Predictive value of number of metastatic lymph nodes and lymph node ratio for prognosis of patients with FIGO 2018 stage IIICp cervical cancer: a multi-center retrospective study.

BACKGROUND: To identify the cut-off values for the number of metastatic lymph nodes (nMLN) and lymph node ratio (LNR) that can predict outcomes in patients with FIGO 2018 IIICp cervical cancer (CC). METHODS: Patients with CC who underwent radical hysterectomy with pelvic lymphadenectomy were identified for a propensity score-matched (PSM) cohort study. A receiver operating characteristic (ROC) curve analysis was performed to determine the critical nMLN and LNR values. Five-year overall survival (OS) and disease-free survival (DFS) rates were compared using Kaplan-Meier and Cox proportional hazard regression analyses. RESULTS: This study included 3,135 CC patients with stage FIGO 2018 IIICp from 47 Chinese hospitals between 2004 and 2018. Based on ROC curve analysis, the cut-off values for nMLN and LNR were 3.5 and 0.11, respectively. The final cohort consisted of nMLN ≤ 3 (n = 2,378) and nMLN > 3 (n = 757) groups and LNR ≤ 0.11 (n = 1,748) and LNR > 0.11 (n = 1,387) groups. Significant differences were found in survival between the nMLN ≤ 3 vs the nMLN > 3 (post-PSM, OS: 76.8% vs 67.9%, P = 0.003; hazard ratio [HR]: 1.411, 95% confidence interval [CI]: 1.108-1.798, P = 0.005; DFS: 65.5% vs 55.3%, P < 0.001; HR: 1.428, 95% CI: 1.175-1.735, P < 0.001), and the LNR ≤ 0.11 and LNR > 0.11 (post-PSM, OS: 82.5% vs 76.9%, P = 0.010; HR: 1.407, 95% CI: 1.103-1.794, P = 0.006; DFS: 72.8% vs 65.1%, P = 0.002; HR: 1.347, 95% CI: 1.110-1.633, P = 0.002) groups. CONCLUSIONS: This study found that nMLN > 3 and LNR > 0.11 were associated with poor prognosis in CC patients.

An electrochemical immunosensor for sensitive and rapid detection of cystatin C based on Fe3O4/AuNPs-MWCNTs@PDA nanocomposite.

Serum Cystatin C (CysC) is an impressive marker for early diagnosis of renal dysfunction. In this work, we established a novel electrochemical immunosensor based on Fe3O4/AuNPs-MWCNTs@PDA nanocomposite for the detection of CysC. The Fe3O4/AuNPs-MWCNTs@PDA nanozyme complex by polydopamine encapsulation can not only carry massive detection antibodies, but also bind the electroactive substance toluidine blue (TB) through electrostatic adsorption. By immobilizing AuNPs onto the electrode to bind the capture antibody (Ab1), we constructed a sandwich electrochemical immunosensor with low cost, high sensitivity, and repeatability. The detection range is 3.9-125.0 ng/mL with a significant linear relationship between the current peak difference (ip) and logarithm of the CysC concentration. Moreover, the detection limit of the immunosensor is 0.157 ng/mL. We have successfully utilized this novel immunosensor to detect CysC in human serum samples, and these results have implications for its potential use in clinical application.

Increased release of microvesicles containing mitochondria is associated with the myeloid differentiation of AML-M5 leukaemia cells.

Extensive literature has demonstrated that acute myeloid leukaemia (AML) cells show enhanced mitochondrial biogenesis and increased reliance on oxidative phosphorylation (OXPHOS) compared with normal hematopoietic progenitors, and one hallmark of AML leukaemia blasts is myeloid differentiation blockade. However, relatively few reports have linked these processes. Recent studies have indicated that therapies that overcome differentiation arrest represent an effective treatment strategy. Here, we identified that the disruption of the mitochondrial mass and energy metabolism promotes leukaemia cellular myeloid differentiation. In this study, we showed that acute monocytic leukaemia (AML-M5) cells package mitochondria in microvesicles (MVs) when MVs shed from membranes. Additionally, during myeloid differentiation, we report for the first time that differentiated leukaemia cells release more MVs than undifferentiated leukaemia cells. Targeting the formation of MVs using a specific inhibitor (Y-27632) restrained myeloid differentiation, suggesting that the increased release level of MVs plays an important role in regulating myeloid differentiation. Furthermore, the intracellular mitochondria and ATP levels were decreased after leukaemia cells overcame the differentiation blockade. Moreover, rotenone, which is used to inhibit the respiratory chain and ATP production, had a strong effect on myeloid differentiation in monocytic leukaemia cells. Collectively, these studies uncovered the relationship between mitochondrial function and myeloid differentiation and may provide more insight into the diagnosis and treatment of AML.

M2 polarization of tumor-associated macrophages is dependent on integrin ß3 via peroxisome proliferator-activated receptor-γ up-regulation in breast cancer.

Macrophages are particularly abundant and play an important role throughout the tumor progression process, namely, tumor-associated macrophages (TAM) in the tumor microenvironment. TAM can be polarized to disparate functional phenotypes, the M1 and M2 macrophages. M1-like type macrophages are defined as pro-inflammatory cells involved in killing cancer cells, while M2-like type cells can specially promote tumor growth and metastasis, tissue remodeling and immunosuppression. In this study, we first found that integrin ß3 was highly expressed on the surface of TAM, both in vivo and in vitro, that displayed the M2-like characteristics. Under intervention of CYC or triptolide, the integrin ß3 inhibitors, the M2 polarization of TAM could be inhibited. Moreover, in the cell model of M2 polarization, either blockade or knockout/knockdown of integrin ß3 could also suppress macrophage M2 polarization, which suggested that the M2 polarization was dependent on integrin ß3. Using knockdown of peroxisome proliferator-activated receptor-γ (PPARγ), an M2 regulator, we found that expression and activation of PPARγ participated in M2 polarization that was mediated by integrin ß3. Finally, to verify the activity of integrin ß3 inhibitors on TAM in vivo, 4T1 tumor-bearing mice were treated with CYC or triptolide; in response, the M1/M2 ratio of TAM was up-regulated, while the infiltration of total lymphocytes into tumor tissue was not altered. In general, our study found a connection between integrin ß3 and macrophage polarization, which provides a strategy for facilitating M2 to M1 repolarization and reconstructing the tumor immune microenvironment.

Identification of Caspase-6 as a New Regulator of Alternatively Activated Macrophages.

Alternatively activated macrophages (AAMs) play essential roles in the promotion of tissue remodeling, vasculogenesis, and tumor progression; however, the detailed mechanisms underlying the activation of AAMs remain largely unknown. Here, by using quantitative proteomic analysis, we identified 62 proteins that were up-regulated in IL-4-induced macrophages. Among these, Caspase-6 was increased significantly. Caspase-6 is important in the apoptotic signaling pathway; however, its role in non-apoptosis is also reported. Here, we first examined the non-apoptotic role of Caspase-6 in the alternative activation of macrophages after administration of IL-4, 4T1 tumor conditional medium, or co-culture with 4T1 cells. Both treatments promoted alternative activation of RAW264.7 cells and primary macrophages, whereas disruption of caspase-6 expression and activity could markedly suppress the biomarker levels of AAMs. Overexpression of Caspase-6 could significantly promote the activation of AAMs. Importantly, we further present evidence that caspase-6 could regulate breast cancer cell invasion by modulating MMP-2 and MMP-9 expression in 4T1 tumor-associated macrophages, as ablation of protein levels or activity of caspase-6 suppressed tumor cell invasion in vitro In conclusion, the observed results markedly expanded our views of the dynamic changes in protein composition during alternative activation of macrophages, and they revealed a critical new role of caspase-6 in regulating this cellular biological process, which suggested that caspase-6 might be a key nod molecule to regulate immunological steady-state and be a therapeutic candidate for tumor immunotherapy.

[Preparation of monoclonal antibody against cystatin C and establishment of its immunoassay system].

OBJECTIVE: To prepare human cystatin C( CysC) recombinant protein and produce monoclonal antibodies with high affinity and specificity. Develop a competitive ELISA detection system to detect of CysC in human serum. METHODS: The CysC gene sequence was found on NCBI. The optimized gene fragments were synthesized and the recombinant CysC protein was expressed in Escherichia coli then used to immunize Balb/c mice. The positive hybridoma cell lines were obtained by hybridoma cell fusion techniques and ascites monoclonal antibody was prepared and purified. Affinity of the antibody was measured by indirect ELISA. Then competitive ELISA detection system was established, and 52 cases of human serum samples were detected by the detection system. RESULTS: Four stable cell lines secreting CysC monoclonal antibodies were obtained. Antibody Ab3 was used as a detection antibody and HRP labeling was performed. Its affinity constant was 4. 26 × 10~6L/mol. The linear range of detection was 0. 011-1. 924 µg/mL. The detection limit was 4. 598 ng/mL and IC_(50) was 0. 145 µg/mL. The established competitiveELISA serum detection system could accurately detect those 52 serum samples. CONCLUSION: The monoclonal antibody against CysC with high affinity and specificity has been successfully obtained. A reliable competitive ELISA serum detection system is established. The method provides a basis for the development of CysC rapid immunoassay kit.

Molecular phylogeny reveals independent origins of body scales in Entomobryidae (Hexapoda: Collembola).

Entomobryidae is the largest family in Collembola but relationships within the family have never been subjected to rigorous phylogenetic analyses. Within the family, body scales are present in many species, and are fundamental in the classification at the subfamilial and tribal levels. A molecular phylogeny was reconstructed using the nuclear 18SrRNA and partial 28SrRNA and the mitochondrial 16SrRNA to examine the evolution of scales across Entomobryidae subfamilies. These datasets were analyzed separately and combined, with parsimony, likelihood and Bayesian algorithms. Monophyly of Orchesellinae was not recovered, and it was split into a scaled clade and an unscaled clade, contradicting to all recent taxonomic conceptions. The monophyly of Entomobryinae, Seirinae and Lepidocyrtinae is well supported however within Entomobryinae, the polyphyly of Entomobryini and Willowsiini implies that classification using the presence/absence of scales is not valid. Analyses of ancestral character state reconstruction in Entomobryidae indicate that the presence of body scales have evolved independently at least five times, with a loss of scales occurring independently at least twice. A revision of the family Entomobryidae on molecular and morphological basis is clearly needed.

Bioinspired Collagen Scaffold Loaded with bFGF-Overexpressing Human Mesenchymal Stromal Cells Accelerating Diabetic Skin Wound Healing via HIF-1 Signal Pathway Regulated Neovascularization.

The healing of severe chronic skin wounds in chronic diabetic patients is still a huge clinical challenge due to complex regeneration processes and control signals. Therefore, a single approach is difficult in obtaining satisfactory therapeutic efficacy for severe diabetic skin wounds. In this study, we adopted a composite strategy for diabetic skin wound healing. First, we fabricated a collagen-based biomimetic skin scaffold. The human basic fibroblast growth factor (bFGF) gene was electrically transduced into human umbilical cord mesenchymal stromal cells (UC-MSCs), and the stable bFGF-overexpressing UC-MSCs (bFGF-MSCs) clones were screened out. Then, an inspired collagen scaffold loaded with bFGF-MSCs was applied to treat full-thickness skin incision wounds in a streptozotocin-induced diabetic rat model. The mechanism of skin damage repair in diabetes mellitus was investigated using RNA-Seq and Western blot assays. The bioinspired collagen scaffold demonstrated good biocompatibility for skin-regeneration-associated cells such as human fibroblast (HFs) and endothelial cells (ECs). The bioinspired collagen scaffold loaded with bFGF-MSCs accelerated the diabetic full-thickness incision wound healing including cell proliferation enhancement, collagen deposition, and re-epithelialization, compared with other treatments. We also showed that the inspired skin scaffold could enhance the in vitro tube formation of ECs and the early angiogenesis process of the wound tissue in vivo. Further findings revealed enhanced angiogenic potential in ECs stimulated by bFGF-MSCs, evidenced by increased AKT phosphorylation and elevated HIF-1α and HIF-1ß levels, indicating the activation of HIF-1 pathways in diabetic wound healing. Based on the superior biocompatibility and bioactivity, the novel bioinspired skin healing materials composed of the collagen scaffold and bFGF-MSCs will be promising for healing diabetic skin wounds and even other refractory tissue regenerations. The bioinspired collagen scaffold loaded with bFGF-MSCs could accelerate diabetic wound healing via neovascularization by activating HIF-1 pathways.

Lipid synthesis, triggered by PPARγ T166 dephosphorylation, sustains reparative function of macrophages during tissue repair.

Macrophages may acquire a reparative phenotype that supports tissue repair and remodeling in response to tissue injury. However, the metabolic requirements underpinning this process are incompletely understood. Here, we show that posttranslational modification (PTM) of PPARγ regulates lipid synthesis in response to wound microenvironmental cues and that metabolic rewiring orchestrates function of reparative macrophages. In injured tissues, repair signaling leads to decreased macrophage PPARγ threonine 166 (T166) phosphorylation, which results in a partially active PPARγ transcriptional program comprised of increased binding activity to the regulator regions of lipid synthesis-associated genes, thereby increased lipogenesis. The accumulated lipids serve as signaling molecules, triggering STAT3-mediated growth factor expression, and supporting the synthesis of phospholipids for the expansion of the endoplasmic reticulum (ER), which is required for protein secretion. Genetic or pharmacological inhibition of PPARγ T166 phosphorylation promotes the reparative function of macrophages and facilitates tissue regeneration. In summary, our work identifies PPARγ T166-regulated lipid biosynthesis as an essential pathway for meeting the anabolic demands of the activation and function of macrophages and provides a rationale for potential therapeutic targeting of tissue repair.

Initial treatment for FIGO 2018 stage IIIC cervical cancer based on histological type: A 14-year multicenter study.

BACKGROUND: To compare the oncological outcomes of radical chemotherapy (R-CT), abdominal radical hysterectomy (ARH), and neoadjuvant chemotherapy and radical surgery (NACT) for International Federation of Gynecology and Obstetrics (FIGO) 2018 stage IIIC cervical cancer, according to histological types: squamous cell carcinoma (SCC) and adenocarcinoma (AC)/adenosquamous cell carcinoma (ASC). METHODS: A comparison of 5-year overall survival (OS) and disease-free survival (DFS) was performed for the SCC and AC/ASC subgroups for the three initial treatments, assessed using Kaplan-Meier and Cox proportional hazards regression analysis and validated using propensity score matching (PSM). RESULTS: The study included 4086 patients: R-CT, n = 1913; ARH, n = 1529; and NACT, n = 644. AC/ASC had a lower survival rate (63.7%) than SCC (73.6%) and a higher recurrence and mortality rate (36.3% and 26.4%, respectively). The 5-year OS and DFS rates were different in the SCC group for R-CT, ARH, and NACT (OS: 69.8% vs. 80.8% vs. 73.0%, p < 0.001; DFS: 66.7% vs. 70.7% vs. 56.4%, p < 0.001), also in the AC/ASC group (OS: 46.1% vs. 70.6% vs. 55.6%, p < 0.001; DFS: 42.7% vs. 64.6% vs. 40.8%, p < 0.001). As for initial treatment, survival outcomes were worse for AC/ASC treated with R-CT and ARH than for SCC (both p < 0.05), with no group differences between the two treated with NACT. CONCLUSION: Initial treatment influences oncological prognosis for patients with FIGO 2018 stage IIIC cervical cancer. ARH is an alternative treatment for stage IIIC cervical SCC and AC/ASC, and NACT needs to be chosen with caution, moreover, R-CT for AC/ASC requires careful selection.

Nano-Film-Forming Plugging Drilling Fluid and Bridging Cross-Linking Plugging Agent Are Used to Strengthen Wellbores in Complex Formations.

Lost circulation is one of the great challenges during the drilling process as it can not only increase the risk of drilling operations but also cause an increase in drilling costs, thus greatly affecting the drilling efficiency. Wellbore strengthening has been widely used to prevent lost circulation, which ultimately expands the mud density window by increasing the formation fracture pressure. This paper proposes a combination of "preventive" wellbore strengthening and "remedial" wellbore strengthening to prevent leakage and plug and stabilize wellbores by means of summarizing the characteristics of lost circulation and wellbore instability in the Hasan area. The formula of the bridging cross-linking plugging agent is determined by experiments as well slurry + 8-10% granular material + 3-5% fiber material + 2-4% elastic material + 0.5-1% cementing material. The formula of the nano-film-forming plugging drilling fluid is determined to be 3% bentonite + 0.2NaOH + 0.2% KPAM + 3% SMP + 1% PB-1 + 2% SMNA-1 + 2% lubricant SMLUB-1 + 1% modified nano-SiO2 particles. Then, the performance evaluation of the composite system is carried out by the high-temperature and high-pressure plugging simulation evaluation device. The results show that the bridging and cross-linking plugging agent can effectively block the 1-3 mm crack, the pressure-bearing capacity is greater than 10 MPa, and the anti-liquid return capacity is greater than 4 MPa. The nano-film-forming plugging drilling fluid has lower fluid loss and better rheological properties.

Changes in the Immunity, Histopathology, and Metabolism of Crayfish (Procambarus clarkii) in Response to Drought.

Freshwater ecosystems are among the most threatened ecosystems on Earth. The freshwater biodiversity crisis has caused widespread global concern. Drought as one of the factors causing freshwater biodiversity is still poorly understood. Crayfish is often used in academic research as a biological indicator. In this study, flow cytometry, hematoxylin-eosin staining, and untargeted metabolomics were used to analyze the immune function, histopathology, and metabolism of crayfish under drought conditions. After drought exposure, the total hemocytes count (THC) was significantly decreased (from 8.9 × 105 mL-1 in the control group to 2.2 × 105 mL-1 at day 5). Phagocytosis decreased by 66% after 5 days of drought. The level of reactive oxygen species (ROS) in the hepatopancreas was upregulated. Moreover, histological disorder and metabolism changes in the hepatopancreas were obvious. These results indicate that drought suppresses immune function, disrupts the balance of oxidative and antioxidative systems, and induces tissue damage and metabolic changes in crayfish.

Stanniocalcin-1 promotes tumor angiogenesis through up-regulation of VEGF in gastric cancer cells.

BACKGROUND: Stanniocalcin-1(STC-1) is up-regulated in several cancers including gastric cancer. Evidences suggest that STC-1 is associated with carcinogenesis and angiogenic process. However, it is unclear on the exact role for STC-1 in inducing angiogenesis and tumorigeneisis. METHOD: BGC/STC cells (high-expression of STC-1) and BGC/shSTC cells (low- expression of STC-1) were constructed to investigate the effect of STC-1 on the xenograft tumor growth and angiogenesis in vitro and in vivo. ELISA assay was used to detect the expression of vascular endothelial growth factor (VEGF) in the supernatants. Neutralizing antibody was used to inhibit VEGF expression in supernatants. The expression of phosphorylated -PKCßII, phosphorylated -ERK1/2 and phosphorylated -P38 in the BGC treated with STC-1protein was detected by western blot. RESULTS: STC-1 could promote angiogenesis in vitro and in vivo, and the angiogenesis was consistent with VEGF expression in vitro. Inhibition of VEGF expression in supernatants with neutralizing antibody markedly abolished angiogenesis induced by STC-1 in vitro. The process of STC-1-regulated VEGF expression was mediated via PKCßII and ERK1/2. CONCLUSIONS: STC-1 promotes the expression of VEGF depended on the activation of PKCßII and ERK1/2 pathways. VEGF subsequently enhances tumor angiogenesis which in turn promotes the gastric tumor growth.

Determination of trace deoxyribonucleic acid by using fluorescein isothiocyanate-phenosafranine as a double-luminescent phosphorescence probe.

Using Pb(2+) as ion perturber, phenosafranine (PF) and fluorescein isothiocyanate (FITC) could emit strong and stable room temperature phosphorescence (RTP) signal on the filter paper, respectively. When they were mixed, the phenomenon that the RTP signal of PF and FITC enhanced significantly was found. And 1.12 ag DNA spot(-1) (sample volume was 0.40 µL, corresponding concentration was 2.8 × 10(-15) g mL(-1)) could cause the RTP signal of both PF and FITC to enhance sharply. The content of DNA was proportional to the ΔI(p) of PF and FITC in the system at 634 and 659 nm. Thus, a new solid substrate room temperature phosphorimetry (SSRTP) for the determination of trace DNA was established by using FITC-PF as double-luminescent phosphorescence probe. The detection limit (LD) of this method calculated by 3S(b)/k was 14 zg DNA spot(-1) for PF and 18 zg DNA spot(-1) for FITC, respectively, showing high sensitivity. It has been applied to the determination of trace DNA in practical samples and the analysis results were in accordance with those of fluorescence probe. The reaction mechanism of SSRTP for the determination of trace DNA was also discussed.

Triple signal amplification strategy for the ultrasensitive electrochemical detection of human papillomavirus 16 E6/E7 mRNA.

Human papilloma virus (HPV) is the primary causative agent of cervical, vaginal, and vulvar cancers. HPV E6/E7 mRNA detection has been proven to improve the specificity and positive predictive value compared with HPV DNA testing in screening, whereby, it may possess higher diagnostic potential. Herein, to establish the ultrasensitive and specific detection of HPV E6/E7 mRNA, we developed a novel triple signal amplification strategy, combined with gold nanoparticles (AuNPs), reverse transcription loop-mediated isothermal amplification (RT-LAMP) and high affinity biotin-avidin system. This novel proposed signal amplification strategy exhibits the desired detection limit of 0.08 fM (approximately 100 copies) and a wide linear range from 0.1 pmol/mL to 100 nmol/mL for HPV16 E6/E7 mRNA detection. Importantly, the present novel biosensor is 10-100 times more sensitive than conventional RT-PCR in detecting HPV16 E6/E7 mRNA positive clinical samples. Conclusively, this biosensor shows good stability, selectivity, and reproducibility, which demonstrates its potential in future clinical diagnosis with desirable sensitivity and specificity.

Solid substrate-room temperature phosphorimetry for the determination of trace lead using p-nitro-phenyl-fluorone-multi-wall carbon nanotubes-Tween-80 micellae compound and diagnosis about human diseases.

The structures of multi-wall carbon nanotubes (MWNTs) were modified by H(2)SO(4)-HNO(3) and H(2)SO(4)-H(2)O(2), respectively. The corresponding products were water-soluble MWNTs-A and MWNTs-B. According to the experiment, it was found that MWNTs-B could emit stable solid substrate-room temperature phosphorescence (RTP) on the surface of paper with Ag(+) as perturber. Under the conditions of 70 degrees C and 15 min, MWNTs-B can react with Tween-80 and p-nitro-phenyl-fluorone (R) to form R-MWNTs-B-Tween-80 micellae compound, which could emit RTP of R and MWNTs-B on the surface of paper, respectively. Pb(2+) could cause the RTP of R and MWNTs-B enhanced sharply, respectively. DeltaI(p) is directly proportional to the content of Pb(2+). A new solid substrate-room temperature phosphorimetry (SS-RTP) for the determination of trace Pb(2+) has been established based on R-MWNTs-B-Tween-80 micellae compound containing double luminescent molecule. The detection limit of this method were 0.035 ag Pb(2+) spot(-1) (8.8 x 10(-17) g Pb(2+) ml(-1), MWNTs-B) and 0.028 ag Pb(2+) spot(-1) (7.1 x 10(-17) g Pb(2+) ml(-1), R). This method is of high sensitivity, good selectivity, high precision and accuracy. It could be applied to determine trace Pb(2+) in serum samples at wavelength of 453.7/623.0 nm (R) or 475.9/645.0 nm (MWNTs-B) with satisfactory results, showing that SS-RTP has flexibility and utility value. Simultaneously, this method can be used to diagnose human diseases. The reaction mechanism for the determination of trace Pb(2+) by SS-RTP based on R-MWNTs-B-Tween-80 micellae compound containing double luminescent molecule was also discussed.

Triptolide-loaded nanoparticles targeting breast cancer in vivo with reduced toxicity.

Triptolide (TP), a diterpenoid triepoxide that is extracted from the plant Tripterygium wilfordii, has been found to be quite effective for treating many malignant tumors. Although TP was initially considered to be a promising chemotherapeutic agent, its poor solubility and high toxicity limited its potential clinical application. Consequently, we synthesized nanoformulated TP coated with hyaluronic acid (HA) for application in treating breast cancer. Our results showed that TP can prevent tumor progression, but at the cost of significant toxicity. By contrast, using the nanoformulated TP, uptake of drugs into the tumor can be facilitated, which leads to a further increase in efficacy while decreasing systemic toxicity.

Protein expression and purification of human Zbtb7A in Pichia pastoris via gene codon optimization and synthesis.

Human Zbtb7A was proved to be an important molecular switch in oncogenesis. However, it is difficult to obtain its protein expression in prokaryotic system, due to high G+C content and rare codons in zbtb7a gene. Therefore, to further research the function and application of this protein, we optimized its coding sequence according to the codon bias of Pichia pastoris, synthesized the sequence with two-step PCR and confirmed the accuracy by DNA sequencing. The assembled fragment was introduced into P. pastoris expression vector pPIC9K and the resultant plasmid pPIC9K-zbtb7a-his(6) was transformed into the P. pastoris strain GS115 by electroporation. The products of the transformants induced by methanol were analyzed by 10% SDS-PAGE and identified by Western Blot assay. The expression conditions of the selected transformant were optimized. Additionally, a two-step purification protocol was applied to purify the recombinant protein. The results showed that the synthetic coding sequence of human Zbtb7A was successfully obtained and inserted into pPIC9K vector. Human Zbtb7A protein was expressed in P. pastoris and identified by western blot. The optimal conditions for its expression in P. pastoris were under a final concentration of 1% methanol and a time-course of 4d. Through the two-step purification, Zbtb7A protein was purified in high purity and its production reached up to as high as 18 mg/L. These results indicated that an effective procedure for expressing and purifying human Zbtb7A in P. pastoris was established.

Over-expressed and truncated midkines promote proliferation of BGC823 cells in vitro and tumor growth in vivo.

AIM: To determine whether midkine (MK) and its truncated form (tMK) contribute to gastric tumorigenesis using in vitro and in vivo models. METHODS: Human MK and tMK plasmids were constructed and expressed in BGC823 (a gastric adenocarcinoma cell line) to investigate the effect of over-expressed MK or tMK on cell growth and turmorigenesis in nude mice. RESULTS: The growth of MK-transfected or tMK-transfected cells was significantly increased compared with that of the control cells, and tMK-transfected cells grew more rapidly than MK-transfected cells. The number of colony formation of the cells transfected with MK or tMK gene was larger than the control cells. In nude mice injected with MK-transfected or tMK-transfected cells, visible tumor was observed earlier and the tumor tissues were larger in size and weight than in control animals that were injected with cells without the transfection of either genes. CONCLUSION: Over-expressed MK or tMK can promote human gastric cancer cell growth in vitro and in vivo, and tMK has greater effect than MK. tMK may be a more promising gene therapeutic target compared with MK for treatment of malignant tumors.

Caspase-1 promotes monocyte-macrophage differentiation by repressing PPARγ.

Monocyte-to-macrophage differentiation is tightly controlled in vivo, as disruption of the normal differentiation program can lead to diverse disorders. Caspase-1, the first identified member of the caspase family, regulates differentiation in various cell types such as Th17 cells and adipocytes. However, the contribution of caspase-1 in monocyte-macrophage differentiation remains elusive. Here we report that caspase-1 is significantly downregulated in leukemia cells from patients with acute monocytic leukemia. By using the phorbol 12-myristate 13-acetate-induced cell differentiation model, we found that caspase-1 activation was required for the differentiation of human monocytes to macrophages. Further analysis of peroxisome proliferator-activated receptor γ (PPARγ) protein levels revealed that the monocyte-macrophage differentiation program could be divided into two stages. Caspase-1-mediated downregulation of PPARγ was important in the late stage of monocyte-macrophage differentiation; however, PPARγ protein levels had little effect on the early stage differentiation. Accumulation of PPARγ protein by troglitazone treatment potently suppressed the late stage of macrophage differentiation, which might be linked to inhibition of nuclear factor-κB activity. The data provide a plausible mechanistic basis by which caspase-1 promotes the differentiation of macrophages from monocytes.