Metabolic Footprinting-Based DNA-AuNP Encoders for Extracellular Metabolic Response Profiling.

Metabolic footprinting as a convenient and non-invasive cell metabolomics strategy relies on monitoring the whole extracellular metabolic process. It covers nutrient consumption and metabolite secretion of in vitro cell culture, which is hindered by low universality owing to pre-treatment of the cell medium and special equipment. Here, we report the design and a variety of applicability, for quantifying extracellular metabolism, of fluorescently labeled single-stranded DNA (ssDNA)-AuNP encoders, whose multi-modal signal response is triggered by extracellular metabolites. We constructed metabolic response profiling of cells by detecting extracellular metabolites in different tumor cells and drug-induced extracellular metabolites. We further assessed the extracellular metabolism differences using a machine learning algorithm. This metabolic response profiling based on the DNA-AuNP encoder strategy is a powerful complement to metabolic footprinting, which significantly applies potential non-invasive identification of tumor cell heterogeneity.

Fibroblast growth factor 21 attenuates ventilator-induced lung injury by inhibiting the NLRP3/caspase-1/GSDMD pyroptotic pathway.

BACKGROUND: Ventilator-induced lung injury (VILI) is caused by overdistension of the alveoli by the repetitive recruitment and derecruitment of alveolar units. This study aims to investigate the potential role and mechanism of fibroblast growth factor 21 (FGF21), a metabolic regulator secreted by the liver, in VILI development. METHODS: Serum FGF21 concentrations were determined in patients undergoing mechanical ventilation during general anesthesia and in a mouse VILI model. Lung injury was compared between FGF21-knockout (KO) mice and wild-type (WT) mice. Recombinant FGF21 was administrated in vivo and in vitro to determine its therapeutic effect. RESULTS: Serum FGF21 levels in patients and mice with VILI were significantly higher than in those without VILI. Additionally, the increment of serum FGF21 in anesthesia patients was positively correlated with the duration of ventilation. VILI was aggravated in FGF21-KO mice compared with WT mice. Conversely, the administration of FGF21 alleviated VILI in both mouse and cell models. FGF21 reduced Caspase-1 activity, suppressed the mRNA levels of Nlrp3, Asc, Il-1ß, Il-18, Hmgb1 and Nf-κb, and decreased the protein levels of NLRP3, ASC, IL-1ß, IL-18, HMGB1 and the cleaved form of GSDMD. CONCLUSIONS: Our findings reveal that endogenous FGF21 signaling is triggered in response to VILI, which protects against VILI by inhibiting the NLRP3/Caspase-1/GSDMD pyroptosis pathway. These results suggest that boosting endogenous FGF21 or the administration of recombinant FGF21 could be promising therapeutic strategies for the treatment of VILI during anesthesia or critical care.

WDR5 facilitates EMT and metastasis of CCA by increasing HIF-1α accumulation in Myc-dependent and independent pathways.

Cholangiocarcinoma (CCA) is a highly aggressive malignancy with extremely poor prognoses. The oncogenic role and prognostic value of c-Myc in CCA is not well elucidated. WD repeat domain 5 (WDR5) is a critical regulatory factor directly interacting with c-Myc to regulate c-Myc recruitment at chromosomal locations, but the interaction of WDR5 and c-Myc in CCA was uncovered. In our study, we detected WDR5 and c-Myc expression in all CCA types, including intrahepatic (iCCA), perihilar (pCCA), and distal (dCCA) CCA, and evaluated their prognostic significance. Consequently, we demonstrated that WDR5 was significantly correlated with poor prognosis of CCA and that WDR5 and c-Myc co-expression was a more sensitive prognostic factor. With in vitro and in vivo experiments and bioinformatics, we showed that WDR5 interacted with the Myc box IIIb (MBIIIb) motif of c-Myc and facilitated Myc-induced HIF1A transcription, thereby promoting the epithelial-mesenchymal transition (EMT), invasion, and metastasis of CCA. Moreover, WDR5 enhanced hypoxia-inducible factor 1 subunit α (HIF-1α) accumulation by binding with histone deacetylase 2 (HDAC2) and increasing histone 3 lysine 4 acetylation (H3K4ac) deacetylation of the prolyl hydroxylase domain protein 2 (PHD2) promoter, resulting in the attenuation of chromatin opening and PHD2 expression, and eventually leading to HIF-1α stabilization and accumulation. In conclusion, WDR5 facilitated EMT and metastasis of CCA by increasing HIF-1α accumulation in a Myc-dependent pathway to promote HIF-1α transcription and a Myc-independent pathway to stabilize HIF-1α.

Characterization of monocytic and granulocytic subsets of myeloid-derived suppressor cells in blood donors with occult hepatitis B virus infection.

Myeloid-derived suppressor cells (MDSCs) accumulate from many diseases. MDSCs are rarely explored in occult hepatitis B virus infection (OBI). The frequency of monocytic MDSCs (M-MDSCs) and granulocytic MDSCs (G-MDSCs) in OBI carriers was analyzed for correlation with clinical parameters, which was no different between OBI and healthy individuals, whereas the frequency of M-MDSCs but G-MDSCs in OBI was significantly lower than that observed in chronic hepatitis B carriers (0.4% vs 0.7%, P = 0.0004). The frequency of MDSCs was not correlated with clinical parameters and viral load of OBI, suggesting that the absence of HBsAg in OBI carriers might not induce the accumulation of MDSCs.

Hepatic stellate cells promote tumor progression by enhancement of immunosuppressive cells in an orthotopic liver tumor mouse model.

The immunosuppressive properties of hepatic stellate cells (HSCs) contribute to the occurrence and development of hepatocellular carcinoma (HCC). The accumulation of cells with immune suppressive activities, such as myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) is a key mechanism for tumor immune evasion. However, the impact of HSCs on immune cell populations in tumor-bearing hosts is unclear. In this study, we established an orthotopic liver tumor mouse model for studying the complex tumor-host interactions in HCC. The activated HSCs promoted HCC growth not only induced tumor angiogenesis and lymphangiogenesis, but also significantly increased the suppressive immune cell population of Tregs and MDSCs in the spleen, bone marrow, and tumor tissues of the tumor-bearing mice. Murine HCC cell line H22-activated HSCs also expanded the expression of Tregs and MDSCs in vitro. In conclusion, our study suggests a novel role for HSCs in the HCC microenvironment. HSCs can promote HCC progression by enhancement of the immunosuppressive cell population. Targeting HSCs, which is a new concept in adjuvant immunotherapy, may be introduced in the near future to improve the outcome of patients with HCC.

18ß-glycyrrhetinic acid inhibits hepatocellular carcinoma development by reversing hepatic stellate cell-mediated immunosuppression in mice.

Hepatic stellate cells (HSCs) have immunosuppressive capabilities and contribute to the occurrence and development of hepatocellular carcinoma (HCC). Thus, activated HSCs may be a suitable target for HCC therapy. Our study used mixed leukocyte reactions (MLR) in vitro to demonstrate that 18ß-glycyrrhetinic acid (GA) could reverse HSC-mediated immunosuppression by reducing T-cell apoptosis and regulatory T (Treg) cells expression, thereby enhancing the ability of T cells to attack tumor cells and attenuating HCC cell invasiveness. Moreover, we established a HCC orthotopic implantation model in immunocompetent C57BL/6 mice, which suggested that GA played a protective role in HCC development by reducing immunosuppression mediated by HSCs in the tumor microenvironment.

Tumor microenvironment-activated Nb2C quantum dots/lactate oxidase nanocatalyst mediates lactate consumption and macrophage repolarization for enhanced chemodynamic therapy.

Chemodynamic therapy (CDT), which takes advantages of CDT agents to selectively induce tumor cells apoptosis via Fenton or Fenton-like reactions, is considered to have great potential for tumor-specific treatment. However, the therapeutic outcome of CDT still faces the challenges of the lack of efficient CDT agents and insufficient supply of endogenous H2O2. Herein, to explore highly efficient CDT agents as well as increase the H2O2 content at tumor sites to enhance the efficiency of CDT, a red blood cell (RBC) membrane encapsulated Nb2C quantum dots/lactate oxidase (LOD) nanocatalyst (Nb2C QDs/LOD@RBC) was proposed. Nb2C quantum dots are quite prospective as efficient CDT agents in CDT application due to the intrinsic merits such as abundant active catalytic sites, satisfactory hydrophilicity, and good biocompatibility. The encapsulation of Nb2C QDs and LOD into RBC membrane was to prolong the in vivo circulation time of the nanocatalyst and increase its tumor sites accumulation. The accumulated Nb2C QDs/LOD@RBC nanocatalyst could efficiently convert the endogenous H2O2 into ·OH, while the overexpressed lactate could be catalyzed into H2O2 by LOD to replenish the depletion of H2O2. The cascaded reaction between Nb2C quantum dots and LOD eventually enhanced the CDT effect of Nb2C QDs/LOD@RBC nanocatalyst for tumors growth inhibition. Moreover, the consumption of lactate at tumor sites induced by Nb2C QDs/LOD@RBC nanocatalyst leads to the increased infiltration of antitumoral M1 tumor-associated macrophages, which alleviated the immunosuppression of the tumor microenvironment and further maximized the therapeutic outcome of CDT. Taken together, the Nb2C QDs/LOD@RBC nanocatalyst provides a promising paradigm for tumor inhibition via catalytic cascaded reaction between Nb2C quantum dots and LOD.

[Effects of Wuling mycelia on pentylenetetrazole-induced epilepsy in rats].

OBJECTIVE: To investigate the effects and mechanisms of Wuling mycelia on seizure development and learning ability induced by pentylenetetrazole-kindling epilepsy in rats. METHODS: SD rats were randomly divided into four groups: pentylenetetrazole-kindling model group (model group), low dose Wuling mycelia (0.3 g*kg(-1)) group (LD-WM group), high dose Wuling mycelia (0.6 g*kg(-1)) group (HD-WM group) and control group. The rats were intraperitoneal injected with a subconvulsive dose (35 mg*kg(-1)) of pentylenetetrazole (saline in control group) every 48 h for 12 times. Wuling mycelia was intragastrically applied 30 min before pentylenetetrazole injection. An 8-arm radial maze ( 4 arms baited) was used to measure the learning ability. Histamine was measured by chemical fluorometric enzyme immunoassay. RESULTS: Compared with the model group, the kindling stage of LD-WM group degraded significantly after 7th injection, the latency to the onset of myoclonic jerks (LTMJ) and the latency to the onset of generalized seizures (LTGS) prolonged after the 6th and 7th injection, respectively (P<0.05). The kindling stage of HD-WM group also degraded markedly after the 6th to 8th injection, and the LTMJ and the LTGS extended after the 8th to 9th and 6th injection, respectively (P<0.05). Compared with the control group, the frequency of working memory error (WME) and reference memory error (RME) of the model group in the 8-arm radial maze increased through 3-d training (P<0.05). The memory tests showed that the impairment induced by pentylenetetrazole was partially reversed by Wuling mycelia. Compared with the control group, brain histamine contents (hippocampus, cortex, thalamus and hypothalamus) were significantly lower in model group (P<0.05). But compared with the model group, hippocampal histamine contents in LD-WM group and hippocampal, thalamic and hypothalamic histamine contents in HD-WM group were elevated (P<0.05). CONCLUSION: Wuling mycelia can delay the kindling and ameliorate the ability of learning in rats with pentylenetetrazole-induced epilepsy and the enhancement of neuronal histamine activity may be one of possible mechanisms.

[Mechanisms of Wuling mycelia powder on memory retrieval impairment in rats with chronic epilepsy].

OBJECTIVE: To investigate the mechanisms of Wuling mycelia powder on memory retrieval impairment in rats with chronic epilepsy. METHOD: SD rats were randomly divided into four groups: the pentylenetetrazole-kindling group (the model control group), the low dose of Wuling mycelia powder (0.3 g x kg(-1), ig) group, the high dose of Wuling mycelia powder (0.6 g x kg(-1), ig) group and the blank control group. After being successfully trained in the 8-arm (4-arm baited) radial maze, the rats were intraperitoneally injected with a subconvulsive dose (35 mg x kg(-1)) of pentylenetetrazole (saline in control group) every 48 h for 12 times. Wuling mycelia powder were orally administered 30 min before every pentylenetetrazole injection. Memory retrieval was tested at the same maze. Phosphorylated CREB were analyzed by Western blot. Brain pathological sections were stained using HE, hippocampal nerve cells were observed under optical microscopes. RESULT: Both of reference and working memory abilities of these chronic epilepsy rats were impaired as expressed in the 8-arm radial maze but reversed by Wuling mycelia powder to some extent. Chronic epilepsy caused a decreasing p-CREB in hippocampal nerve cells and injury in hippocampal CA1 region and CA3 region among rats. Wuling mycelia powder inhibited hippocampal p-CREB from decreasing and protected hippocampal nerve cells. CONCLUSION: Wuling mycelia powder could ameliorate memory impairment induced by epilepsia. Its mechanism may be related to the increase in p-CREB expression in brain and the protective effect on hippocampal nerve cells.

A Knowledge Transfer Framework for General Alloy Materials Properties Prediction.

Biomedical metal implants have many applications in clinical treatment. Due to a variety of application requirements, alloy materials with specific properties are being designed continuously. The traditional alloy properties testing experiment is faced with high-cost and time-consuming challenges. Machine learning can accurately predict the properties of materials at a lower cost. However, the predicted performance is limited by the material dataset. We propose a calculation framework of alloy properties based on knowledge transfer. The purpose of the framework is to improve the prediction performance of machine learning models on material datasets. In addition to assembling the experiment dataset, the simulation dataset is also generated manually in the proposed framework. Domain knowledge is extracted from the simulation data and transferred to help train experiment data by the framework. The high accuracy of the simulation data (above 0.9) shows that the framework can effectively extract domain knowledge. With domain knowledge, the prediction performance of experimental data can reach more than 0.8. And it is 10% higher than the traditional machine learning method. The explanatory ability of the model is enhanced with the help of domain knowledge. In addition, five tasks are applied to show the framework is a general method.

The role of microglial exosomes in brain injury.

Microglia are involved in immune responses to central nervous system (CNS) injury. Meanwhile, exosomes derived from microglia are important mediators of information and material exchange in brain, which play an important role in neuroprotective or damaging effects. Microglial exosomes contain a variety of molecular cargos, including microRNAs, soluble proteins, and lipids, which have regulatory effects on other types of cells and microenvironment in brain. In this review, we summarized microglial exosome characteristics, release patterns, pro-proliferative and pro-apoptotic effects on neurons and other glial cells, immunomodulatory effects, and regulation of the extracellular microenvironment. Understanding the relationship between microglia exosomes and brain injury can provide new targets for clinical treatment.

Microglial exosomal miR-466i-5p induces brain injury via promoting hippocampal neuron apoptosis in heatstroke.

Background: Brain injury is the main cause of poor prognosis in heatstroke (HS) patients due to heat-stress-induced neuronal apoptosis. However, as a new cross-talk way among cells, whether microglial exosomal-microRNAs (miRNAs) are involved in HS-induced neuron apoptosis has not been elucidated. Methods: We established a heatstroke mouse model and a heat-stressed neuronal cellular model on HT22 cell line. Then, we detected neuron apoptosis by histopathology and flow cytometry. The microglial exosomes are isolated by standard differential ultracentrifugation and characterized. Recipient neurons are treated with the control and HS exosomes, whereas in vivo, the exosomes were injected into the mice tail vein. The internalization of HS microglial exosomes by neurons was tracked. Apoptosis of HT22 was evaluated by flow cytometry and Western blot in vitro, TUNEL assay, and immunohistochemistry in vivo. We screened miR-466i-5p as the mostly upregulated microRNAs in HS exosomes by high-throughput sequencing and further conducted gene ontology (GO) pathway analysis. The effect and mechanism of HS exosomal miR-466i-5p on the induction of neuron apoptosis are demonstrated by nasal delivery of miR-466i-5p antagomir in vivo and transfecting miR-466i-5p mimics to HT22 in vitro. Results: HS induced an increase in neurons apoptosis. Microglial exosomes are identified and taken up by neurons, which induced HT22 apoptosis in vivo and vitro. HS significantly changed the miRNA profiles of microglial exosomes based on high-throughput sequencing. We selected miR-466i-5p as a target, and upregulated miR-466i-5p induced neurons apoptosis in vivo and vitro experiments. The effects are exerted by targeting Bcl-2, activating caspase-3 to induce neurons apoptosis. Conclusions: We demonstrate the effect of microglial exosomal miR-466i-5p on neurons apoptosis and reveal potentially Bcl-2/caspase-3 pathway in heatstroke.

The Smad4-MYO18A-PP1A complex regulates ß-catenin phosphorylation and pemigatinib resistance by inhibiting PAK1 in cholangiocarcinoma.

Cholangiocarcinoma (CCA), consisting of three subtypes-intrahepatic (iCCA), perihilar (pCCA), and distal (dCCA), is a highly aggressive cancer arising from the bile duct and has an extremely poor prognosis. Pemigatinib is the only FDA-approved targeted drug for CCA, and the CCA treatment options are substantially insufficient considering its poor prognosis and increasing morbidity. Here, we performed next-generation sequencing (NGS) of 15 pCCAs and 16 dCCAs and detected the expression of SMAD4, a frequently mutated gene, in 261 CCAs. By univariate and multivariate analyses, we identified Smad4 as a favorable prognostic biomarker in iCCA and pCCA. With in vitro and in vivo experiments, we demonstrated that Smad4 suppressed CCA proliferation, migration and invasion by inhibiting ß-catenin-S675 phosphorylation and intranuclear translocation. We applied LC-MS/MS and multiple biochemical techniques and identified PP1A as the phosphatase in Smad4-mediated dephosphorylation of PAK1-T423, which is responsible for ß-catenin-S675 phosphorylation. Moreover, we demonstrated that MYO18A is the PP1-interacting protein of PP1A for substrate recognition in CCA. MYO18A interacts with PP1A via its RVFFR motif and interacts with Smad4 via CC domain. Patients with coexpression of MYO18A and Smad4 have a more favorable prognosis than other patients. Smad4 enhances Pemigatinib efficiency, and Smad4 knockdown results in Pemigatinib resistance. In conclusion, coexpression of Smad4 and MYO18A is a favorable prognostic indicator for iCCA and pCCA. The Smad4-MYO18A-PP1A complex dephosphorylates PAK1-T423 and thus inhibits ß-catenin-S675 phosphorylation and its intranuclear localization. Smad4 suppresses CCA proliferation, migration, invasion, and sensitivity to Pemigatinib by governing the phosphorylation and intracellular localization of ß-catenin.

Activated hepatic stellate cells promote hepatocellular carcinoma development in immunocompetent mice.

Activated hepatic stellate cells (HSCs) play a central role in the hepatic fibrosis and cirrhosis. Recently, HSCs were reported to have strong immune modulatory activities. However, the role of HSCs in hepatocellular carcinoma (HCC) remains unclear. In this study, we showed that HSCs could promote HCC growth both in vitro and in vivo. We examined the HSC-mediated inhibition of T-cell proliferation and the ability of conditioned medium from activated HSCs to promote the growth of murine HCC cell lines in vitro. We also assessed the immune suppression by HSCs during the development of HCC in immunocompetent mice. Cotransplantation of HSCs promoted HCC growth and progression by enhancing tumor angiogenesis and tumor cell proliferation and by creating an immunosuppressed microenvironment. Cotransplanted HSCs inhibited the lymphocyte infiltration in tumors and the spleens of mice bearing tumors, induced apoptosis of infiltrating mononuclear cells, and enhanced the expression of B7H1 and CD4(+) CD25(+) Treg cells. The immune modulation by HSCs seemed to be systemic. In conclusion, our data provide new information to support an integral role for HSCs in promoting HCC progression in part via their immune regulatory activities, and suggest that HSCs may serve as a therapeutic target in HCC.

Risk Factors for the 90-Day Prognosis Of Severe Heat Stroke: a Case-Control Study.

BACKGROUND: Severe heat stroke is a clinical syndrome caused by host stress dysfunction due to heat stress and subsequent life-threatening organ dysfunction. We aimed to explore the early risk factors affecting the 90-day prognosis of severe heat stroke patients. METHODS: A case-control study was used to retrospectively analyze the clinical data of 117 severe heat stroke patients admitted to the intensive care unit of the General Hospital of Southern Theater Command from April 2014 to May 2019. The risk factors affecting the 90-day mortality of the patients were analyzed, and subgroup analysis was performed comparing the complete recovery and the sequelae subgroups of survivors. RESULTS: Thirteen patients (11.1%) died within 90 days. The multivariate Cox risk regression model showed that cooling time (HR 4.87; 95% CI: 1.94-12.18; P = 0.001), heart rate (HR 1.04; 95% CI: 1.01-1.09; P = 0.027), and Sequential Organ Failure (SOFA) score (HR 1.41; 95% CI: 1.21-1.65; P < 0.001) were independent risk factors affecting the survival of patients. The area under the Receiver Operating Characteristic (ROC) curve of the combination of cooling time, heart rate, and SOFA score for the prediction of mortality due to severe heat stroke was 98.1% (95% CI 0.957-1.000, P < 0.001), the sensitivity was 96.2%, and the specificity was 92.3%. CONCLUSIONS: The longer the cooling duration, the faster the heart rate at admission, and the higher the SOFA score, the lower the 90-day survival rate was. These three indicators can be used in combination to predict 90-day mortality and poor prognosis in patients with severe heat stroke.

Comparison of acute pneumonia caused by SARS-COV-2 and other respiratory viruses in children: a retrospective multi-center cohort study during COVID-19 outbreak.

BACKGROUND: Until January 18, 2021, coronavirus disease-2019 (COVID-19) has infected more than 93 million individuals and has caused a certain degree of panic. Viral pneumonia caused by common viruses such as respiratory syncytial virus, rhinovirus, human metapneumovirus, human bocavirus, and parainfluenza viruses have been more common in children. However, the incidence of COVID-19 in children was significantly lower than that in adults. The purpose of this study was to describe the clinical manifestations, treatment and outcomes of COVID-19 in children compared with those of other sources of viral pneumonia diagnosed during the COVID-19 outbreak. METHODS: Children with COVID-19 and viral pneumonia admitted to 20 hospitals were enrolled in this retrospective multi-center cohort study. A total of 64 children with COVID-19 were defined as the COVID-19 cohort, of which 40 children who developed pneumonia were defined as the COVID-19 pneumonia cohort. Another 284 children with pneumonia caused by other viruses were defined as the viral pneumonia cohort. The epidemiologic, clinical, and laboratory findings were compared by Kolmogorov-Smirnov test, t-test, Mann-Whitney U test and Contingency table method. Drug usage, immunotherapy, blood transfusion, and need for oxygen support were collected as the treatment indexes. Mortality, intensive care needs and symptomatic duration were collected as the outcome indicators. RESULTS: Compared with the viral pneumonia cohort, children in the COVID-19 cohort were mostly exposed to family members confirmed to have COVID-19 (53/64 vs. 23/284), were of older median age (6.3 vs. 3.2 years), and had a higher proportion of ground-glass opacity (GGO) on computed tomography (18/40 vs. 0/38, P < 0.001). Children in the COVID-19 pneumonia cohort had a lower proportion of severe cases (1/40 vs. 38/284, P = 0.048), and lower cases with high fever (3/40 vs. 167/284, P < 0.001), requiring intensive care (1/40 vs. 32/284, P < 0.047) and with shorter symptomatic duration (median 5 vs. 8 d, P < 0.001). The proportion of cases with evaluated inflammatory indicators, biochemical indicators related to organ or tissue damage, D-dimer and secondary bacterial infection were lower in the COVID-19 pneumonia cohort than those in the viral pneumonia cohort (P < 0.05). No statistical differences were found in the duration of positive PCR results from pharyngeal swabs in 25 children with COVID-19 who received antiviral drugs (lopinavir-ritonavir, ribavirin, and arbidol) as compared with duration in 39 children without antiviral therapy [median 10 vs. 9 d, P = 0.885]. CONCLUSION: The symptoms and severity of COVID-19 pneumonia in children were no more severe than those in children with other viral pneumonia. Lopinavir-ritonavir, ribavirin and arbidol do not shorten the duration of positive PCR results from pharyngeal swabs in children with COVID-19. During the COVID-19 outbreak, attention also must be given to children with infection by other pathogens infection.

Association between SNP rs10569304 on the second expressed region of hole gene and the congenital heart disease.

The correlation of single nucleotide polymorphism (SNP) rs10569304 on the second expressed region of hole gene and congenital heart disease (CHD) of human being, and the effect of hole gene on CHD were investigated. 179 patients with CHD as CHD group and 183 healthy people as control group were selected in the case-control study. DNA was abstracted from the peripheral blood by phenol-chloroform method. Primer was designed for the flanking sequence of SNP rs10569304 on the second expressed region of hole gene. The genotype was identified by PCR degenerative acrylamide electrophoresis with amplification products. Then the three amplification products received sequencing. By chi-square test, the genotype frequency and allele frequency in CHD group and control group were analyzed. There was insertion-deletion (GCC/-) of SNP rs10569304 which corresponded to alleles of A and B in Southern Chinese people. The genotype frequency and allele frequency in control group and CHD group were met the Hardy-Weinberg equilibrium. By chi-square test, in control group and CHD group, the genotype frequency of AA (insertion homozygous), AB (insertion-deletion heterozygous) and BB (deletion homozygous) was 21.31%, 54.09%, 24.59% and 16.75%, 46.36%, 36.87%, respectively. The distributional difference of genotype frequency had statistical significance (chi2=6.51, P<0.05); The allele frequency of A and B was 48.36% and 51.64% in control group, 39.94% and 60.06% in CHD group, respectively. The distributional difference of allele frequency had statistical significance (chi2=5.20, P<0.05). Meanwhile, by contrast with the control group, the BB genotype frequency and B allele frequency in CHD group was higher, but the AA and AB frequency was lower. There was higher risk to suffer from CHD involving B allele. BB genotype had 1.907-fold increased risk of developing CHD according to AA genotype (P<0.05). It is concluded that there is insertion-deletion (GCC/-) of SNP rs10569304 in the Southern Chinese people, and the people whose hole gene involving BB genotype have higher risk to suffering from CHD.

The short hairpin RNA driven by polymerase II suppresses both wild-type and lamivudine-resistant hepatitis B virus strains.

BACKGROUND: Chronic infection with hepatitis B virus (HBV) is widespread because of the limited availability of therapeutic treatments. Although previous reports have suggested that RNA interference has promise as a treatment for HBV infection, further studies of long-term and off-target drug effects on HBV, especially on drug-resistant strains of HBV, are needed. Therefore, seven vectors that express short hairpin RNAs (shRNAs), driven by the polymerase II promoter, pSilencer4.1/HBV, were constructed to target open reading frames (ORFs) of the HBV C and S genes from wild-type and drug-resistant strains. Treatment efficiency was also assessed. METHODS: The pSilencer4.1/HBV vectors were investigated in HepG2.2.15 cells and transgenic mice that consistently produce wild-type HBV. Additionally, vectors that produce a lamivudine-resistant strain of HBV were developed and cotransfected, along with pSilencer/HBV, into both HepG2 cells and mice. The effects of polymerase-II-driven pSilencer4.1/HBV were compared with those of polymerase-III-driven pSilencer3.1/HBV at both the gene and protein level. RESULTS: pSilencer4.1/HBV inhibited the expression of viral protein, DNA and HBV subtype ayw mRNA in both HepG2.2.15 cells and transgenic mice. Toxicity, as well as off-target effects, did not occur after a short- to medium-term examination. Moreover, an HBV strain resistant to lamivudine, subtype adr, was suppressed by shRNA in both HepG2 cells and mice. In contrast to polymerase III, vectors that used polymerase II could drive efficient silencing without off-target effects. CONCLUSIONS: Silencing by shRNA dramatically inhibited HBV expression and replication regardless of strain type. ShRNA could therefore be a promising treatment for HBV infection.

Effects of insulin-like growth factor I on the development of osteoblasts in hyperglycemia.

The delayed wound healing of tooth extraction, the activation of alveolar absorption and the being hindered bone formation around the implants in diabetes are difficult to be solved for dentists. So, the aim of the study was to investigate the influences of hyperglycemia and insulin-like growth factor I (IGF-I) on osteoblasts. Osteoblasts were cultured in different conditions: normal glucose, mimic hyperglycemia, hyperglycemia with IGF-I, hyperglycemia with insulin. The proliferation and mineralization of osteoblasts were observed. As abnormal transport of glucose involved in the development of chronic complications in diabetes. The expression of glucose transporter 1 (GLUT1) was further evaluated by RT-PCR, immunofluorescence and Western blot in different groups. These results showed that hyperglycemia increased the proliferation and inhibited the mineralization of osteoblasts, while IGF-I seemed to reverse these effects. The levels of GLUT1 mRNA and protein in hyperglycemia were elevated by 51% and 35%, respectively, compared with that in normal glucose, while the levels in hyperglycemia with IGF-I were almost the same as that in normal glucose. In conclusion, the increased expression of GLUT1 may contribute to the delayed mineralization of osteoblasts in hyperglycemia. Also IGF-I may be a new drug for diabetic bone disease through normalizing the expression of GLUT1.

Changes in innate and permissive immune responses after hbv transgenic mouse vaccination and long-term-siRNA treatment [corrected].

BACKGROUND: Currently, no licensed therapy can thoroughly eradicate hepatitis B virus (HBV) from the body, including interferon α and inhibitors of HBV reverse-transcription. Small interfering RNA (siRNA) seem to be a promising tool for treating HBV, but had no effect on the pre-existing HBV covalently closed circular DNA. Because it is very difficult to thoroughly eradicate HBV with unique siRNAs, upgrading the immune response is the best method for fighting HBV infection. Here, we aim to explore the immune response of transgenic mice to HBV vaccination after long-term treatment with siRNAs and develop a therapeutic approach that combines siRNAs with immunopotentiators. METHODOLOGY/PRINCIPAL FINDINGS: To explore the response of transgenic mice to hepatitis B vaccine, innate and acquired immunity were detected after long-term treatment with siRNAs and vaccination. Antiviral cytokines and level of anti-hepatitis B surface antigen antibody (HBsAg-Ab) were measured after three injections of hepatitis B vaccine. RESULTS: Functional analyses indicated that toll-like receptor-mediated innate immune responses were reinforced, and antiviral cytokines were significantly increased, especially in the pSilencer4.1/HBV groups. Analysis of CD80+/CD86+ dendritic cells in the mouse liver indicated that dendritic cell antigen presentation was strengthened. Furthermore, the siRNA-treated transgenic mice could produce detectable HBsAg-Ab after vaccination, especially in the CpG oligonucleotide vaccine group. CONCLUSIONS/SIGNIFICANCE: For the first time, our studies demonstrate that siRNAs with CpG HBV vaccine could strengthen the immune response and break the immune tolerance status of transgenic mice to HBV. Thus, siRNAs and HBV vaccine could provide a sharp double-edged sword against chronic HBV infection.