Binary optical encoding strategy for multiplex assay.

A binary optical encoding strategy is proposed to meet the increasing requirements of multiplex bioassays. As illustrated in fluorescence immunodetection of multiplex antigen molecules, photonic crystal beads (PCBs) and quantum dots (QDs) can be used as biomolecular microcarriers and fluorescence labels, respectively. The categories of antigens were deciphered by the binary combination of optical spectra of PCBs and QDs as independent encoding elements. The number of categories that could be detected was theoretically m × n, where m and n represent the number of encoding PCBs and QDs, respectively. In addition, the concentrations of the antigens were determined by the fluorescence signals of the QDs. Results of sensitivity analysis indicate that a low-level detection of 58 pg/mL was achieved. Because of the special nanostructures of these two encoding elements, the binary encoding strategy demonstrated its superiority and practicability when compared with single PCB or QD encoding. This supports potential application in multiplex bioassays.

miR-497-5p induces apoptosis in K562 cells by downregulating ROCK1.

OBJECTIVE: To validate the role of miR-497-5p in apoptosis in K562 cells by targeting Rho-associated kinase isoform 1 (ROCK1). METHODS: From January, 2017 to February, 2019, 57 patients with chronic myeloid leukemia (CML) treated in our hospital were included in patient group, and 50 healthy individuals were recruited as control group. miR-497-5p level in peripheral blood was quantitated using qRT-PCR. After transfecting with miR-497-5p overexpression vector and ROCK1 inhibitor, K562 cells were monitored in terms of proliferation (CCK8 assay), migration and invasion (Transwell), and apoptosis (flow cytometry). Binding loci between miR-497-5p and ROCK1 were predicted, and the targeting relationship was confirmed (dual-luciferase reporter (DLR) assay). RESULTS: miR-497-5p was poorly expressed in CML (P < 0.05). Forced overexpression of miR-497-5p or inhibition of ROCK1 suppressed malignant processes (proliferation, proliferation, migration and invasion) in K562 cells and induced apoptosis (P < 0.05). DLR assay revealed a decreased luciferase activity after miR-497-5p binding to ROCK1 (P < 0.05). CONCLUSION: miR-497-5p induces apoptosis in K562 cells by downregulating ROCK1.

miR-506 in patients with chronic myeloid leukemia and its effect on apoptosis of K562 cells.

OBJECTIVE: To explore the expression of miR-506 in chronic myeloid leukemia (CML) and its influence on the biological function of CML cells. METHODS: Altogether 84 CML patients from February 2012 to September 2014 were obtained as the observation group (OG), and 71 healthy people were taken as the control group (CG). miR-506 was tested using RT-qPCR, and the 5-year survival of patients with high and low expression of miR-506 was compared with the median value of miR-506 as the limit. ROC curve was applied to detect the value of miR-506 in diagnosing CML and predicting the 5-year survival of patients, and K562 cell line was transfected with miR-506 inhibitor and miR-506 mimic for observing its effects on the cell proliferation and apoptosis. RESULTS: miR-506 in CML patients was evidently lower than that in healthy people, the AUC of diagnosis of miR-506 was 0.883, the total survival of patients with low miR-506 was evidently lower than those with high miR-506, and the AUC of predicted survival of patients was 0.778. The proliferation of cells transfected with miR-506 inhibitor was promoted, the apoptosis and the survival rate reduced. CONCLUSION: miR-506 is evidently reduced in CML, and may be applied as a diagnostic and predictive treatment for CML and 5-year related survival; it can also can hinder the viability of K562 cells and promote apoptosis.

Study on the relationship between G1057D variants of IRS2 gene and obese T2DM in Chinese Han subjects.

OBJECTIVE: To investigate the relationship between the G1057D variants of insulin receptor substrate-2(IRS2) gene and type 2 diabetes mellitus (T2DM) in subjects. METHODS: Four hundred and thirty-nine Chinese Han subjects, including 218 patients with T2DM and 221 normal controls, were selected from the Hans in the Liaoning area, and each group was divided into two subgroups according to body mass index. The G1057D variants of IRS2 were detected by polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) and their relationships with T2DM were analyzed. RESULTS: (1) The frequency of G1057D variant was 29% in all subjects. The frequency of DD genotype was significantly lower in non-obese DM group than in non-obese control group. The Logistic regression analysis showed that the odds ratio of DD genotype was 0.265. The frequency of DD genotype was significantly higher in obese DM group than in obese control group. The Logistic regression analysis showed that the odds ratio of DD genotype was 3.991. (2) In the non-obese control group, the FPG and 2hCP of DD genotypes were lower than those of GG genotypes (P< 0.05, P< 0.01), the HOMA-B of DD genotypes was higher than that of GG genotype (P< 0.01). In the non-obese DM group, the waistline/hip ratio (WHR) of DD genotypes was higher than that of GG genotypes(P< 0.01). In the obese DM group, the WHR, HOMA-IR, 2hPG, 2hINS and 2hCP levels of DD genotypes were higher than those of GG genotypes, while the level of HOMA-B of DD genotypes was lower than that of GG genotypes. In the obese control group, the WHR, HOMA-IR, 2hPG, 2hINS and 2hCP levels of DD genotype were higher than those of GG genotype, and the HOMA-B level of DD genotype was lower than that of GG genotypes (P< 0.05). CONCLUSION: The relationships between G1057D variants of IRS2 and T2DM are mediated by obesity.

Interaction between Rice stripe virus disease-specific protein and host PsbP enhances virus symptoms.

Rice stripe virus (RSV) causes severe diseases in Oryza sativa (rice) in many Eastern Asian countries. Disease-specific protein (SP) of RSV is a non-structural protein and its accumulation level in rice plant was shown to determine the severity of RSV symptoms. Here, we present evidence that expression of RSV SP alone in rice or Nicotiana benthamiana did not produce visible symptoms. Expression of SP in these two plants, however, enhanced RSV- or Potato virus X (PVX)-induced symptoms. Through yeast two-hybrid screening, GST pull-down, and bimolecular fluorescence complementation assays, we demonstrated that RSV SP interacted with PsbP, a 23-kDa oxygen-evolving complex protein, in both rice and N. benthamiana. Furthermore, our investigation showed that silencing of PsbP expression in both plants increased disease symptom severity and virus accumulation. Confocal microscopy using N. benthamiana protoplast showed that PsbP accumulated predominantly in chloroplast in wild-type N. benthamiana cells. In the presence of RSV SP, most PsbP was recruited into cytoplasm of the assayed cells. In addition, accumulation of SP during RSV infection resulted in alterations of chloroplast structure and function. Our findings shed light on the molecular mechanism underlying RSV disease symptom development.

Broad bean wilt virus 2 encoded VP53, VP37 and large capsid protein orchestrate suppression of RNA silencing in plant.

Viruses encode RNA silencing suppressors to counteract host RNA silencing-mediated defense responses. In this study, we demonstrate that VP53, VP37 and LCP encoded by RNA2 of broad bean wilt virus 2 (BBWV-2), a member of the genus Fabavirus, are strong suppressors of RNA silencing triggered by single-stranded sense RNA. They, however, had no effect on suppression of RNA silencing induced by double-stranded RNA. We provide evidence that these three suppressors can significantly limit the accumulation of small interfering RNAs (siRNAs) in tissues where the GFP gene has been silenced, and prevent the long distance spread of the induced silencing signal. Gel mobility shift assays showed that all three suppressors could bind ssRNA in a size-specific manner. Interestingly, VP37 and LCP, but not VP53, could reverse the silencing of a GFP gene in leaf tissue. Furthermore, these three proteins are capable of enhancing pathogenicity of potato virus X. Collectively, our findings indicate that viruses employ a more sophisticated strategy to overcome the host defense response mediated through suppression of RNA silencing during virus infection. As far as we are aware, this is the first report of RNA silencing suppressors encoded by a virus in the genus Fabavirus.

Cerium oxide nanoparticles induce cytotoxicity in human hepatoma SMMC-7721 cells via oxidative stress and the activation of MAPK signaling pathways.

BACKGROUND: Lanthanide cerium oxide (CeO2) nanoparticles have extensive applications in industrial fields, and concerns regarding their potential toxicity in humans and their environmental impact have increased. We investigated the underlying molecular mechanisms by which CeO2 nanoparticles induce toxicity in human hepatoma SMMC-7721 cells. RESULTS: Our results demonstrated that CeO2 nanoparticles reduced viability, caused dramatic morphological damage, and induced apoptosis in SMMC-7721 cells. CeO2 nanoparticles significantly increased the production of reactive oxygen species (ROS) and malondialdehyde (MDA), and significantly reduced the activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-px) and catalase (CAT). The phosphorylation levels of ERK1/2, JNK and p38 MAPK were significantly elevated after treatment with CeO2 nanoparticles. Pretreatment with the antioxidant N-acetyl-cysteine (NAC): reduced the induction of ROS and MDA by CeO2 nanoparticles; recovered the activity of SOD, GSH-px and CAT; reduced the phosphorylation levels of ERK1/2, JNK and p38; and attenuated CeO2 nanoparticles-induced damage and apoptosis in SMMC-7721 cells. CONCLUSIONS: Our data demonstrated that CeO2 nanoparticles induced damage and apoptosis in human SMMC-7721 cells via oxidative stress and the activation of MAPK signaling pathways.