Mitigating bisphenol A-induced apoptosis in KGN cells: the therapeutic role of 1,25-dihydroxyvitamin D3 through upregulation of PGC-1α expression and inhibition of the mitochondrial cytochrome c pathway.

PURPOSE: This study investigated the potential of 1,25-dihydroxyvitamin D3 (1,25(OH)2VD3) to mitigate bisphenol A (BPA)-induced apoptosis in human ovarian granulosa KGN cells with the aim of establishing a theoretical foundation for understanding of how vitamin D improved ovarian function in patients with polycystic ovary syndrome (PCOS). METHODS: The impact of varying concentrations of BPA and 1,25(OH)2VD3 on KGN cell viability was elucidated. It was established that BPA-induced apoptosis in KGN cells. Subsequently, KGN cells underwent pretreatment with 1,25(OH)2VD3, followed by exposure to BPA. The apoptosis rate, reactive oxygen species (ROS) levels, and mitochondrial function of the cells were meticulously assessed, along with the expression levels of genes associated with apoptosis as well as antioxidant and mitochondrial biogenesis. RESULTS: BPA induced a notable increase in apoptosis (P < 0.001) and oxidative stress (P < 0.001) in KGN cells, accompanied by a significant reduction in mitochondrial membrane potential (P < 0.001) and severe impairment of mitochondrial function. Following pretreatment of KGN cells with 1,25(OH)2VD3, there was a significant decrease in the apoptosis rate (P = 0.004), coupled with a reduction in ROS production (P = 0.002). Concomitantly, the upregulation of PGC-1α (P = 0.009) and SOD (P = 0.018) was observed, while mRNA expression of BAX (P = 0.011), Cyt c (P = 0.001), Apaf-1 (P = 0.012), caspase-9 (P < 0.001), and caspase-3 (P = 0.011) was downregulated. Notably, the mitigation of mitochondrial damage was evident through restored mitochondrial membrane potential (P < 0.001), as corroborated by electron microscope results. CONCLUSIONS: 1,25(OH)2VD3 mitigated BPA-induced damage and apoptosis in KGN cells by upregulating the expression of PGC-1α and impeding the mitochondrial cytochrome c (Cyt c) apoptotic pathway. This study established a novel theoretical foundation for utilizing vitamin D in the treatment of PCOS patients.

Effects of dulaglutide combined with insulin degludec on glucose fluctuations and appetite in type 2 diabetes.

Introduction: The aim of this study was to describe appetite and glucose fluctuation in type 2 diabetes mellitus patients initiating treatment with dulaglutide combined with insulin degludec. Methods: This retrospective study of patients identified adults starting treatment with once-weekly (QW) dulaglutide combined with insulin degludec (experimental group) or insulin degludec alone (control group). Patients were followed for up to 6 months from treatment initiation. The clinical characteristics of patients, treatment patterns, CGM data, and appetite scores were obtained for the two groups. Results: A total of 236 patients were included in this study. SDBG, MAGE, LAGE, and PPGE of the experimental group were lower than the control group's (P < 0.05). The proportions of patients achieving a time in range (TIR) of ≥70% in the experimental group were higher than in the control group, with 43% and 10% on the second day, 88% and 47% on the fourth day, 95% and 47% on the seventh day, and 100% and 67% on the tenth day, respectively. Significant associations existed between TIR and the prevalence of islet function. At six months, 89.2% of patients in the experimental group were still using dulaglutide. Appetite decreased significantly at 1 week and increased at 3 months after treatment with dulaglutide. Conclusion: Dulaglutide combined with insulin degludec significantly reduces glucose fluctuations in patients with type 2 diabetes mellitus and improves the TIR rate. However, the treatment on appetite could decrease in the first three months.

1,25-Dihydroxyvitamin D inhibits hepatic diacyglycerol accumulation and ameliorates metabolic dysfunction in polycystic ovary syndrome rat models.

Introduction: We aimed to evaluate the influence of 1,25-dihydroxyvitamin D (1,25(OH)2D) on metabolic dysfunction and elucidate its underlying mechanism using a rat model of polycystic ovary syndrome (PCOS). Methods: Twenty-four Sprague-Dawley rats were randomly divided into four groups: control group (CON, 2 ml/kg of oral 0.5% CMC), 1,25VD group (oral 0.5% CMC and 2.5 ug/kg intraperitoneal 1,25(OH)2D), PCOS group (1 mg/kg oral letrozole), PCOS+1,25VD group (1 mg/kg oral letrozole orally 2.5 ug/kg intraperitoneal 1,25(OH)2D). The treatments were administered for 8 weeks. Body weight, estrus cycle, insulin tolerance, and oral glucose tolerance of the rats in the different groups were assessed. The rats were euthanized at the 8th weeks, and plasma, ovarian, and liver samples were collected and analyzed. The hepatic lipid profile was characterized using HPLC/MRM. Results: Letrozole-induced PCOS rats exhibited increased weight, insulin resistance, postprandial glucose abnormalities, and dyslipidemia. Compared with the PCOS group rats, the PCOS+1,25VD group rats showed reduced body weight, increased sensitivity to insulin, decreased postprandial glucose, and elevated levels of high-density lipoprotein cholesterol. Moreover, abnormally increased liver concentrations of total diacylglycerol (DG) and DG species in the PCOS rats were reversed by treatment with 1,25(OH)2D. Additionally, hepatic DG and insulin sensitivity were correlated. Conclusion: 1,25(OH)2D inhibited hepatic DG accumulation and ameliorated metabolic dysfunction in PCOS rat models.

1,25-Dihydroxyvitamin D3 alleviates hyperandrogen-induced ferroptosis in KGN cells.

PURPOSE: Hyperandrogenism, one of the most frequent causes of anovulation in women, increases the risk of metabolic disorders in patients with polycystic ovary syndrome (PCOS). Ferroptosis, characterized by iron-dependent lipid peroxidation, has provided new insight into the progression of PCOS. 1,25-dihydroxyvitamin D3 (1,25D3) may play a role in reproduction because its receptor, VDR, which contributes to the inhibition of oxidative stress, is primarily located in the nuclei of granulosa cells. This study has therefore investigated whether 1,25D3 and hyperandrogenism affect granulosa-like tumor cells (KGN cells) through ferroptosis. METHODS: KGN cells were treated with dehydroepiandrosterone (DHEA) or pretreated with 1,25D3. Cell viability was evaluated with the cell counting kit-8 (CCK-8) assay. The mRNA and protein expression levels of ferroptosis-related molecules, including glutathione peroxidase 4 (GPX4), solute carrier family 7 member (SLC7A11), and long-chain acyl-CoA synthetase 4 (ACSL4), were assessed via qRT-PCR and western blot. The concentration of malondialdehyde (MDA) was measured by ELISA. The rates of reactive oxygen species (ROS) production and lipid peroxidation were assessed via photometric methods. RESULTS: Decreased cell viability, suppression of GPX4 and SLC7A11 expression, increased expression of ACSL4, elevated levels of MDA, accumulation of ROS, and increased lipid peroxidation, which are changes representative of ferroptosis, were observed in KGN cells after treatment with DHEA. Pretreatment with 1,25D3 in KGN cells significantly prevented these changes. CONCLUSIONS: Our findings demonstrate that 1,25D3 attenuates hyperandrogen-induced ferroptosis of KGN cells. This finding might lead to new insights into the pathophysiology and therapy of PCOS and provides new evidence for the treatment of PCOS with 1,25D3.

T follicular helper 17 (Tfh17) cells are superior for immunological memory maintenance.

A defining feature of successful vaccination is the ability to induce long-lived antigen-specific memory cells. T follicular helper (Tfh) cells specialize in providing help to B cells in mounting protective humoral immunity in infection and after vaccination. Memory Tfh cells that retain the CXCR5 expression can confer protection through enhancing humoral response upon antigen re-exposure but how they are maintained is poorly understood. CXCR5+ memory Tfh cells in human blood are divided into Tfh1, Tfh2, and Tfh17 cells by the expression of chemokine receptors CXCR3 and CCR6 associated with Th1 and Th17, respectively. Here, we developed a new method to induce Tfh1, Tfh2, and Tfh17-like (iTfh1, iTfh2, and iTfh17) mouse cells in vitro. Although all three iTfh subsets efficiently support antibody responses in recipient mice with immediate immunization, iTfh17 cells are superior to iTfh1 and iTfh2 cells in supporting antibody response to a later immunization after extended resting in vivo to mimic memory maintenance. Notably, the counterpart human Tfh17 cells are selectively enriched in CCR7+ central memory Tfh cells with survival and proliferative advantages. Furthermore, the analysis of multiple human cohorts that received different vaccines for HBV, influenza virus, tetanus toxin or measles revealed that vaccine-specific Tfh17 cells outcompete Tfh1 or Tfh2 cells for the persistence in memory phase. Therefore, the complementary mouse and human results showing the advantage of Tfh17 cells in maintenance and memory function supports the notion that Tfh17-induced immunization might be preferable in vaccine development to confer long-term protection.

Association Of Blood Lipocalin-2 Levels with Gestational Diabetes Mellitus: A Systematic Review and Meta-Analysis.

Lipocalin-2 (LCN2) is becoming recognized as a pleiotropic mediator of metabolic disorders. However, the relationship between LCN2 and gestational diabetes mellitus (GDM) is not well understood. We performed a systematic review and meta-analysis to explore it. A systematic search of Cochrane Library, PubMed, Embase, Scopus, Web of Science, Chinese National Knowledge Infrastructure, and Wan-fang Database was done for relevant articles published up to September 29, 2021. Standardized mean difference (SMD) with 95% confidence intervals (CI) was calculated to explore the association of LCN2 levels with GDM using Revman 5.3 and Stata 15.1. Fifteen case-control studies were included in this meta-analysis. The patients with GDM had significantly higher levels of blood LCN2 than parturients with normal glucose tolerance (SMD=3.41, 95% CI=2.24 to 4.58). Meta-regression and subgroup analysis were conducted to investigate the source of heterogeneity. Likely sources of heterogeneity were age and testing methods. This study found that GDM showed higher blood LCN2 levels than controls. However, caution is warranted on the interpretation of these findings. Standardized LCN2 measurement methods and longitudinal studies are required to disentangle and better understand the relationships observed.

Zebrafish Heme Oxygenase 1a Is Necessary for Normal Development and Macrophage Migration.

Heme oxygenase function is highly conserved between vertebrates where it plays important roles in normal embryonic development and controls oxidative stress. Expression of the zebrafish heme oxygenase 1 genes is known to be responsive to oxidative stress suggesting a conserved physiological function. In this study, we generate a knockout allele of zebrafish hmox1a and characterize the effects of hmox1a and hmox1b loss on embryonic development. We find that loss of hmox1a or hmox1b causes developmental defects in only a minority of embryos, in contrast to Hmox1 gene deletions in mice that cause loss of most embryos. Using a tail wound inflammation assay we find a conserved role for hmox1a, but not hmox1b, in normal macrophage migration to the wound site. Together our results indicate that zebrafish hmox1a has clearly a partitioned role from hmox1b that is more consistent with conserved functions of mammalian Heme oxygenase 1.

Rough and smooth variants of Mycobacterium abscessus are differentially controlled by host immunity during chronic infection of adult zebrafish.

Prevalence of Mycobacterium abscessus infections is increasing in patients with respiratory comorbidities. After initial colonisation, M. abscessus smooth colony (S) variants can undergo an irreversible genetic switch into highly inflammatory, rough colony (R) variants, often associated with a decline in pulmonary function. Here, we use an adult zebrafish model of chronic infection with R and S variants to study M. abscessus pathogenesis in the context of fully functioning host immunity. We show that infection with an R variant causes an inflammatory immune response that drives necrotic granuloma formation through host TNF signalling, mediated by the tnfa, tnfr1 and tnfr2 gene products. T cell-dependent immunity is stronger against the R variant early in infection, and regulatory T cells associate with R variant granulomas and limit bacterial growth. In comparison, an S variant proliferates to high burdens but appears to be controlled by TNF-dependent innate immunity early during infection, resulting in delayed granuloma formation. Thus, our work demonstrates the applicability of adult zebrafish to model persistent M. abscessus infection, and illustrates differences in the immunopathogenesis induced by R and S variants during granulomatous infection.

Transplantation of high fat fed mouse microbiota into zebrafish larvae identifies MyD88-dependent acceleration of hyperlipidaemia by Gram-positive cell wall components.

Gut dysbiosis is an important modifier of pathologies including cardiovascular disease but our understanding of the role of individual microbes is limited. Here, we have used transplantation of mouse microbiota into microbiota-deficient zebrafish larvae to study the interaction between members of a mammalian high fat diet-associated gut microbiota with a lipid rich diet challenge in a tractable model species. We find zebrafish larvae are more susceptible to hyperlipidaemia when exposed to the mouse high fat-diet-associated microbiota and that this effect can be driven by two individual bacterial species fractionated from the mouse high fat-diet-associated microbiota. We find Stenotrophomonas maltophilia increases the hyperlipidaemic potential of chicken egg yolk to zebrafish larvae independent of direct interaction between S. maltophilia and the zebrafish host. Colonization by live, or exposure to heat-killed, Enterococcus faecalis accelerates hyperlipidaemia via host MyD88 signaling. The hyperlipidaemic effect is replicated by exposure to the Gram-positive toll-like receptor agonists peptidoglycan and lipoteichoic acid in a MyD88-dependent manner. In this work, we demonstrate the applicability of zebrafish as a tractable host for the identification of gut microbes that can induce conditional host phenotypes via microbiota transplantation and subsequent challenge with a high fat diet.

Haem oxygenase limits Mycobacterium marinum infection-induced detrimental ferrostatin-sensitive cell death in zebrafish.

Iron homeostasis is essential for both sides of the host-pathogen interface. Restricting access of iron slows bacterial growth while iron is also a necessary cofactor for host immunity. Haem oxygenase 1 (HMOX1) is a critical regulator of iron homeostasis that catalyses the liberation of iron during degradation of haem. It is also a stress-responsive protein that can be rapidly upregulated and confers protection to the host. Although a protective role of HMOX1 has been demonstrated in a variety of diseases, the role of HMOX1 in Mycobacterium tuberculosis infection is equivocal across experiments with different host-pathogen combinations. Here, we use the natural host-pathogen pairing of the zebrafish-Mycobacterium marinum infection platform to study the role of zebrafish haem oxygenase in mycobacterial infection. We identify zebrafish Hmox1a as the relevant functional paralog of mammalian HMOX1 and demonstrate a conserved role for Hmox1a in protecting the host from M. marinum infection. Using genetic and chemical tools, we show zebrafish Hmox1a protects the host against M. marinum infection by reducing infection-induced iron accumulation and ferrostatin-sensitive cell death.

Pidotimod increases inflammation in wounded zebrafish embryos.

Pidotimod (PDT) is a synthetic dipeptide molecule which can improve immune responses in mice and humans, protecting hosts from infection. However, the exact mechanism of protection remains ill-defined. The effect of pidotimod has not yet been investigated in the inflammatory response of zebrafish. In this study, we used tail wound and infection models of zebrafish to study the effect of PDT on inflammation. We found that zebrafish larvae were sensitive to PDT immersion causing toxicity at doses above 50 µg/mL. The tail wound assay showed that PDT increased the recruitment of neutrophils and macrophages to the wound site and promoted the transcription of the pro-inflammatory cytokine il1b. However, we did not observe protection of uropathogenic Escherichia coli or Mycobacterium marinum infected zebrafish larvae following PDT treatment. This study provides a new platform for PDT research, which is worthy of further research to identify further effects of PDT therapy.

The metabolic hormone leptin promotes the function of TFH cells and supports vaccine responses.

Follicular helper T (TFH) cells control antibody responses by supporting antibody affinity maturation and memory formation. Inadequate TFH function has been found in individuals with ineffective responses to vaccines, but the mechanism underlying TFH regulation in vaccination is not understood. Here, we report that lower serum levels of the metabolic hormone leptin associate with reduced vaccine responses to influenza or hepatitis B virus vaccines in healthy populations. Leptin promotes mouse and human TFH differentiation and IL-21 production via STAT3 and mTOR pathways. Leptin receptor deficiency impairs TFH generation and antibody responses in immunisation and infection. Similarly, leptin deficiency induced by fasting reduces influenza vaccination-mediated protection for the subsequent infection challenge, which is mostly rescued by leptin replacement. Our results identify leptin as a regulator of TFH cell differentiation and function and indicate low levels of leptin as a risk factor for vaccine failure.

Exosomal hsa_circ_0006859 is a potential biomarker for postmenopausal osteoporosis and enhances adipogenic versus osteogenic differentiation in human bone marrow mesenchymal stem cells by sponging miR-431-5p.

BACKGROUND: As one of the most common chronic diseases in the world, osteoporosis occurs especially in postmenopausal women. Circular RNAs (circRNAs) are emerging as major drivers in human disease. The aim of the present study was to analyse circRNA expression profiles in osteoporosis and to explore the clinical significance and the regulatory molecular mechanism of hsa_circ_0006859 during osteoporosis. METHODS: Exosomes were isolated from clinically collected serum samples. A circRNA microarray was performed to screen differentially expressed circRNAs. Quantitative real-time PCR (qRT-PCR) and western blot were performed to analyse target gene mRNA expression and protein expression. Alizarin red staining (ARS) was performed to evaluate the mineralization ability of human bone marrow mesenchymal stem cells (hBMSCs). Oil Red O staining was performed to evaluate the lipid droplet formation ability of hBMSCs. Bioinformatics analysis and the luciferase reporter assay were performed to investigate the interaction between two genes. RESULTS: Hsa_circ_0006859 was identified as one of the most upregulated circRNAs in the microarray analysis. Hsa_circ_0006859 in exosomes was upregulated in osteoporosis patients compared to healthy controls. Hsa_circ_0006859 differentiated osteopenia or osteoporosis patients from healthy controls with high sensitivity and specificity. Hsa_circ_0006859 suppressed osteoblastic differentiation and promoted adipogenic differentiation of hBMSCs. Hsa_circ_0006859 directly bound to miR-431-5p, and ROCK1 was identified as a novel target gene of miR-431-5p. Hsa_circ_0006859 is a competing endogenous RNA (ceRNA) of miR-431-5p that promotes ROCK1 expression. Hsa_circ_0006859 suppressed osteogenesis and promoted adipogenesis by sponging miR-431-5p to upregulate ROCK1. CONCLUSIONS: Exosomal hsa_circ_0006859 is a potential biomarker for postmenopausal osteoporosis and controls the balance between osteogenesis and adipogenesis in hBMSCs by sponging miR-431-5p.

Circular RNA hsa_circ_0001445 in plasma as a novel biomarker for osteoporosis in postmenopausal women.

Background: The present study evaluated the potential of hsa_circ_0001445 as a biomarker in plasma for postmenopausal patients with osteoporosis (OPO). Materials & methods: The expression levels of hsa_circ_0001445 in plasma were detected in healthy controls, patients with osteopenia (OPE) and patients with OPO by using quantitative reverse-transcriptase PCR. Results: The expression levels of hsa_circ_0001445 in plasma were much lower in OPO patients compared with OPE patients and healthy controls. Its expression was positively correlated with the T-score and was negatively correlated with ß-isomerized C-terminal telopeptides (ß-CTx). It could distinguish OPE or/and OPO patients from healthy controls. Moreover, its expression was significantly upregulated in the plasma of OPO patients after anti-osteoporotic treatment. Conclusion: Hsa_circ_0001445 in plasma may be a novel potential diagnostic biomarker for OPO.

p62 acts as an oncogene and is targeted by miR-124-3p in glioma.

BACKGROUND: Glioma is the most common central nervous system (CNS) tumour. p62, an important autophagy adaptor, plays a crucial role in cancer. However, the role of p62 in the progression of glioma is poorly characterized. METHODS: We examined the expression of p62 in glioma tissues and cell lines. Then we investigated the function of p62 in vitro, and clarified the mechanism underlying the regulation of p62 expression. RESULTS: We revealed that p62 was upregulated at both the mRNA and protein levels in human glioma tissues irrelevant to isocitrate dehydrogenase (IDH) status. Then, we found that overexpression of p62 promoted glioma progression by promoting proliferation, migration, glycolysis, temozolomide (TMZ) resistance and nuclear factor κB (NF-κB) signalling pathway, and repressing autophagic flux and reactive oxygen species (ROS) in vitro. In accordance with p62 overexpression, knockdown of p62 exerted anti-tumour effects in glioma cells. Subsequently, we demonstrated that miR-124-3p directly targeted the 3'-UTR of p62 mRNA, leading to the downregulation of p62. Finally, we found that p62 function could be partially reversed by miR-124-3p overexpression. CONCLUSIONS: Our results demonstrate that p62 can be targeted by miR-124-3p and acts as an oncogene in glioma, suggesting the potential value of p62 as a novel therapeutic target for glioma.

miR-454-3p Is an Exosomal Biomarker and Functions as a Tumor Suppressor in Glioma.

Glioma is the most common type of primary malignant brain tumor in adults. Our previous work discovered that plasma miR-454-3p may have some advantages in glioma prognosis, but the clinical significance and the regulatory mechanism of miR-454-3p in glioma have not been systematically investigated, especially regarding the relationship between circulating and tissue miR-454-3p. The expression level of miR-454-3p in glioma serum and tissues was analyzed through quantitative real-time PCR (qRT-PCR). Cell-Counting Kit 8 (CCK-8), wound healing, transwell invasion, apoptosis, and immunofluorescence assays were used to assess the role of miR-454-3p in glioma cancer cells. ATG12 was selected as the target gene of miR-454-3p by bioinformatic analysis. The relationship between ATG12 and miR-454-3p was further validated by luciferase reporter assays and Western blot analysis. miR-454-3p was significantly downregulated in tumor tissues, while it was remarkably upregulated in exosomes from the same patients with glioma. The area under curve (AUC) of exosomal miR-454-3p for glioma diagnosis was 0.8663. The exosomal miR-454-3p was prominently lower in the postoperative serums than that in the preoperative serums. High miR-454-3p expression in exosomes or low miR-454-3p expression in tissue was associated with poor prognosis. Restored expression of miR-454-3p suppressed cell proliferation, migration, invasion, and autophagy in glioma. ATG12 was validated as a direct target of miR-454-3p. The overexpression of ATG12 could partially reverse the effects induced by miR-454-3p suppression. Our data indicate that miR-454-3p may serve as an exosomal biomarker and may be developed into a novel treatment for glioma.

miR-10b Downregulated by DNA Methylation Acts as a Tumor Suppressor in HPV-Positive Cervical Cancer via Targeting Tiam1.

BACKGROUND/AIMS: microRNAs (miRNAs) are known to act as oncogenes or tumor suppressors in diverse cancers. Although miR-10b is an oncogene implicated in many tumors, its role in cervical cancer (CC) remains largely unclear. Here, we investigated the function and underlying mechanisms of miR-10b in human CC. METHODS: Quantitative RT-PCR was used to measure miR-10b expression in CC and normal tissues, and its association with clinicopathologic features was analyzed. Methylation of CpG sites in the miR-10b promoter was analyzed by methylation sequencing. Cell proliferation, apoptosis, migration, and invasion assays were used to elucidate the biological effects of miR-10b and expression of the target gene was assayed with Western blot. RESULTS: miR-10b was downregulated in CC tissues compared with normal tissues, and less miR-10b expression was associated with larger tumors, vascular invasion and HPV-type 16 positivity. miR-10b expression decreased in HeLa (HPV18-positive) and SiHa (HPV16-positive) cells compared with C-33A (HPV-negative), but increased after treatment with 5-Aza-CdR. Methylation ratio of site -797 in the miR-10b promoter in C-33A was lower than that in HeLa and SiHa. Further analysis indicates that site -797 is located within a transcription factor AP-2A (TFAP2A) binding element. Functionally, overexpression of miR-10b in HeLa and SiHa suppressed cell proliferation, migration and invasion, and induced apoptosis and miR-10b downregulation had opposite effects. Mechanistically, T-cell lymphoma invasion and metastasis 1 (Tiam1) was identified as a direct and functional target of miR-10b. CONCLUSION: miR-10b acts as a tumor suppressor in CC by suppressing oncogenic Tiam1, and its expression may be downregulated through methylation of TFAP2A binding element by HPV.

Apolipoprotein M increases the expression of vitamin D receptor mRNA in colorectal cancer cells detected with duplex fluorescence reverse transcription-quantitative polymerase chain reaction.

Apolipoprotein M (ApoM) and the vitamin D receptor (VDR) are apolipoproteins predominantly presenting in high-density lipoprotein (HDL) and a karyophilic protein belonging to the steroid­thyroid receptor superfamily, respectively. Previous studies have demonstrated that ApoM and VDR are associated with cholesterol metabolism, immune and colorectal cancer regulation. In order to investigate whether ApoM affected the expression of VDR in colorectal cancer cells, a single­tube duplex fluorescence reverse transcription­quantitative polymerase chain reaction (RT­qPCR) system was developed to simultaneously detect the mRNA levels of VDR and GAPDH in HT­29 cells overexpressing ApoM. The results demonstrated that the amplification products were confirmed as the specific fragment of VDR/GAPDH using the DNA sequencing instrument. The sensitivity, linear range, correlation coefficient, amplification efficiency, intra­assay and inter­assay coefficients of variation were 40 copies/µl, 4.00x101­4.00x105 copies/µl, 0.999, 92.42%, 0.09­0.34% and 0.32­0.65% for VDR, and 40 copies/µl, 4.00x101­4.00x105 copies/µl, 0.999, 98.07%, 0.19­0.43% and 0.40­0.75% for GAPDH, respectively. The results indicated that the expression of VDR mRNA was significantly higher in HT­29 cells overexpressing ApoM, compared with the negative control group (P<0.05). In conclusion, the current study successfully developed the single­tube duplex RT­qPCR to simultaneously detect VDR and GAPDH expression in colorectal cancer cells. The methodology results demonstrated that the duplex RT­qPCR system with high sensitivity and specificity could ensure the objectivity and credibility of the detection. The present study confirmed that ApoM significantly increased the expression of VDR in HT­29 cells. In addition, it was hypothesized that ApoM may be involved in antineoplastic activity via the upregulation of VDR expression, which may provide novel directions for the investigation of ApoM in cancer.

Type I Interferons Triggered through the Toll-Like Receptor 3-TRIF Pathway Control Coxsackievirus A16 Infection in Young Mice.

UNLABELLED: Coxsackievirus A16 (CVA16) is one of the major etiological agents of hand, foot, and mouth disease (HFMD) in children. The host defense mechanisms against CVA16 infection remain almost entirely unknown. Unlike previous observations with enterovirus 71 (EV71) infection, here we show that gamma interferon (IFN-γ) or invariant NK T cell deficiency does not affect disease development or the survival of CVA16-infected mice. In contrast, type I interferon receptor deficiency resulted in the development of more severe disease in mice, and the mice had a lower survival rate than wild-type mice. Similarly, a deficiency of Toll-like receptor 3 (TLR3) and TRIF, but not other pattern recognition receptors, led to the decreased survival of CVA16-infected mice. TLR3-TRIF signaling was indispensable for the induction of type I interferons during CVA16 infection in mice and protected young mice from disease caused by the infection. In particular, TRIF-mediated immunity was critical for preventing CVA16 replication in the neuronal system before disease occurred. IFN-ß treatment was also found to compensate for TRIF deficiency in mice and decreased the disease severity in and mortality of CVA16-infected mice. Altogether, type I interferons induced by TLR3-TRIF signaling mediate protective immunity against CVA16 infection. These findings may shed light on therapeutic strategies to combat HFMD caused by CVA16 infection. IMPORTANCE: Hand, foot, and mouth disease (HFMD) is a major threat to public health in the Asia-Pacific region. Both CVA16 and EV71 are major pathogens that are responsible for HFMD. The majority of research efforts have focused on the more virulent EV71, but little has been done with CVA16. Thus far, host immune responses to CVA16 infection have not yet been elucidated. The present study discovered an initial molecular mechanism underlying host protective immunity against CVA16 infection, providing the first explanation for why CVA16 and EV71 cause different clinical outcomes upon infection of humans. Therefore, different therapeutic strategies should be developed to treat HFMD cases caused by these two viruses.