Construction of a New Probe Based on Copper Chaperone Protein for Detecting Cu2+ in Cells.

Biomacromolecular probes have been extensively employed in the detection of metal ions for their prominent biocompatibility, water solubility, high selectivity, and easy modification of fluorescent groups. In this study, a fluorescent probe FP was constructed. The probe FP exhibited high specificity recognition for Cu2+. With the combination of Cu2+, the probe was subjected to fluorescence quenching. The research suggested that the probe FP carried out the highly sensitive detection of Cu2+ with detection limits of 1.7 nM. The fluorescence quenching of fluorescamine was induced by Cu2+ perhaps due to the PET (photoinduced electron transfer) mechanism. The FP-Cu2+ complex shows weak fluorescence, which is likely due to the PET quenching effect from Cu2+ to fluorescamine fluorophore. Moreover, the probe FP can be employed for imaging Cu2+ in living cells. The new fluorescent probe developed in this study shows the advantages of good biocompatibility and low cytotoxicity. It can be adopted for the targeted detection of Cu2+ in cells, and it has promising applications in the mechanism research and diagnosis of Cu2+-associated diseases.

Clinical Evaluation and Test of a Modified Lp-PLA2 Kit in Diagnosing Atherosclerosis.

BACKGROUND: Lipoprotein-associated phospholipase A2 (Lp-PLA2) has been identified as an inflammatory marker tightly correlated with the onset of atherosclerosis. Although several methodologies have been developed to detect Lp-PLA2, including enzyme-linked immunosorbent assay, Lp-PLA2 detection is still time- and resource-consuming with poor antiinterference ability and low sensitivity. Thus, it is urgent to explore new methodology for Lp-PLA2 detection. METHODS: In the current study, we evaluated the clinical performance of a modified Lp-PLA2 quantitative assay kit based on magnetic particle chemiluminescence, and analyzed the levels of Lp-PLA2 in atherosclerosis patients using this kit. RESULTS: Our results showed that the magnetic particle chemiluminescence method could effectively dissociate Lp-PLA2 from lipoprotein and finish the test within 20 minutes with high accuracy and good repeatability, as demonstrated by the results of linear measurement range, precision, and recovery rate. Furthermore, our preliminary data revealed that serum Lp-PLA2 levels were correlated to the presence and degree of atherosclerotic plaques. CONCLUSIONS: Lp-PLA2 could be helpful in diagnosing atherosclerosis.

Cx43 and NMDA receptors changes in UL122 genetically modified mice hippocampus: a mechanism for spatial memory impairment.

Congenital HCMV infection has been reported to be involved in learning and memory impairment, but whether HCMV IE2 plays a key role in the process remains unknown. The purpose of this study was to study the effects of IE2 on the expression levels of NMDA receptors and CX43 in the hippocampal neurons of ul122 transgenic mice. Firstly, the ul122 genetically modified mice models that can steadily and continuously express IE2 protein were established. Then, the mice were divided into the experimental group (positive mice identified) and the control group (wild type mice. n = 24 in each group). The establishment of ul122 genetically modified mice was identified by PCR technology. The learning and memory ability were measured using the Morris water-maze test. Western blot and immunohistochemical study were performed to detect the expression level of Cx43 and NMDA receptors. The results of PCR indicated that the ul122 genetically modified model was successfully constructed. Morris water maze test result showed that in the experimental group, less platform crossings and Quadrant time (%) compared to the control group, but there was no difference in escape latency. The expression level of Cx43 in the hippocampus CA1 of the experimental group was significantly reduced in keeping with NMDA receptors in immunohistochemistry. The significant decreased expression level of Cx43 and NMDA receptors in the ul122 genetically modified mice hippocampus may be connection with the mechanism for spatial memory impairment.

TLR3 regulates PD-L1 expression in human cytomegalovirus infected glioblastoma.

Glioblastoma, the most common primary brain tumor of adults, is characterized by poor survival rates. Programmed death ligand 1 (PD-L1, CD274) has been implicated in the immune escape of glioblastoma. The presence of human cytomegalovirus (HCMV) in glioblastoma multiforme (GBM) has sparked considerable interest and controversy. The exposure of toll-like receptor 3 (TLR3) to pathogens induces an antiviral state in cells or in animals. In the current study, the expression of PD-L1 and TLR3 in HCMV-infected glioma specimens was observed to be higher compared to the control. We therefore investigated if PD-L1 expression in glioblastoma is mediated by TLR3 triggering in HCMV infected glioblastoma. TLR3 siRNA transfections were utilized to identify the induction of PD-L1 via TLR3 triggering in HCMV infected cell lines. Also, IL-8 and TGF-ß were detected by ELISA for the antitumor role of TLR3. Thus, we propose a novel immune treatment using a combination of PD-L1 blockade with TLR3 triggering against HCMV infected glioblastoma.

HCMV-encoded IE2 promotes NAFLD progression by up-regulation of SREBP1c expression in UL122 genetically modified mice.

Non-alcoholic fatty liver disease (NAFLD), a liver manifestation of metabolic syndrome, is associated with considerable health and socioeconomic burdens in many populations worldwide. Recent studies suggest that human cytomegalovirus (HCMV) infection might play a role in the pathogenesis of metabolic diseases, including NAFLD, but it is still unclear whether HCMV-encoded IE2 plays an important role in this process. Interestingly, SREBP1c was recently reported to play critical roles in the development of hepatic steatosis. In this study, we aimed to study the IE2 effect on the expression levels of SREBP1c and on lipid metabolism in the liver of UL122 genetically modified mice. First, UL122 genetically modified mice models that can steadily and continuously express IE2 protein were established. Then, the mice were divided into the experimental group (positive mice identified) and the control group (wild-type mice, n=16 per group). The establishment of UL122 genetically modified mice was identified by PCR technology. The triglyceride content in their livers was measured using a colorimetric assay and oil red O-stain. Real-time PCR and immunohistochemistry were performed to detect the expression levels of SREBP1c mRNA and protein after HCMV infection. We found that SREBP1c expression was significantly elevated in the experimental group, and its overexpression in the liver cells can promote triglyceride accumulation and hepatic steatosis. Taken together, our data collectively demonstrate that HCMV infection is highly associated with NAFLD, SREBP1c overexpression promotes hepatic steatosis, and this up-regulation is most likely mediated by IE2.