Human papillomavirus is an important risk factor for esophageal carcinoma in a Chinese population.

PURPOSE: Different types of HPV have been associated with cancer in humans, but the role of HPV in esophageal cancer (EC) is controversial. The purpose of this study was to evaluate the correlation between HPV infection and EC in the Chinese population and to provide the scientific basis for the future prevention, control, early diagnosis, and treatment strategies of EC in China. METHODS: PCR detected HPV infection in 1112 esophageal cancer tissue samples, and 89 HPV-positive samples were detected by genotyping. Proximity ligation assays (PLAs) and immunohistochemistry were used to detect the expression of HPV E6 and E7 proteins. Real-time fluorescent quantitative PCR was used to detect the integration of HPV16 E6. The level of HPV-specific antibody IgG in serum was detected by ELISA and PLA. RESULTS: The positive rates of HPV L1, HPV16, HPV18, hpv16 + 18 E6 and hpv16/18 E6 in 1,112 EC tissue samples were 77.6%, 41.4%, 27.2%, 14.2% and 55.4% respectively. Multiple HPV subtypes were detected in HPV-positive EC samples. PLA showed that E6 and E7 were expressed in EC109 and formed complexes with p53 and pRb, respectively. Immunohistochemistry showed that the positive rates of hpv16 + 18 E6 and E7 in HPV-positive EC samples were 56.4% and 37.0%, respectively. HPV-DNA integration rate in HPV-positive EC tissues (88.79%) was higher than that in adjacent tissues (54.17%). HPV antibody was found in the serum of EC patients by a serological test. CONCLUSION: The study suggests that HPV, especially HPV16 and HPV18, the infection may be a risk factor for EC in the Chinese population and that the E6 protein may play a key role in HPV-associated malignancies. These results may be important for the prevention and treatment of HPV-positive EC in China.

Nanostructured Lipid Carrier-Based Delivery of Pioglitazone for Treatment of Type 2 Diabetes.

Pioglitazone (PGZ) is utilized as a therapeutic agent in the management of (type 2) diabetes to control blood glucose levels. The existing research work was intended to make and optimize PGZ-containing NLCs (nanostructured lipid carriers). The fabricated nanostructured lipid carrier preparation was optimized by using different concentrations of the surfactants (Tween 80 and Span 80) and solid lipid (Compritol® 888 ATO) and liquid lipid (Labrasol®) while keeping the concentration of drug (PGZ), and co-surfactants (poloxamer 188) the same. The optimized NLC formulation (PGZ-NLCs) was further assessed for physical and chemical characterization, in vitro PGZ release, and stability studies. The optimized PGZ-NLCs have shown an average diameter of 150.4 nm, EE of 92.53%, PDI value of 0.076, and zeta-potential of -29.1 mV, correspondingly. The DSC thermal analysis and XRD diffractograms had not presented the spectrum of PGZ, confirming the comprehensive encapsulation of PGZ in the lipid core. PGZ-NLCs showed significantly extended release (51% in 24 h) compared to the unformulated PGZ. Our study findings confirmed that PGZ-NLCs can be a promising drug delivery system for the treatment of type 2 diabetes.

MAGEA6 positively regulates MSMO1 and promotes the migration and invasion of oesophageal cancer cells.

The melanoma antigen gene family A (MAGEA) family of proteins comprises of cancer-testis antigens that are highly expressed in a number of tumours but are minimally expressed in normal cells. Due to its expression characteristics, this protein family has become a popular target for anti-cancer drugs and immunotherapy research over recent years. Although, elevated expression levels of MAGEA6 has been found in different types of tumours, there remains to be insufficient information on the function of MAGEA6 and its associated gene regulation pathways. The present study used Transwell, Cell Counting Kit-8 and wound healing assays to analyse the effects of MAGEA6 on Eca109 cell invasion, migration and proliferation. The main functions and pathways involved in MAGEA6 were predicted by Illumina Hiseq screening for mutually regulated genes and core genes. Eca109 cell line with a high expression of MAGEA6 was a stable cell line obtained by transfection in the early stage, and this cell line was used in subsequent experiments. Transcriptome sequencing was performed on this cell line and the Eca109 cell line that normally expressed MAGEA6. It was revealed that a high expression of MAGEA6 conferred a significant stimulating effect on cell proliferation whilst also significantly increasing cell invasion and migration. Transcriptomic analysis identified 14 differentially expressed genes and 13 core regulatory genes closely associated with MAGEA6 expression regulation, such as methylsterol monooxygenase 1 (MSMO1). The present study suggest that MAGEA6 positively regulated MSMO1 expression, which may serve an oncogenic role in cells through this regulatory effect. Overall, this provided a novel route of investigation for an in-depth study of the regulatory function of MAGEA6.

Apoptotic Effect of 1800 MHz Electromagnetic Radiation on NIH/3T3 Cells.

To investigate the effect of 1800 MHz electromagnetic radiation (EMR) on apoptosis, we exposed NIH/3T3 cells at 1800 MHz with a specific absorption rate (SAR) of 2 W/kg intermittently for 12, 24, 36, and 48 h. After exposure, Cell Counting Kit-8 (CCK-8) and flow cytometry were used to detect cell viability and apoptosis; the expression of p53, a molecule with the key role in apoptosis, was measured by real-time qPCR, western blot, and immunofluorescence; and images of the structure of the mitochondria, directly reflecting apoptosis, were captured by electron microscopy. The results showed that the viability of cells in the 12, 36, and 48 h exposure groups significantly decreased compared with the sham groups; after 48 h of exposure, the percentage of late apoptotic cells in the exposure group was significantly higher. Real-time qPCR results showed that p53 mRNA in the 48 h exposure group was 1.4-fold of that in the sham group; significant differences of p53 protein fluorescence expression were observed between the exposure groups and the sham groups after 24 h and 48 h. The mitochondrial swelling and vesicular morphology were found in the electron microscopy images after 48 h exposure. These findings demonstrated 1800 MHz, SAR 2 W/kg EMR for 48 h may cause apoptosis in NIH/3T3 cells and that this apoptosis might be attributed to mitochondrial damage and upregulation of p53 expression.

Investigation into the expression levels of MAGEA6 in esophageal squamous cell carcinoma and esophageal adenocarcinoma tissues.

Esophageal carcinoma (EC) is the sixth most deadly of all cancers. It is among the most malignant cancers due to its highly aggressive nature and low survival rate. The incidence of EC is high in Asia, particularly in Southern areas including China, Iran and Japan. There is a large body of evidence to suggest an association between the melanoma antigen gene (MAGE) family and the initiation of cancer; however, there is no clear evidence to suggest an association between EC and MAGE. Discovery of the chemical and physiological processes relevant to the occurrence of EC is vital for clinicians to diagnose and treat this highly aggressive cancer. The present study focused on the association of EC with the expression of MAGE family member A6 (MAGEA6) at the mRNA and protein levels using gene chip, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry. The expression of MAGEA6 in human esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC) tissue samples were compared with those in paracancerous tissue. The result of the gene chip assay revealed that as the generation grew, there was a significant increase in MAGEA6 transcription in the esophageal epithelial cell line, SHEE Different ESC cell lines also exhibited a significantly higher transcription of MAGEA6 compared with the HaCaT cell line, as determined via reverse transcription-quantitative PCR. An higher positive rate of MAGEA6 expression in ESCC and EAC tissues was also revealed when compared with paracancerous tissues, as determined via immunohistochemistry. The results indicated that MAGEA6 is highly transcribed and expressed in the development of EC and may therefore serve as a novel biomarker for the diagnosis or treatment of EC.

Recombinant DENV 2 NS5: An effective antigen for diagnosis of DENV infection.

Dengue fever is a mosquito-borne viral disease with dramatically increasing morbidity rate worldwide in decades. Since there is no specific treatment to date, early diagnosis is important for providing proper timely medical care to minimize mortality, and for the prompt initiation of public health control measures. NS5 is a potential biomarker for dengue virus infection due to its highly conserved and immunogenic properties. In this study, the DENV 2 NS5 full-length and the DENV 2 NS5 C-terminus RNA-dependent RNA polymerase domain fragment (NS5-C70) expression plasmids were constructed, and the 104 kDa full-length NS5 and the 70 kDa NS5-C70 were respectively expressed in Escherichia coli. These two purified recombinant products were found to react with the sera of patients infected with dengue virus when analyzed by an enzyme-linked immunosorbent assay (ELISA), which resulted in significantly higher absorption values than those of control sera. The recombinant DENV 2 NS5 exhibited strong reactivity to each of the four types of sera, whereas the NS5-C70 showed strong reactivity only to DENV 2 and 4. In comparison, the positive agreement value of recombinant NS5-based assay with either MyBioSource or Panbio assay was higher than that of the two commercially available IgG indirect ELISA kits. These results suggest that the recombinant DENV 2 NS5 be an effective antigen for detection of dengue virus infection. The recombinant NS5-C70 may also be used as an auxiliary antigen for diagnostic purposes.

Long Non-coding RNA LINC01234 Regulates Proliferation, Invasion and Apoptosis in Esophageal Cancer Cells.

Esophageal cancer is one of the leading malignancies globally and long non-coding RNAs (lncRNAs) have been proved to have an important role in different malignancies including esophageal cancer. However their role in disease progression is still not clear. The objective of the study was to investigate the expression and role of LINC01234 in progression of esophageal cancer cells. LncRNA LINC01234 was found to be upregulated in esophageal cancer cells by chip sequencing. The expression level of LINC01234 was detected from different esophageal cancer cell lines by qRT-PCR. After this, the LINC01234 knockdown effects on cell proliferation, migration, invasion, and apoptosis were evaluated by cell proliferation assay, wound healing assay, invasion assay, and flow cytometric analysis in vitro. Expression of lncRNA LINC01234 was found to be markedly upregulated in the CEC2 cell line. Furthermore, cell proliferation, migration and invasion were significantly (P < 0.05) suppressed as compared to negative control while apoptotic rate was also found increased as a result of the knockdown of LINC01234. Significantly upregulated expression of LINC01234 in CEC2 cells and downregulated expression after knockdown is observed. The impact of LINC01234 knockdown on cell migration, invasion, proliferation and apoptosis indicated that LINC01234 may represent a new marker and a potential therapeutic target for esophageal cancer.

Diagnostic value of platelet-derived microparticles in pulmonary thromboembolism: A population-based study.

An early and accurate diagnosis of pulmonary thromboembolism (PTE) remains challenging. The present study aimed to evaluate the diagnostic value of platelet-derived microparticles in PTE based on a population study. A total of 102 patients with PTE, 102 healthy controls and 40 patients suspected with PTE were enrolled in this study. The platelet count, mean platelet volume and platelet distribution width were assessed using an automated hematology analyzer, P-selectin was assessed using an ELISA kit and PMPs were explored using flow cytometry using Megamix beads. Receiver operating characteristic curves were established to evaluate the diagnostic values of PMPs, D-dimer, PMPs combined with D-dimer, and multiple parameters (including PMPs, platelet distribution width, P-selectin and D-dimer in PTE). The PMP levels were significantly higher in the patients with PTE (609.10/µl) compared with those in the healthy controls (230.60/µl) and patients with suspicious PTE (166.70/µl; P<0.01). The accuracy (72.06%) of PMPs in the diagnosis of PTE was similar to those of D-dimer (P>0.05). The combination of D-dimer and PMPs significantly increased the sensitivity (86.27%) of D-dimer and the specificity of PMP for the diagnosis of PTE (P<0.01). The combination of PMPs, platelet distribution width, P-selectin and D-dimer exhibited high sensitivity (88.24%), specificity (91.18%) and accuracy (89.71%) in the diagnosis of PTE. These findings suggest that elevated PMP levels are an effective predictor of PTE. The combination of PMPs, platelet distribution width, P-selectin and D-dimer may be used in the diagnosis of PTE with high sensitivity and specificity.

The Protective Effect of Autophagy on DNA Damage in Mouse Spermatocyte-Derived Cells Exposed to 1800 MHz Radiofrequency Electromagnetic Fields.

BACKGROUND/AIMS: The effects of exposure to radiofrequency electromagnetic fields (RF-EMFs) on the male reproductive system have raised public concern and studies have shown that exposure to RF-EMFs can induce DNA damage and autophagy. However, there are no related reports on the role of autophagy in DNA damage in spermatocytes, especially after exposure to RF-EMFs. The aim of the present study was to determine the mechanism and role of autophagy induced by RF-EMFs in spermatozoa cells. METHODS: Mouse spermatocyte-derived cells (GC-2) were exposed to RF-EMFs 4 W/kg for 24 h. The level of reactive oxygen species (ROS) was determined by ROS assay kit. Comet assay was utilized to detect DNA damage. Autophagy was detected by three indicators: LC3II/LC3I, autophagic vacuoles, and GFP-LC3 dots, which were measured by western blot, transmission electron microscopy, and transfection with GFP-LC3, respectively. The expression of the molecular signaling pathway AMP-activated protein kinase (AMPK)/mTOR was determined by western blot. RESULTS: The results showed that RF-EMFs induced autophagy and DNA damage in GC-2 cells via ROS generation, and the autophagy signaling pathway AMPK/mTOR was activated by ROS generation. Furthermore, following inhibition of autophagy by knockdown of AMPKα, increased DNA damage was observed in GC-2 cells following RF-EMFs exposure, and overexpression of AMPKα promoted autophagy and attenuated DNA damage. CONCLUSIONS: These findings demonstrated that the autophagy which was induced by RF-EMFs via the AMPK/mTOR signaling pathway could prevent DNA damage in spermatozoa cells.

Water Carcinogenicity and Prevalence of HPV Infection in Esophageal Cancer Patients in Huaihe River Basin, China.

OBJECTIVE: The incidence of the upper gastrointestinal tumor has increased rapidly during recent decades. The relationship between local water pollution and the tumor is still not much clear, so this study was conducted to further investigate the local water pollution and its influence on the malignant cell transformation. Prevalence of human papillomavirus (HPV) in local esophageal cancer (EC) patients was also analyzed in Shenqiu County for the first time. METHODS: Two-step cell transformation was used to study different sources of water in the malignant cell transformation, and the existence of 3-methylcholanthrene (3-MC) in water was analyzed from the river and shallow and deep wells. HPV DNA in tissue samples of EC patients was detected by polymerase chain reaction (PCR) and HPV diagnostic kit. RESULTS: The river water has higher cytotoxicity than the shallow well water and induced significant cell malignant transformation, while deep well water has not shown the malignant cell transformation. In Huaihe River water, the 3-MC concentration was found higher than shallow and deep wells. An HPV infection rate was found high in patients with esophageal cancer. CONCLUSION: Long-term consumption of polluted water can induce malignant cell transformation, and the presence of HPV may be an important cause of cancer.

Oxidative changes and apoptosis induced by 1800-MHz electromagnetic radiation in NIH/3T3 cells.

To investigate the potential adverse effects of mobile phone radiation, we studied reactive oxygen species (ROS), DNA damage and apoptosis in mouse embryonic fibroblasts (NIH/3T3) after intermittent exposure (5 min on/10 min off, for various durations from 0.5 to 8 h) to an 1800-MHz GSM-talk mode electromagnetic radiation (EMR) at an average specific absorption rate of 2 W/kg. A 2',7'-dichlorofluorescin diacetate fluorescence probe was used to detect intracellular ROS levels, immunofluorescence was used to detect γH2AX foci as a marker for DNA damage, and flow cytometry was used to measure apoptosis. Our results showed a significant increase in intracellular ROS levels after EMR exposure and it reached the highest level at an exposure time of 1 h (p < 0.05) followed by a slight decrease when the exposure continued for as long as 8 h. No significant effect on the number of γH2AX was detected after EMR exposure. The percentage of late-apoptotic cells in the EMR-exposed group was significantly higher than that in the sham-exposed groups (p < 0.05). These results indicate that an 1800-MHz EMR enhances ROS formation and promotes apoptosis in NIH/3T3 cells.

Oral intake of hydrogen-rich water ameliorated chlorpyrifos-induced neurotoxicity in rats.

Chronic exposure to low-levels of organophosphate (OP) compounds, such as chlorpyrifos (CPF), induces oxidative stress and could be related to neurological disorders. Hydrogen has been identified as a novel antioxidant which could selectively scavenge hydroxyl radicals. We explore whether intake of hydrogen-rich water (HRW) can protect Wistar rats from CPF-induced neurotoxicity. Rats were gavaged daily with 6.75mg/kg body weight (1/20 LD50) of CPF and given HRW by oral intake. Nissl staining and electron microscopy results indicated that HRW intake had protective effects on the CPF-induced damage of hippocampal neurons and neuronal mitochondria. Immunostaining results showed that the increased glial fibrillary acidic protein (GFAP) expression in astrocytes induced by CPF exposure can be ameliorated by HRW intake. Moreover, HRW intake also attenuated CPF-induced oxidative stress as evidenced by enhanced level of MDA, accompanied by an increase in GSH level and SOD and CAT activity. Acetylcholinesterase (AChE) activity tests showed significant decrease in brain AChE activity after CPF exposure, and this effect can be ameliorated by HRW intake. An in vitro study demonstrated that AChE activity was more intense in HRW than in normal water with or without chlorpyrifos-oxon (CPO), the metabolically-activated form of CPF. These observations suggest that HRW intake can protect rats from CPF-induced neurotoxicity, and the protective effects of hydrogen may be mediated by regulating the oxidant and antioxidant status of rats. Furthermore, this work defines a novel mechanism of biological activity of hydrogen by directly increasing the AChE activity.

Protective effect of hydrogen-rich saline on ischemia/reperfusion injury in rat skin flap.

OBJECTIVE: Skin damage induced by ischemia/reperfusion (I/R) is a multifactorial process that often occurs in plastic surgery. The mechanisms of I/R injury include hypoxia, inflammation, and oxidative damage. Hydrogen gas has been reported to alleviate cerebral I/R injury by acting as a free radical scavenger. Here, we assessed the protective effect of hydrogen-rich saline (HRS) on skin flap I/R injury. METHODS: Abdominal skin flaps of rats were elevated and ischemia was induced for 3 h; subsequently, HRS or physiological saline was administered intraperitoneally 10 min before reperfusion. On postoperative Day 5, flap survival, blood perfusion, the accumulation of reactive oxygen species (ROS), and levels of cytokines were evaluated. Histological examinations were performed to assess inflammatory cell infiltration. RESULTS: Skin flap survival and blood flow perfusion were improved by HRS relative to the controls. The production of malondialdehyde (MDA), an indicator of lipid peroxidation, was markedly reduced. A multiplex cytokine assay revealed that HRS reduced the elevation in the levels of inflammatory cytokines, chemokines and growth factors, with the exception of RANTES (regulated on activation, normal T-cell expressed and secreted) growth factor. HRS treatment also reduced inflammatory cell infiltration induced by I/R injury. CONCLUSIONS: Our findings suggest that HRS mitigates I/R injury by decreasing inflammation and, therefore, has the potential for application as a therapy for improving skin flap survival.

Cellular neoplastic transformation induced by 916 MHz microwave radiation.

There has been growing concern about the possibility of adverse health effects resulting from exposure to microwave radiations, such as those emitted by mobile phones. The purpose of this study was to investigate the cellular neoplastic transformation effects of electromagnetic fields. 916 MHz continuous microwave was employed in our study to simulate the electromagnetic radiation of mobile phone. NIH/3T3 cells were adopted in our experiment due to their sensitivity to carcinogen or cancer promoter in environment. They were divided randomly into one control group and three microwave groups. The three microwave groups were exposed to 916 MHz EMF for 2 h per day with power density of 10, 50, and 90 w/m(2), respectively, in which 10 w/m(2) was close to intensity near the antenna of mobile phone. The morphology and proliferation of NIH/3T3 cells were examined and furthermore soft agar culture and animal carcinogenesis assay were carried out to determine the neoplastic promotion. Our experiments showed NIH/3T3 cells changed in morphology and proliferation after 5-8 weeks exposure and formed clone in soft agar culture after another 3-4 weeks depending on the exposure intensity. In the animal carcinogenesis study, lumps developed on the back of SCID mice after being inoculated into exposed NIH/3T3 cells for more than 4 weeks. The results indicate that microwave radiation can promote neoplastic transformation of NIH/3T3cells.

[Detection of SARS-CoV and RNA on aerosol samples from SARS-patients admitted to hospital].

OBJECTIVE: To assess the risk of aerosol transmission in severe acute respiratory syndrome (SARS) patients admitted to Hospital through testing the air samples. METHODS: Air samples were collected from 7 wards and 1 balcony of the Hospital, 3 times a day for 3 continuous days, using bioaerosol sampler type FA-2. Bioaerosol particles were then washed down from the samples by serum-free Dulbecco's Modified Eagle Medium (DMEM) culture medium. Nested-reverse transcription-polymerase chain reaction (RT-PCR) was used to amplify the N protein gene of the SARS associated coronavirus (SARS-CoV) from these washing solutions. The residual solutions were inoculated into prepared cell cultures to isolate live virus. The positive samples were then identified by indirect immunofluorescence assay and sequence analysis of the PCR products. RESULTS: Positive rates of RT-PCR test on air samples were 29.03% in the wards and 20.0% in balcony respectively. Results from sequential analysis showed that the homology of amplified cDNA fragments to previously known SARS-CoV stains was 98%. A strain of live pathogen was isolated from one of the 36 samples. The isolate could cause typical cytopathic effects, similar to those SARS-CoV on Vero-E6 cells and the effects could be stably passed. Indirect immunofluorescence assay showed positive from serum of a SARS patient. CONCLUSION: SARS-CoV existed in the air hospital, where SARS patients were admitted to, but the activity of SARS-CoV in air samples was rather low. SARS patients could still shed SARS-CoV even during the recovery phase. Potential possibility of aerosol transmission might exist within 1 meter square area around SARS patients.