAT1 in EC have not been fully clarified. We conducted this study to reveal the function of NEAT1 in EC tissues and cell lines. Materials and methods Cancer and adjacent tissues were collected from EC patients. HEC-1A and Ishikawa cells were cultured in vitro. NEAT1 expression was downregulated by transfecting small hairpin RNA (shRNA) and miR-144-3p was overexpressed by transfecting miR-144-3p mimics. Cell proliferation was detected by MTT assay and colony formation assay. Cell migration and invasion abilities were assessed by transwell assay. A dual-luciferase reporter assay was used to verify the relationship among NEAT1, EZH2, and miR-144-3p. The expression level of EZH2 was measured by Western blot and qPCR. Results NEAT1 was highly expressed in EC tissues and cells. Knockdown of NEAT1 inhibited the proliferation, migration and invasion of EC cells. Additionally, NEAT1 acted as a ceRNA of miR-144-3p, leading to EZH2 upregulation. Overexpression of miR-144-3p suppressed the proliferation and invasion of EC cells. Conclusions NEAT1 promotes EC cells proliferation and invasion by regulating the miR-144-3p/EZH2 axis.

Bardoxolone methyl (CDDO-Me or RTA402) induces cell cycle arrest, apoptosis and autophagy via PI3K/Akt/mTOR and p38 MAPK/Erk1/2 signaling pathways in K562 cells.

Chronic myeloid leukemia (CML) treatment remains a challenge due to drug resistance and severe side effect, rendering the need on the development of novel therapeutics. CDDO-Me (Bardoxolone methyl), a potent Nrf2 activator and NF-κB inhibitor, is a promising candidate for cancer treatment including leukemia. However, the underlying mechanism for CDDO-Me in CML treatment is unclear. This study aimed to evaluate the molecular interactome of CDDO-Me in K562 cells using the quantitative proteomics approach stable-isotope labeling by amino acids in cell culture (SILAC) and explore the underlying mechanisms using cell-based functional assays. A total of 1,555 proteins responded to CDDO-Me exposure, including FANCI, SRPK2, XPO5, HP1BP3, NELFCD, Na+,K+-ATPase 1, etc. in K562 cells. A total of 246 signaling pathways and 25 networks regulating cell survival and death, cellular function and maintenance, energy production, protein synthesis, response to oxidative stress, and nucleic acid metabolism were involved. Our verification experiments confirmed that CDDO-Me down-regulated Na+,K+-ATPase α1 in K562 cells, and significantly arrested cells in G2/M and S phases, accompanied by remarkable alterations in the expression of key cell cycle regulators. CDDO-Me caused mitochondria-, death receptor-dependent and ER stress-mediated apoptosis in K562 cells, also induced autophagy with the suppression of PI3K/Akt/mTOR signaling pathway. p38 MAPK/Erk1/2 signaling pathways contributed to both apoptosis- and autophagy-inducing effects of CDDO-Me in K562 cells. Taken together, these data demonstrate that CDDO-Me is a potential anti-cancer agent that targets cell cycle, apoptosis, and autophagy in the treatment of CML.

The combination of digoxin and GSK2606414 exerts synergistic anticancer activity against leukemia in vitro and in vivo.

Digoxin is a member of cardiac glycosides and recent studies show that digoxin plays anticancer role in several types of cancer. However, the anticancer effects and mechanism of digoxin in leukemia is largely unknown. Her, our data show that digoxin treatment significantly inhibits leukemia cell viability. In addition, digoxin treatment significantly induced apoptosis and G2/M cell cycle arrest in leukemia cells. Furthermore, we demonstrated that digoxin treatment inactivate that oncogenic pathway Akt/mTOR signaling in leukemia cells. In addition, our data show that digoxin treatment induces activation of unfolded protein response (UPR) signaling in leukemia cells. Interestingly, our in vitro and in vivo experiments show that combination treatment of digoxin and UPR inhibitor can synergistically suppress leukemia growth and induces apoptosis and cell cycle arrest compared to single drug treatment. In summary, our findings indicate that digoxin has potential anticancer effects on leukemia. The combination of digoxin and UPR signaling inhibitor can exerts synergistic anticancer activity against leukemia. © 2017 BioFactors, 43(6):812-820, 2017.

Identification and expression analysis of CYS-A1, CYS-C1, NIT4 genes in rice seedlings exposed to cyanide.

Involvement of genes (CYS-A1, CYS-C1 and NIT4) encoded with cysteine synthase, ß-cyanoalanine synthase, nitrilase and cyanide metabolisms are evident in Arabidopsis. In the present study, identifications of CYS-A1, CYS-C1 and NIT4, predictions of conserved motifs, and constructions of phylogenetic relationships, based on their amino acid sequences in rice, were conducted. In order to elucidate the transcriptional responses of these cyanide-degrading genes, two candidate homologues were selected for each gene to test their expression changes upon exposure to exogenous KCN in rice seedlings using RT-PCR. Results showed that all selected candidate homologous genes were differentially expressed at different exposure points in roots and shoots of rice seedlings, suggesting their distinct roles during cyanide assimilation. Both candidate homologues for CYS-A1 constantly exhibited more abundant transcripts in comparison to control. However, only one candidate homologue for CYS-C1 and NIT4 showed a remarkable up-regulation during KCN exposure. Analysis of both tissue and solution cyanide indicated that rice seedlings were quickly able to metabolize exogenous KCN with minor accumulation in plant tissues. In conclusion, significant up-regulation of CYS-A1 suggested that the endogenous pool of cysteine catalyzed by cysteine synthase does not restrict the conversion of exogenous KCN into cyanoalanine through the ß-cyanoalanine pathway. However, insufficient responses of the transcription level of NIT4 suggested that NIT enzyme may be a limiting factor for cyanoalanine assimilation by rice seedlings.

[Smoothing Gan Reinforcing Shen Method Adjuvantly Treated Poor Response of Diminished Ovari- an Reserve Patients in in vitro Fertilization and Embryo Transfer: a Clinical Study].

OBJECTIVE: To study clinical efficacy of smoothing Gan reinforcing Shen (SGRS) method in treating poor response of diminished ovarian reserve (DOR) patients in in vitro fertilization and embryo, transfer (IVF-ET). METHODS: Totally 84 DOR patients undergoing IVF-ET were assigned to the experimental group (SGRS Chinese herbs as adjuvant therapy) and the control group according to random digit table, 42 in each group. Patients in the control group received controlled ovarian hyperstimulation (COH) and IVF-ET. Those in the experimental group additionally received basic formula of SGRS method, one dose per day. The dose and use time of recombinant follicle-stimulating hormone (r-FSH) were recorded during ovarian stimulation process. On the injection day of human chorionic gonadotropin (HCG) and serum levels of estradiol (E2) on the oocyte retrieval day were determined using chemiluminescent method. E2 contents in the follicular fluid on the oocyte retrieval day were detected using ELISA. The total number of retrieved oocytes, the number of mature oocytes in metaphase II (M II), the number of normal fertilization [with two pronucleus (2PN)], the number of portable embryos, and the number of good quality embryos were recorded. The correlation between Chinese medical adjuvant therapy and the aforesaid indices were observed. The clinical pregnancy rate and the abortion rate were finally compared between the two groups. RESULTS: The total dose of r-FSH, the E2 level on HCG injection day, the serum E2 level on the oocyte retrieval day, the number of retrieved oocyte, the number of oocytes in M II the number of oocytes with 2PN, the number of portable embryos, and the number of good quality embryos were all positively correlated with Chinese medical adjuvant therapy (P < 0.05, P < 0.01). Compared with the control group, serum E2 levels on the HCG injection day and the oocyte retrieval day obviously increased, the number of retrieved oocytes, the number of oocytes in M II, and the number of portable embryos were increased more in the experimental group with statistical difference (P < 0.05, P < 0.01). There was no statistical significance in the clinical pregnancy rate or the abortion rate between the two groups (P > 0.05). CONCLUSION: SGRS Chinese herbs as adjuvant therapy could improve ovarian responsiveness of DOR patients undergoing IVF-ET, increase the number of retrieved oocytes, elevate the quality of oocytes and the number of embryos.

Unexplained infertility patients have increased serum IL-2, IL-4, IL-6, IL-8, IL-21, TNFα, IFNγ and increased Tfh/CD4 T cell ratio: increased Tfh and IL-21 strongly correlate with presence of autoantibodies.

Infertility is a disease of the reproductive system that affects millions of people globally. Reproductive failure is a major medical issue adversely affecting human health in the 21st century. Many factors contribute to infertility, including immune conditions which may lead to immune infertility (immunologic infertility). It is known that specific T helper cells (Th) and their cytokines are involved in the stages of infertility. The aim of this study is to provide a new diagnostic approach to immunologic infertility by investigating the correlation of follicular helper T cells (Tfh) and their secreted cytokines with the autoantibodies in peripheral blood samples from immunologically infertile patients. Thirty (30) patients suffering from immune infertility and 20 control subjects were selected as the sample base for this study. The levels of Tfh, 20 cytokines and 4 antibodies were evaluated for this investigation and evaluated using flow cytometry, antibody chip and ELISA technologies. It was found that, in immunologically infertile patients, Tfh cell numbers were significantly higher than those in the control group. Likewise, seven (7) serum cytokines were expressed to a greater degree in infertile patients compared to the control group. Finally, four (4) antibodies were found to be higher in immunologically infertile patients. The results show that, among patients with immunologic infertility, the levels of Tfh cells and IL-21 were increased significantly in peripheral blood samples and correlate positively with the autoantibodies. IL-12 was positively correlated with the two antibodies, while TNFα was negatively correlated with two additional antibodies. The detection and quantification of Tfh cells, IL-21, IL-12 and TNFα may provide new diagnostic indicators to screen for immunologic infertility.

Vitamin B6 and colorectal cancer: current evidence and future directions.

Colorectal cancer remains the third most common cancer in both women and men worldwide. Identifying modifiable dietary factors is crucial in developing primary prevention strategies. Vitamin B6 is involved in more than 100 coenzyme reactions, and may influence colorectal cancer risk in multiple ways including through its role in one-carbon metabolism related DNA synthesis and methylation and by reducing inflammation, cell proliferation, and oxidative stress. Observational studies of dietary or dietary plus supplementary intake of vitamin B6 and colorectal cancer risk have been inconsistent with most studies reporting nonsignificant positive or inverse associations. However, published studies of plasma pyridoxal 5'-phosphate (the active form of vitamin B6) levels consistently support an approximately 30%-50% reduction in risk of colorectal cancer comparing high with low concentrations. The reasons for the discrepancy in the results between dietary-based and plasma-based studies remain unresolved. Other unresolved questions include the effects of vitamin B6 intake in early life (i.e., childhood or adolescence) and of suboptimal vitamin B6 status on colorectal cancer risk, whether the associations with vitamin B6 differ across molecular subtypes of colorectal cancer, and whether the vitamin B6-colorectal cancer association is modified by genetic variants of one-carbon metabolism.

[Association between hand-foot-and-mouth disease in Xi'an and enterovirus 71].

OBJECTIVE: To isolate the prevalent strain of enterovirus 71 (EV71) in Xi'an area in 2008, and compare the concordance of viral isolation, reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescent technique in detecting EV71, find the fast and effective method for detection, and analyze the differences between the EV71 strains isolated from Xi'an and Fuyang, Anhui. METHOD: Virus isolation and RT-PCR were carried out on vesicle fluid and throat swab specimens that were collected from the patients with hand-foot-and-mouth disease, RD and HEp-2 cell lines were used for viral isolation. The virus was identified by using immunofluorescence technique. Nucleotide sequencing was performed on positive product of RT-PCR, and compared with EV71 isolated from Fuyang in 2008, then submitted to Genbank. RESULT: Among the 56 samples of throat swab inoculated on RD and HEp-2 cells, the positive rates were 5.4% (3/56) and 1.8% (1/56), respectively. Among the 56 samples of vesicle fluid inoculated on RD and HEp-2 cells, the positive rates were 12.5% ( 7/56 ) and 5.4% (3/56), respectively. Cytopathic effect of RD and HEp-2 cells appeared on days 7 and 10, respectively. The positive rates of RT-PCR on throat swab and vesicle fluid samples were 21.4% (12/56) and 33.9% (19/56), respectively. Cytopathic effect was found in cell culture for 14 cases and immunofluorescence, showed that 9 of them were infected with EV71. The authors obtained the EV71 strain prevalent in Xi'an during 2008. The nucleotide sequence was submitted to the NCBI Genbank and gained the accession number EU812461. CONCLUSION: The EV71 in Xi'an prevalent during 2008 may have a weaker epithelial tropism. Comparison of the EV71 strain isolated from Xi'an with EU703812, EU703813 and EU703814 isolated from Fuyang, Anhui showed that the homology was 97%-98%. RT-PCR is an important method for rapid detection of EV71.

[Preparation, characterization and application of anti-human adenovirus monoclonal antibody with neutralization activity.].

AIM: To prepare, identify and apply anti-human adenoviru(HAdv)neutralization monoclonal antibody(mAb). METHODS: BALB/c mice were immunized with live human adenovirus type3(HAdv-3) strain intranarially. Sp2/0 cells were fused with the spleen cells harvested from BALB/c mice. The chromosomal amounts of the hybridoma cells were analyzed by colchicine. A commercially available mouse mAb isotyping kit was used to identify the isotype of this mAb. Clones secreting specific monoclonal antibody were screened by indirect enzyme linked immunosorbent assay (ELISA), Western blot and indirect immunofluorescent assay. Infected animal model was established, and the protective effect of mAb was studied. RESULTS: The fusion rate was 86%, and the positive rate was 51.4%. One of the hybridoma cell was identified(1A4), the chromosomal amounts of was 98, the subtype mAb type belonged to IgG2a/kappa, and the titer of the mAb secreted by the strain in ascite reached more than 10(-5);. The specificity of the mAb was proved by ELISA , Western blot and indirect immunofluorescent assay. This mAb had protective effect on animal infected by HAdv-3. CONCLUSION: The anti-adenovirus mAb which have neutralization activities was successfully prepared. The mAb recognized the hexon subunit and had protective effect on animal infected by HAdv-3.

Tea drinking and the risk of biliary tract cancers and biliary stones: a population-based case-control study in Shanghai, China.

Biliary tract cancers, encompassing tumors of the gallbladder, extrahepatic bile ducts and ampulla of Vater, are rare but highly fatal malignancies. Apart from gallstones, etiologic factors for biliary tract cancer are not clearly defined. Several epidemiologic studies have suggested that consumption of tea, especially green tea, is protective against a variety of cancers, including gastrointestinal malignancies. As part of a large population-based case-control study of biliary tract disease in Shanghai, China, we evaluated the effects of tea consumption on the risk of biliary tract cancers and biliary stones. The study included 627 incident cases with biliary tract cancer, 1,037 cases with biliary stones and 959 randomly selected controls. Study subjects were interviewed to ascertain data on demographic, medical and dietary factors, including tea consumption. Forty-one percent of the controls were ever tea drinkers, defined as those who consumed at least 1 cup of tea per day for at least 6 months. After adjustment for age, education and body mass index, among women, ever tea drinkers had significantly reduced risks of biliary stones (OR = 0.73, 95% CI = 0.54-0.98) and gallbladder cancer (OR = 0.56, 95% CI = 0.38-0.83). The inverse relationship between tea consumption and gallbladder cancer risk was independent of gallstone disease. Among men, tea drinkers were more likely to be cigarette smokers, and the risk estimates were generally below 1.0, but were not statistically significant. Further studies are needed to confirm these results in other populations and clarify the hormonal and other mechanisms that may be involved.

[The study on the construction of expression plasmid containing rHO-1 cDNA, analysis of rHO-1 activity and function of anti-apoptosis in HUVEC].

Xenograft rejection are due to the activation of endothelial cells and the expression of a series of proinflammatory genes encoding adhesion molecules, cytokines/chemokines and procoagulant molecules,leading to endothelial cell injury and apoptosis. HO-1 which acts as a cytoprotective gene can suppress a variety of inflammatory responses to prevent graft rejection. In this study, The plasmid containing HO-1 cDNA was constructed and transfected into human vascular endothelial cells (HUVEC) for expression using DOTAP lipsomal transfection reagents. Cells were homogenized in cell culture lysis reagent (CCLR) and the activity of HO-1 was measured. The apoptosis of HUVEC induced by tumor necrosis factor (TNF)-alpha was analyzed by flow cytometry. Meanwhile, Heme and Sn-protoporphyrin (SnPP) were added respectively to evaluate the apoptosis rate of HUVEC. The results showed that the expression of HO-1 gene can be significantly up-regulated in HUVEC transfected with pcDNA3HO1. The apoptosis rate of cells treated with Heme was less than 20% but significantly increased when cells treated with SnPP, the maximum arrived at 95%. The apoptosis rate in heme induced cells was 5-20 times lower than that in SnPP inhibited cells. The apoptosis rate was a negative relation to HO-1 expression. HO-1 protein can inhibit apoptosis in HUVEC. The results provide evidence to support the essential role of HO-1 in the cytoprotective function.