OBJECTIVETo construct recombinant baculoviruses co-expressing three structural genes vp2, vp6 and vp7 of rotavirus, and assemble rotavirus-like particles (VLPs) in BmN cells.

METHODSHuman group A rotavirus was cultivated in MA104 cells, and the RNA was extracted and the three genes were obtained by RT-PCR. The PCR products were inserted into the transfer vectors pFBDM and pUCDM, respectively. A enhanced green fluorescent protein gene (egfp) driven by IE1 promoter was introduced into pFBDM to investigate the efficiency of infection. The expression baculoviruse was constructed by Tn7 and Cre-LoxP recombinant and transfected into BmN cells. The gene expression was determined by detecting 6-His tag fused into VP7 C-terminus, and the assembled VLPs were observed by transmission electron micrography.

RESULTSThree genes of rotavirus were cloned and BmMultiBac was constructed. The genes were expressed and the rotavirus-like particles assembled in BmN cells successfully as verified by ELISA and electron microscope.

CONCLUSIONWe have successfully constructed the recombinant baculovirus co-expressing the 3 structural genes of rotavirus, which provide the basis for producing protein complex containing multiple subunits and investigation of the structure of the macromolecules.

Animals ; Baculoviridae ; genetics ; metabolism ; Bombyx ; virology ; Capsid Proteins ; genetics ; Cell Line ; Gene Expression ; Genetic Vectors ; Rotavirus ; genetics ; metabolism

After bathing at a hot spring resort, a 75-year-old man presented to the emergency department because of seizure-like attack with loss of conscious. This is the first case of primary amebic meningoencephalitis (PAM) caused by Naegleria fowleri in Taiwan. PAM was diagnosed based on detection of actively motile trophozoites in cerebrospinal fluid using a wet-mount smear and the Liu's stain. The amoebae were further confirmed by PCR and gene sequencing. In spite of administering amphotericin B treatment, the patient died 25 days later.
Aged ; Amebiasis/*diagnosis/parasitology/*pathology ; Central Nervous System Protozoal Infections/*diagnosis/parasitology/*pathology ; Cerebrospinal Fluid/parasitology ; DNA, Protozoan/chemistry/genetics ; Fatal Outcome ; Humans ; Male ; Microscopy ; Naegleria fowleri/classification/genetics/*isolation & purification ; Polymerase Chain Reaction ; Sequence Analysis, DNA ; Taiwan

We have previously demonstrated the expression of GATA-DNA-binding protein (GATA)-3, a transcription factor, in osteoblasts and have verified its function in transducing cell survival signaling. This translational study was further designed to evaluate the roles of GATA-3 in regulating bone healing and to explore its possible mechanisms. A metaphyseal bone defect was created in the left femurs of male ICR mice. Analysis by micro-computed topography showed that the bone volume, trabecular bone number and trabecular thickness were augmented and that the trabecular pattern factor decreased. Interestingly, immunohistological analyses showed specific expression of GATA-3 in the defect area. In addition, colocalized expression of GATA-3 and alkaline phosphatase was observed at the wound site. As the fracture healed, the amounts of phosphorylated and non-phosphorylated GATA-3 concurrently increased. Separately, GATA-3 mRNA was induced during bone healing, and, levels of Runx2 mRNA and protein were also increased. The results of confocal microscopy and co-immunoprecipitation showed an association between nuclear GATA-3 and Runx2 in the area of insult. In parallel with fracture healing, Bcl-XL mRNA was significantly triggered. A bioinformatic search revealed the existence of a GATA-3-specific DNA-binding element in the promoter region of the bcl-x(L) gene. Analysis by chromatin immunoprecipitation assays further demonstrated transactivation activity by which GATA-3 regulated bcl-x(L) gene expression. Therefore, this study shows that GATA-3 participates in the healing of bone fractures via regulating bcl-xL gene expression, owing to its association with Runx2. In the clinic, GATA-3 may be used as a biomarker for diagnoses/prognoses or as a therapeutic target for bone diseases, such as bone fractures.
Alkaline Phosphatase ; Animals ; Bone Diseases ; Cell Survival ; Chromatin Immunoprecipitation ; Computational Biology ; Femur ; Fracture Healing ; Fractures, Bone ; Gene Expression ; Humans ; Immunoprecipitation ; Male ; Mice ; Mice, Inbred ICR ; Microscopy, Confocal ; Osteoblasts ; Promoter Regions, Genetic ; RNA, Messenger ; Transcription Factors ; Transcriptional Activation ; Up-Regulation* ; Wounds and Injuries

PURPOSE: Lovastatin is an effective inhibitor of cholesterol synthesis. A previous study demonstrated that lovastatin can also suppress airway hyperresponsiveness (AHR) in murine model of asthma. We aimed to investigate the effect of lovastatin on mucus secretion and inflammation-associated gene expression in the lungs of murine model of asthma. METHODS: Female BALB/c mice were sensitized and challenged with ovalbumin (OVA) by intraperitoneal injection, and orally administered lovastatin from days 14 to 27 post-injection. Gene expression in lung tissues was analyzed using real-time polymerase chain reaction. AHR and goblet cell hyperplasia were also examined. BEAS-2B human bronchial epithelial cells were used to evaluate the effect of lovastatin on the expression of cell adhesion molecules, chemokines, and proinflammatory cytokines in vitro. RESULTS: We showed that lovastatin inhibits the expression of Th2-associated genes, including eotaxins and adhesion molecules, in the lungs of murine model of asthma. Mucin 5AC expression, eosinophil infiltration and goblet cell hyperplasia were significantly decreased in the lung tissue of murine model of asthma treated with lovastatin. Furthermore, lovastatin inhibited AHR and expression of Th2-associated cytokines in bronchoalveolar lavage fluid. However, a high dose (40 mg/kg) of lovastatin was required to decrease specific IgE to OVA levels in serum, and suppress the expression of Th2-associated cytokines in splenocytes. Activated BEAS-2B cells treated with lovastatin exhibited reduced IL-6, eotaxins (CCL11 and CCL24), and intercellular adhesion molecule-1 protein expression. Consistent with this, lovastatin also suppressed the ability of HL-60 cells to adhere to inflammatory BEAS-2B cells. CONCLUSIONS: These data suggest that lovastatin suppresses mucus secretion and airway inflammation by inhibiting the production of eotaxins and Th2 cytokines in murine model of asthma.
Animals ; Asthma* ; Bronchoalveolar Lavage Fluid ; Cell Adhesion Molecules ; Chemokines ; Cholesterol ; Cytokines ; Eosinophils ; Epithelial Cells ; Female ; Gene Expression ; Goblet Cells ; HL-60 Cells ; Humans ; Hyperplasia ; Immunoglobulin E ; Inflammation* ; Injections, Intraperitoneal ; Intercellular Adhesion Molecule-1 ; Interleukin-6 ; Lovastatin* ; Lung ; Mice ; Mucin 5AC ; Mucus* ; Ovalbumin ; Ovum ; Real-Time Polymerase Chain Reaction

OBJECTIVETo study the effects of lutein on apoptosis and its mechanism.

METHODThe cells of human esophageal carcinoma EC9706 were grown in RPMI medium containing 10% bovine serum and were treated with lutein at 100 microg x mL(-1) concentration. Flow cytometry was employed to investigate the effects of lutein on cell apoptosis of EC9706 cells. Histochemistry was performed to determine apoptosis-related protein expresion.

RESULTFlow cytometry analyses revealed that lutein increased EC9706 cell apoptosis ratio when treated with lutein 100 microg x mL(-1) at 96 h. Lutein decreased the expression of Bcl-2 protein and increased the expression of Bax protein in EC9706 cells.

CONCLUSIONLutein could inhibit mitosis and stimulate apoptosis of EC9706 cells. The apoptotic effect may result from the down-regulation of expression of Bcl-2 and up-regulation expression of Bax.

Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; drug effects ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Cell Line, Tumor ; Esophageal Neoplasms ; metabolism ; pathology ; Humans ; Lutein ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo investigate the protective effect of Yigan Fuzheng Paidu Capsules (YC) combined with medical ozone against hepatic injury in dogs induced by hepatotoxic drug.

METHODSTwenty-four dogs were randomized equally into 4 groups (n=6), namely the model group, oleanolic acid tablet (OAT) group, YC group and YC+O(3) group, given either no particular treatment, oral OAT at 10 mg/day, oral YC at 0.2 g/day, or YC at 0.2 g/day plus 150 ml medical ozone transrectal insufflation every other day, respectively, for totally 30 consecutive days. Acute hepatic injury was induced after the treatment in the dogs with a sing-dose intraperitoneal injection of 0.9 ml/kg CCl(4) and peanut oil mixture (1:1, W/W). The general condition, survival time, alanine aminotransferase (ALT), aspartate aminotransferase/alanine aminotransferase (AST/ALT), serum total bilirubin (TBIL), prothrombin time (PT), blood ammonia (AMMO), and blood urea nitrogen (BUN) were recorded or measured. The hepatic pathological changes were observed upon death or on day 15 following CCl(4) injection.

RESULTSCompared with the other 3 treatment protocols, YC plus O(3) showed favorable effects on the activity, mental state, diet, urination and defecation of the dogs, which had significantly higher survival rate and higher levels of ALT, TBIL, PT, and AMMO than the model and OAT groups (P<0.05). AST/ALT remained normal in YC+O(3) group, which had also milder hepatic injury than the other 3 groups.

CONCLUSIONSYC combined with medical ozone may decrease transaminase and blood ammonia levels, relieve jaundice, prolong the survival time of dogs with CCl(4)-induced hepatic injury.

Alanine Transaminase ; blood ; Ammonia ; blood ; Animals ; Aspartate Aminotransferases ; blood ; Bilirubin ; blood ; Blood Urea Nitrogen ; Capsules ; Carbon Tetrachloride ; toxicity ; Dogs ; Drug Therapy, Combination ; Female ; Liver ; drug effects ; pathology ; Liver Diseases ; blood ; etiology ; prevention & control ; Male ; Medicine, Chinese Traditional ; Oxidants, Photochemical ; therapeutic use ; Ozone ; therapeutic use ; Survival Analysis

OBJECTIVETo study the effects of low level manganese (Mn) exposure on the serum neuroendocrine hormones levels of the welders.

METHODSThe exposure group consisted of 41 male welders, 40 male workers without exposing to harmful agents served as controls. The serum contents of prolactin (PRL), luteinizing hormone (LH), follicle stimulating hormone (FSH), testosterone (TST) and thyroid stimulating hormone (TSH) of 81 subjects were detected by chemiluminescence immunoassay.

RESULTSThe geometric mean value of airborne Mn concentrations was 0.03 mg/m(3) (0.003 - 0.519 mg/m(3)) in the welding circumstances. The levels of Mn in red blood cells (RBCs) and urinary Mn of the exposure group were significantly higher than those of control group (P < 0.01). The contents of serum LH and TSH of the exposure group were 2.89 ± 0.69 mIU/ml and 1.45 ± 0.56 uIU/ml, which were significantly lower than those (3.82 ± 1.61 mIU/ml and 2.19 ± 1.28 µIU/ml) of control group (P < 0.01). The serum contents of LH, FSH and TSH of the group exposed to Mn for < 5 years were significantly lower than those of the control group, The serum TST level of the group exposed to Mn for < 5 years was significantly higher than those of the control group and group exposed to Mn for 5 ∼ years, the serum FSH level of the group exposed to Mn for < 5 years was significantly lower than that of the group exposed to Mn for 10 years (P < 0.05 or P < 0.01). The serum contents of LH and TSH of the group exposed to Mn for 5 ∼ years were significantly lower than those of the control group (P < 0.05 or P < 0.01). The serum contents of PRL, LH and TSH of the group exposed to Mn for 10 years were significantly lower than those of the control group (P < 0.05). There was negative correlation between blood (RBC) Mn and urinary Mn (r = -0.310, P < 0.05), also there was negative correlation between serum PRL and serum TST (r = -0.409, P < 0.01), the positive correlation between serum LH and serum FSH was observed (r = 0.361, P < 0.05).

CONCLUSIONThe results of present study showed that the long exposure to low level of Mn may decrease the levels of serum PRL, LH and TSH in workers occupationally exposed to Mn, which can influence the metabolism of neuroendocrine hormones to certain extent.

Adult ; Air Pollutants, Occupational ; Follicle Stimulating Hormone ; blood ; Humans ; Luteinizing Hormone ; blood ; Male ; Manganese ; Occupational Exposure ; Prolactin ; blood ; Testosterone ; blood ; Thyrotropin ; blood ; Welding

INTRODUCTION: Advanced breast cancer (ABC) remains common in Singapore. In 2019, 22.1% of breast cancer patients presented with ABC in our institution. Despite increasing affluence and the advent of national mammographic screening, the incidence of ABC has not changed significantly. This suggests inherent differences in women who present late. We aim to explore the socio-economic background, knowledge and attitudes of women who present with ABC. METHODS: Between December 2013 and July 2015, 100 patients who presented consecutively with ABC in a tertiary institution in Singapore were recruited to participate in an interviewer-led questionnaire exploring psychosocial and economic issues. RESULTS: Among the 100 patients, 63 and 37 presented with stages 3 and 4 breast cancer respectively. Median age was 57 (27-86), 52% had at least secondary education, 53% had no formal employment and 71% were married; 88% were aware of breast cancer symptoms, 82% were aware that mammography can help detect cancer, 82% believed that current treatment modality for breast cancer is effective, 96% had never undergone a mammography and 52.9% felt mammograms were unnecessary. A total of 64% presented symptomatic from the breast tumour, with a median duration of 3 months. Many of the patients were aware of breast cancer symptoms and the utility of mammography. However, a group of patients did not comply with screening. This may be due to poor understanding about breast screening and detection in its asymptomatic phase. CONCLUSION Further public education to improve understanding of breast cancer and screening mammography may help to improve rates for earlier detection of breast cancer.

Background: Feline mammary carcinoma (FMC) is one of the most prevalent malignancies of female cats. FMC is highly metastatic and thus leads to poor disease outcomes. Among all metastases, liver metastasis occurs in about 25% of FMC patients. However, the mechanism underlying hepatic metastasis of FMC remains largely uncharacterized. Results: Herein, we demonstrate that FMC-derived extracellular vesicles (FMC-EVs) promotes the liver metastasis of FMC by activating hepatic stellate cells (HSCs) to prime a hepatic premetastatic niche (PMN). Moreover, we provide evidence that sphingosine kinase 1 (SK1) delivered by FMC-EV was pivotal for the activation of HSC and the formation of hepatic PMN. Depletion of SK1 impaired cargo sorting in FMC-EV and the EV-potentiated HSC activation, and abol‑ ished hepatic colonization of FMC cells. Conclusions Taken together, our findings uncover a previously uncharacterized mechanism underlying liver-metas‑ tasis of FMC and provide new insights into prognosis and treatment of this feline malignancy.

Background/Aims: Nonalcoholic fatty liver disease (NAFLD) is closely associated with diabetes. The cumulative impact of both diseases synergistically increases risk of adverse events. However, present population analysis is predominantly conducted with reference to non-NAFLD individuals and has not yet examined the impact of prediabetes. Hence, we sought to conduct a retrospective analysis on the impact of diabetic status in NAFLD patients, referencing non-diabetic NAFLD individuals. Methods: Data from the National Health and Nutrition Examination Survey 1999–2018 was used. Hepatic steatosis was defined with United States Fatty Liver Index (US-FLI) and FLI at a cut-off of 30 and 60 respectively, in absence of substantial alcohol use. A multivariate generalized linear model was used for risk ratios of binary outcomes while survival analysis was conducted with Cox regression and Fine Gray model for competing risk. Results: Of 32,234 patients, 28.92% were identified to have NAFLD. 36.04%, 38.32% and 25.63% were non-diabetic, prediabetic and diabetic respectively. Diabetic NAFLD significantly increased risk of cardiovascular disease (CVD), stroke, chronic kidney disease, all-cause and CVD mortality compared to non-diabetic NAFLD. However, prediabetic NAFLD only significantly increased the risk of CVD and did not result in a higher risk of mortality. Conclusions Given the increased risk of adverse outcomes, this study highlights the importance of regular diabetes screening in NAFLD and adoption of prompt lifestyle modifications to reduce disease progression. Facing high cardiovascular burden, prediabetic and diabetic NAFLD individuals can benefit from early cardiovascular referrals to reduce risk of CVD events and mortality.