This study was conducted to determine the mRNA and protein expression levels of peroxisome proliferator-activated receptors (PPARs) in visceral adipose tissue, as well as serum adipokine levels, in Sprague Dawley rats. The rats were fed either a normal (control rats) or excessive (experimental rats) intake of food for 8 or 16 weeks, then sacrificed, at which time visceral and subcutaneous adipose tissues, as well as blood samples, were collected. The mRNA and protein expression levels of PPARs in the visceral adipose tissues were determined using reverse transcription-polymerase chain reaction and Western blotting, respectively. In addition, the levels of adipokines in the serum samples were determined using commercial ELISA kits. The results revealed that at 8 weeks, the mass of subcutaneous adipose tissue was higher than that of the visceral adipose tissue in the experimental rats, but the reverse occurred at 16 weeks. Furthermore, at 16 weeks the experimental rats exhibited an upregulation of PPARγ mRNA and protein expression levels in the visceral adipose tissues, and significant increases in the serum levels of CCL2 and interleukin (IL)-6 were observed, compared with those measured at 8 weeks. In conclusion, this study demonstrated that the PPARγ expression level was likely correlated with serum levels of CCL2 and IL-6, molecules that may facilitate visceral adipose tissue accumulation. In addition, the levels of the two adipokines in the serum may be useful as surrogate biomarkers for the expression levels of PPARγ in accumulated visceral adipose tissues.
This study aims to elucidate the effects of chrysin on human ER-negative breast cancer cell line, MDA-MB-231. The study demonstrated that treatment of MDA-MB-231 cells with 20 microM chysin for 48 h significantly inhibited the growth of MDA-MB-231 cells and induced cytoplasmic lipid accumulation in the cells, but that the observed of cell death was not caused by apoptosis. The expression of PPARalpha mRNA in chrysin-treated MDA-MB-231 cells was significantly increased, which was likely associated to the proliferation of the cells post chrysin treatment.
Encephalomyelitis is a well-known complication of hand, foot, and mouth disease (HFMD) due to Enterovirus 71 (EV71) infection. Viral RNA/antigens could be detected in the central nervous system (CNS) neurons in fatal encephalomyelitis but the mechanisms of neuronal cell death is not clearly understood. We investigated the role of absent in melanoma 2 (AIM2) inflammasome in neuronal cell death, and its relationship to viral replication. Our transcriptomic analysis, RT-qPCR, Western blot, immunofluorescence and flow cytometry studies consistently showed AIM2 gene up-regulation and protein expression in EV-A71-infected SK-N-SH cells. Downstream AIM2-induced genes, CARD16, caspase-1 and IL-1β were also up-regulated and caspase-1 was activated to form cleaved caspase-1 p20 subunits. As evidenced by 7-AAD positivity, pyroptosis was confirmed in infected cells. Overall, these findings have a strong correlation with decreases in viral titers, copy numbers and proteins, and reduced proportions of infected cells. AIM2 and viral antigens were detected by immunohistochemistry in infected neurons in inflamed areas of the CNS in EV-A71 encephalomyelitis. In infected AIM2-knockdown cells, AIM2 and related downstream gene expressions, and pyroptosis were suppressed, resulting in significantly increased virus infection. These results support the notion that AIM2 inflammasome-mediated pyroptosis is an important mechanism of neuronal cell death and it could play an important role in limiting EV-A71 replication.
Enterovirus A71 (EV-A71) and coxsackievirus A16 (CV-A16) are closely related enteroviruses that cause hand, foot and mouth disease (HFMD) in children. Serious neurological complications almost always occur in EV-A71 infection, but are rare in CV-A16 infection. Based on the hypothesis that this may be because EV-A71 infects neuronal cells more easily than CV-A16, we compared virus infection, replication and spread of EV-A71 and CV-A16 in SK-N-SH cells. We found that CV-A16 invariably showed significantly lower replication and caused less necrotic cell death in SK-N-SH cells, compared with EV-A71. This was not due to a lower proportion of CV-A16-infected cells, since both viruses showed similar proportions of infected cells at all time points analyzed. Furthermore, reduced replication of CV-A16 in SK-N-SH cells does not appear to be due to limited viral receptor availability, which might limit viral entry, because experiments with viral RNA-transfected cells showed the same results as for live virus infections. On the other hand, no differences were observed between EV-A71 and CV-A16 in RD cells and results were generally similar in RD cells for both viruses. Taken together, our findings suggest that the poor growth of CV-A16 and EV-A71in SK-N-SH cells, compared with RD cells, may be due to cell type-specific restrictions on viral replication and spread. Furthermore, the lower viral replication and necrotic cell death in CV-A16-infected SK-N-SH cells, compared with EV-A71-infected SK-N-SH cells, is consistent with the lower prevalence of neurotropism observed in CV-A16-associated HFMD outbreaks. Nonetheless, in vivo data and more extensive comparisons of different viral strains are essential to confirm our findings.
Coxsackievirus A16 (CV-A16) and enterovirus A71 (EV-A71) are closely related enteroviruses that cause the same hand, foot, and mouth disease (HFMD), but neurological complications occur only very rarely in CV-A16 compared to EV-A71 infections. To elucidate host responses that may be able to explain these differences, we performed transcriptomic analysis and real-time quantitative PCR (RT-qPCR) in CV-A16-infected neuroblastoma cells (SK-N-SH), and the results showed that the radical S-adenosylmethionine domain containing 2 (RSAD2) was the highest upregulated gene in the antimicrobial pathway. Increased RSAD2 expression was correlated with reduced viral replication, while RSAD2 knockdown cells were correlated with increased replication. EV-A71 replication showed no apparent correlation to RSAD2 expressions. Absent in melanoma 2 (AIM2), which is associated with pyroptotic cell death, was upregulated in EV-A71-infected neurons but not in CV-A16 infection, suggesting that the AIM2 inflammasome played a significant role in suppressing EV-A71 replication. Chimeric viruses derived from CV-A16 and EV-A71 but containing swapped 5' nontranslated regions (5' NTRs) showed that RSAD2 expression/viral replication and AIM2 expression/viral replication patterns may be linked to the 5' NTRs of parental viruses. Differences in secondary structure of internal ribosomal entry sites within the 5' NTR may be responsible for these findings. Overall, our results suggest that CV-A16 and EV-A71 elicit different host responses to infection, which may help explain the apparent lower incidence of CV-A16-associated neurovirulence in HFMD outbreaks compared to EV-A71 infection.IMPORTANCE Although coxsackievirus A16 (CV-A16) and enterovirus A17 (EV-A71) both cause hand, foot, and mouth disease, EV-A71 has emerged as a leading cause of nonpolio, enteroviral fatal encephalomyelitis among young children. The significance of our research is in the identification of the possible differing and novel mechanisms of CV-A16 and EV-A71 inhibition in neuronal cells that may impact viral neuropathogenesis. We further showed that viral 5' NTRs may play significant roles in eliciting different host response mechanisms.
Excessive adipose tissue accumulation is an increasing health problem worldwide. The present study aimed to determine differentially expressed genes (DEGs) that are associated with the excessive accumulation of adipose tissues by PCR arrays in an excess dietary intake animal model. For this purpose, male Sprague Dawley rats were randomly assigned to 2 groups: Control (given an ordinary diet) and experimental (given twice the amount of the ordinary diet). After 2 months of feeding, the abdominal cavities of the rats from each group were opened, then subcutaneous and visceral adipose tissues were removed. The adipose tissues collected were then used for total RNA extraction and then reverse transcribed to cDNA, which was then used as a template to identify the DEGs of 84 transcripts for rat obesity by RT2 Profiler PCR Arrays. The results showed significant downregulation of bombesin‑like receptor 3 (BRS3) and uncoupling protein 1 (UCP1) in visceral adipose tissues of experimental rats compared with those of the control rats, and differential gene expression analysis showed an association with fat cell differentiation and regulation of triglyceride sequestration, as well as fatty acid binding. The gene expression patterns observed in the present study, which may be associated with peroxisome proliferator‑activated receptor‑γ (PPARG) on excessive visceral adipose tissue accumulation, may be useful in identifying a group of surrogate biomarkers for the early diet‑induced accumulation of visceral adipose tissue detection in humans. The biomarkers can also be the specific targets for drug development to reduce excessive visceral adipose tissue accumulation in the body and its associated diseases.
Enterovirus D68 (EV-D68) is an emerging respiratory pathogen since the 2014 outbreak in USA. A low level of virus circulation has been reported in Kuala Lumpur, Malaysia in the past. However, the extent of the infection in Malaysia is not known. In the present study, we determine the seroepidemiology of EV-D68 in Kuala Lumpur, Malaysia before and after the USA outbreak in Aug 2014. A luciferase-based seroneutralization test was developed using a clone-derived prototype Fermon strain carrying a nanoluciferase marker. We screened the neutralization capacity of 450 serum samples from children and adults (1-89 years old) collected between 2013 to 2015. EV-D68 seropositivity increased with age, with children aged 1-3 showing significantly lower seroprevalence compared to adults. Multivariate analysis showed that older age groups 13-49 years (odds ratio [OR] 4.78 [95% CI 2.69-8.49], p<0.0001) and >50 years (OR 3.83 [95% CI 2.19-6.68], p<0.0001) were more likely to be EV-D68 seropositive than children <13 years. Sampling post-Sept 2014 compared to pre-Sept 2014 also predicted seropositivity (OR 1.66 [95% CI 1.04-2.65]). Presence of neutralizing antibodies against EV-D68 in the study population suggests that EV-D68 was circulating prior to 2014. A higher seropositivity post-Sept 2014 suggests that Malaysia also experienced an upsurge in EV-D68 infections after the USA outbreaks in Aug 2014. A low seropositivity rate observed in children, especially those aged 1-3 years old, suggests that they are at risk and should be prioritized for future vaccination. This article is protected by copyright. All rights reserved.
Enterovirus A71 (EV-A71) causes hand, foot and mouth disease epidemics with neurological complications and fatalities. However, the neuropathogenesis of EV-A71 remains poorly understood. In mice, adaptation and virulence determinants have been mapped to mutations at VP2-149, VP1-145 and VP1-244. We investigate how these amino acids alter heparin-binding phenotype and shapes EV-A71 virulence in one-day old mice. We constructed six viruses with varying residues at VP1-98, VP1-145 (which are both heparin-binding determinants) and VP2-149 (based on the wild type 149K/98E/145Q, termed KEQ) to generate KKQ, KKE, KEE, IEE and IEQ variants. We demonstrated that the weak heparin-binder IEE was highly lethal in mice. The initially strong heparin-binding IEQ variant acquired an additional mutation VP1-K244E, which confers weak heparin-binding phenotype resulting in elevated viremia and increased virus antigens in mice brain, with subsequent high virulence. IEE and IEQ-244E variants inoculated into mice disseminated efficiently and displayed high viremia. Increasing polymerase fidelity and impairing recombination of IEQ attenuated the virulence, suggesting the importance of population diversity in EV-A71 pathogenesis in vivo. Combining in silico docking and deep sequencing approaches, we inferred that virus population diversity is shaped by electrostatic interactions at the five-fold axis of the virus surface. Electrostatic surface charges facilitate virus adaptation by generating poor heparin-binding variants for better in vivo dissemination in mice, likely due to reduced adsorption to heparin-rich peripheral tissues, which ultimately results in increased neurovirulence. The dynamic switching between heparin-binding and weak heparin-binding phenotype in vivo explained the neurovirulence of EV-A71.