Development and analytical validation of a novel nasopharynx swab-based Epstein-Barr virus C promoter methylation quantitative assay for nasopharyngeal carcinoma detection.

OBJECTIVES: Epstein-Barr virus (EBV) C promoter (Cp) hypermethylation, a crucial factor for EBV latent infection of nasopharyngeal epithelial cells, has been recognized as a promising biomarker for nasopharyngeal carcinoma (NPC) detection. In this study, we develop a novel EBV Cp methylation quantification (E-CpMQ) assay and evaluate its diagnostic performance for NPC detection. METHODS: A novel qPCR assay for simultaneous quantification of methylated- and unmethylated EBV Cp was developed by the combinational modification of MethyLight and QASM, with an innovative calibrator to improve the detection accuracy and consistency. The NP swab samples and synthetic standards were used for the analytical validation of the E-CpMQ. The diagnostic efficacy of the developed E-CpMQ assay was validated in 137 NPC patients and 137 non-NPC controls. RESULTS: The E-CpMQ assay can detect the EBV Cp methylation ratio in one reaction system under 10 copies with 100 % recognition specificity, which is highly correlated to pyrosequencing with a correlation coefficient over 0.99. The calibrated E-CpMQ assay reduces the coefficient of variation by an average of 55.5 % with a total variance of less than 0.06 units standard deviation (SD). Linear methylation ratio detection range from 4.76 to 99.01 %. The sensitivity and specificity of the E-CpMQ respectively are 96.4 % (95 % CI: 91.7-98.8 %), 89.8 % (95 % CI: 83.5-94.3 %). CONCLUSIONS: The developed E-CpMQ assay with a calibrator enables accurate and reproducible EBV Cp methylation ratio quantification and offers a sensitive, specific, cost-effective method for NPC early detection.

Associations between serum trace elements and the risk of nasopharyngeal carcinoma: a multi-center case-control study in Guangdong Province, southern China.

Background: Associations between trace elements and nasopharyngeal carcinoma (NPC) have been speculated but not thoroughly examined. Methods: This study registered a total of 225 newly diagnosed patients with NPC and 225 healthy controls matched by sex and age from three municipal hospitals in Guangdong Province, southern China between 2011 and 2015. Information was collected by questionnaire on the demographic characteristics and other possibly confounding lifestyle factors. Eight trace elements and the level of Epstein-Barr virus (EBV) antibody were measured in casual (spot) serum specimens by inductively coupled plasma-mass spectrometry (ICP-MS) and enzyme-linked immunosorbent assay (ELISA), respectively. Restricted cubic splines and conditional logistic regression were applied to assess the relationship between trace elements and NPC risk through single-and multiple-elements models. Results: Serum levels of chromium (Cr), cobalt (Co), nickel (Ni), arsenic (As), strontium (Sr) and molybdenum (Mo) were not associated with NPC risk. Manganese (Mn) and cadmium (Cd) were positively associated with NPC risk in both single-and multiple-element models, with ORs of the highest tertile compared with the reference categories 3.90 (95% CI, 1.27 to 7.34) for Mn and 2.30 (95% CI, 1.26 to 3.38) for Cd. Restricted cubic splines showed that there was a linear increasing trend between Mn and NPC risk, while for Cd there was a J-type correlation. Conclusion: Serum levels of Cd and Mn was positively related with NPC risk. Prospective researches on the associations of the two trace elements with NPC ought to be taken into account within the future.

Effect of Jianpi Yichang Powder on NLRP3/ASC/Caspase-l Signaling Pathway in Rats with Ulcerative Colitis / 中国实验方剂学杂志

ObjectiveTo explore the effect of Jianpi Yichang power on the nucleotide-binding oligomerization domain-like receptor protein 3 （NLRP3） inflammasome signaling pathway in a rat model of ulcerative colitis （UC）. MethodSixty Sprague-Dawley rats were randomly divided into a normal group （n=10） and an experimental group （n=50）. The experimental group received 5% dextran sulfate sodium （DSS） solution freely for 7 days to induce UC， and then they were further randomly divided into model group， sulfasalazine （0.3 g·kg-1） group， and high-， medium-， and low-dose Jianpi Yichang power groups （54.4， 27.2， 13.6 g·kg-1） for continuous treatment of 14 days. The general condition of the rats was observed and recorded daily， and the disease activity index （DAI） was scored before and after treatment. Enzyme-linked immunosorbent assay （ELISA） was used to detect the levels of interleukin-1β （IL-1β） and interleukin-18 （IL-18） in the serum of rats in each group. Hematoxylin-eosin （HE） staining was performed to observe the histopathological changes in the colon tissue. Immunohistochemistry， Western blot， and Real-time polymerase chain reaction （Real-time PCR） were used to detect the positive protein expression， protein expression， and mRNA expression of NLRP3， apoptosis-associated speck-like protein containing a CARD （ASC）， and cysteine aspartate-special proteases-1（Caspase-1） in the colon tissue. ResultCompared with the condition in the normal group， the general condition of rats in the model group was relatively poor， with increased DAI scores （P<0.01）， pathological changes in the colon， increased levels of IL-1β and IL-18 in the serum （P<0.01）， and enhanced positive protein expression， protein expression， and mRNA expression of NLRP3， ASC， and Caspase-1 in the colon tissue （P<0.01）. Compared with the condition in the model group， the general condition of rats in the Jianpi Yichang power groups at various doses improved significantly， with reduced DAI scores （P<0.05， P<0.01）， alleviated pathological changes in the colon as revealed by HE staining， and reduced protein expression levels of NLRP3 and Caspase-1 in the colon tissue （P<0.05， P<0.01）. The serum levels of IL-1β and IL-18， and ASC protein expression in the colon， as well as the mRNA expression levels of NLRP3， ASC， and Caspase-1， decreased in the high- and medium-dose Jianpi Yichang power groups （P<0.05， P<0.01）. The positive protein expression levels of NLRP3， ASC， and Caspase-1 were reduced in the high-dose Jianpi Yichang power group （P<0.01）. The positive protein expression levels of ASC and Caspase-1 were reduced in the medium-dose Jianpi Yichang power group （P<0.05）. The mRNA expression level of ASC was reduced in the low-dose Jianpi Yichang power group （P<0.05）. ConclusionJianpi Yichang power can reduce colon immune inflammatory damage by regulating the NLRP3 inflammasome signaling pathway， thereby exerting a role in treating UC.

Effect of recombinant human amelogenin-loaded PCLA-PEG-PCLA hydrogels on biological properties of human periodontal ligament fibroblasts / 上海交通大学学报（医学版）

Objective • To determine the effect of recombinant human amelogenin (rhAm)-loaded PCLA-PEG-PCLA hydrogels on cell proliferation, immigration, attachment and osteogenic differentiation of human periodontal ligament fibroblasts (HPDLFs). Methods • HPDLFs were obtained by tissue block method in vitro from extracted premolars and the 3rd-5th passages of HPDLFs were treated with DMEM medium (control group), 20 μg/mL rhAm (rhAm group) or rhAm-loaded PCLA-PEG-PCLA hydrogels (rhAm-loaded hydrogel group). Proliferation activity was measured by CCK-8, while cell migration was assayed both by wound-healing experiment in vitro and Transwell experiment. Cell attachment was measured by hemocytometer and observed by scanning electron microscope. Osteogenic differentiation was measured by real-time PCR, with ALP, Runx2, CEMP1 and CAP as the target genes. Results • RhAm-loaded PCLA-PEG-PCLA hydrogels had no significant effect on cell growth curve of HPDLFs, but promoted cell proliferation after 3 days (P<0.05). RhAm accelerated cell migration mostly both in wound-healing experiment and Transwell experiment, with rhAm-loaded hydrogels in the second place. RhAm-loaded hydrogels promoted cell attachment, and in the 4th hour the promotion was of statistic significance (P<0.05). Meanwhile cells of rhAm group and rhAm-loaded hydrogel group had a better stretch condition than control group under the scanning electron microscope. After culture with rhAm-loaded hydrogels, ALP, Runx2, CEMP1, and CAP mRNA were upregulated in different time points. Conclusion • Recombinant human amelogenin-loaded PCLA-PEG-PCLA hydrogels can significantly improve proliferation, attachment and osteogenic differentiation of HPDLFs, but has no effect on cell migration on a statistical scale.

[Variation of SpO2 during a Step-wise Paced Breathing Procedure].

Rhythmic respiratory movement in a deep and slow pattern can be beneficial to cardiovascular system, this paper investigates the effect of step-wise paced breathing procedure on blood oxygen saturation (SpO2). Experiment objects were divided into two groups, the normobaric hypoxia (simulated altitude of 4 500 meters hypoxia environment, 8 persons), normoxia and normobaric group (laboratory environment, 49 persons). The respiratory movements were performed by a high-to-low progressive change in two groups respectively. During the experiment, each object's blood oxygen saturation and heart rate were recorded. Results showed that progressive guided breathing could significantly increase the subjects' blood oxygen saturation level from 90% to 95% under the hypoxic condition. Even under the normobaric and normoxic condition, progressive guided breathing with stable blood oxygen saturation level can also enhance the blood oxygen saturation level. In both groups, mean heart rate declined in the progressive guided breathing. The research showed that the step-wise paced breathing technique could regulate the blood oxygen saturation and effectively improve the level of blood oxygen saturation by adjusting the respiratory motion in the low oxygen environment.

Tilapia Piscidin 4 (TP4) Stimulates Cell Proliferation and Wound Closure in MRSA-Infected Wounds in Mice.

Antimicrobial peptides (AMPs) are endogenous antibiotics that directly affect microorganisms, and also have a variety of receptor-mediated functions. One such AMP, Tilapia piscidin 4 (TP4), was isolated from Nile tilapia (Oreochromis niloticus); TP4 has antibacterial effects and regulates the innate immune system. The aim of the present study was to characterize the role of TP4 in the regulation of wound closure in mice and proliferation of a keratinocyte cell line (HaCaT) and fibroblast cell line (Hs-68). In vitro, TP4 stimulated cell proliferation and activated collagen I, collagen III, and keratinocyte growth factor (KGF) gene expression in Hs-68 cells, which induces keratin production by HaCaT cells. This effect was detectable at TP4 concentrations of 6.25 µg/mL in both cell lines. In vivo, TP4 was found to be highly effective at combating peritonitis and wound infection caused by MRSA in mouse models, without inducing adverse behavioral effects or liver or kidney toxicity. Taken together, our results indicate that TP4 enhances the survival rate of mice infected with the bacterial pathogen MRSA through both antimicrobial and wound closure activities mediated by epidermal growth factor (EGF), transforming growth factor (TGF), and vascular endothelial growth factor (VEGF). The peptide is likely involved in antibacterial processes and regulation of tissue homeostasis in infected wounds in mice. Overall, these results suggest that TP4 may be suitable for development as a novel topical agent for wound dressing.

A significant increase of RNAi efficiency in human cells by the CMV enhancer with a tRNAlys promoter.

RNA interference (RNAi) is the process of mRNA degradation induced by double-stranded RNA in a sequence-specific manner. Different types of promoters, such as U6, H1, tRNA, and CMV, have been used to control the inhibitory effect of RNAi expression vectors. In the present study, we constructed two shRNA expression vectors, respectively, controlled by tRNA(lys) and CMV enhancer-tRNA(lys) promoters. Compared to the vectors with tRNA(lys) or U6 promoter, the vector with a CMV enhancer-tRNA(lys) promoter silenced pokemon more efficiently on both the mRNA and the protein levels. Meanwhile, the silencing of pokemon inhibited the proliferation of MCF7 cells, but the induction of apoptosis of MCF7 cells was not observed. We conclude that the CMV enhancer-tRNA(lys) promoter may be a powerful tool in driving intracellular expression of shRNA which can efficiently silence targeted gene.

Transcutaneous immunization by lipoplex-patch based DNA vaccines is effective vaccination against Japanese encephalitis virus infection.

Skin, the biggest organ of human body, contains antigen presenting cells such as Langerhans cells (LCs) that modulate various immune responses. The skin therefore is an ideal venue to effect the transcutaneous immunization (TCI). Most current immunization procedures make use of needles and syringes for vaccine administration, which however have raised many safety concerns. To overcome the stratum corneum barrier of the skin without carrying out any skin penetration, cationic liposomes, DC-Chol/DOPE and DOTAP, were employed as vehicles for the transdermal antigen DNA delivery in this study. The optimal ratio of liposomes to DNAs for maximal transfection efficiency was determined to be 5:1 (w/w) for both formulas in BHK-21 cell transfection assays. This ratio was applied to lipoplex in tests on the dorsal skin of hair-removed mice. Reporter genes were found expressed in epidermis and spleen over 3 days. C3H/HeN mice transcutaneously immunized with the skin patch containing liposome-pCJ-3/ME (lipoplex-patch; pCJ-3/ME expressing the whole membrane and envelope protein genes of Japanese encephalitis virus (JEV)) can induce effective and protective antibodies against the infection with 50 times the 50% lethal dose (LD(50)) of JEV. The developed lipoplex-patch DNA vaccines have proven to be simple and noninvasive, by which the antibodies incurred provide marked therapeutic effects in test animals.