Enzyme Cascade Amplification-Based Immunoassay Using Alkaline Phosphatase-Linked Single-Chain Variable Fragment Fusion Tracer and MnO2 Nanosheets for Detection of Deoxynivalenol in Corn Samples.

Deoxynivalenol (DON) is a common mycotoxin that contaminates cereals. Therefore, the development of sensitive and efficient detection methods for DON is essential to guarantee food safety and human health. In this study, an enzyme cascade amplification-based immunoassay (ECAIA) using a dual-functional alkaline phosphatase-linked single-chain fragment variable fusion tracer (scFv-ALP) and MnO2 nanosheets was established for DON detection. The scFv-ALP effectively catalyzes the hydrolysis of ascorbyl-2-phosphate (AAP) to produce ascorbic acid (AA). This AA subsequently interacts with MnO2 nanosheets to initiate a redox reaction that results in the loss of oxidizing properties of MnO2. In the absence of ALP, MnO2 nanosheets can oxidize 3,3',5,5'-tetramethylbenzidine (TMB) to produce the blue oxidized product of TMB, which exhibits a signal at a wavelength of 650 nm for quantitative analysis. After optimization, the ECAIA had a limit of detection of 0.45 ng/mL and a linear range of 1.2-35.41 ng/mL. The ECAIA exhibited good accuracy in recovery experiments and high selectivity for DON. Moreover, the detection results of the actual corn samples correlated well with those from high-performance liquid chromatography. Overall, the proposed ECAIA based on the scFv-ALP and MnO2 nanosheets was demonstrated as a reliable tool for the detection of DON in corn samples.

Generation of a nanobody-alkaline phosphatase fusion and its application in an enzyme cascade-amplified immunoassay for colorimetric detection of alpha fetoprotein in human serum.

Sensitive detection of liver disease biomarkers can facilitate the diagnosis of primary hepatoma and other benign liver diseases, and the alpha fetoprotein (AFP) was selected as the model macromolecule in this work. Herein an enzyme cascade-amplified immunoassay (ECAIA) based on the nanobody-alkaline phosphatase fusion (Nb-ALP) and MnO2 nanoflakes was developed for detecting AFP. The bifunctional biological macromolecule Nb-ALP serves as the detection antibody and the reporter molecule. The MnO2 nanoflakes mimic the oxidase for catalyzing the 3,3',5,5'-tetramethylbenzidine (TMB) into the blue oxidized TMB, which has a quantitative signal at the wavelength of 650 nm. Moreover, the Nb-ALP could dephosphorylate the ascorbic acid-2-phosphate (AAP) to form the ascorbic acid (AA) that can disintegrate the nanoflakes to reduce their oxidation capacity with the content decrease of the oxidized TMB. Using the constructed TMB-MnO2 colorimetric sensing system for Nb-ALP and the optimized experimental parameters, the ECAIA has a limit of detection (LOD) of 0.148 ng/mL which is 18.7-fold lower than that of the p-nitrophenylphosphate (pNPP)-based method (LOD = 2.776 ng/mL). The ECAIA showed good selectivity for AFP with observed negligible cross-reactions with several common cancer biomarkers. The recovery rate for AFP spiked in human serum ranged from 94.8% to 113% with the relative standard deviation from 0.3% to 6.5%. For analysis of the actual human serum samples, a good linear correlation was found between the results tested by the ECAIA and the automatic chemiluminescence analyzer. Thus, the ECAIA was demonstrated to be a promising tool for highly sensitive and selective detection of AFP, providing a reference for analysis of other macromolecule biomarkers.

LncRNA-mediated ceRNA network was identified as a crucial determinant of differential effects in periodontitis and periimplantitis by high-throughput sequencing.

BACKGROUND AND OBJECTIVE: Although periimplantitis and periodontitis share similar features, particularly clinical features, they are two different diseases and should be analyzed separately. Thus far, few omics-level differences in periimplantitis and periodontitis have been reported. This study was aimed at exploring the differential effects of expression mRNAs, lncRNAs, and miRNAs in periodontitis and periimplantitis by high-throughput sequencing and competitive endogenous RNA (ceRNA) analysis. METHODS: Gingival tissues of healthy individuals (HI) and periimplantitis (PI) and periodontitis (P) patients were collected and used for genome-wide sequencing. The differentially expressed genes (DEGs) were screened and visualized by R software. The functions and pathways of DEGs were analyzed using Metascape, and the ceRNA network was constructed using the Cytoscape software. Finally, gene set enrichment analysis (GSEA) was used to predict the function of key nodes in ceRNA. RESULTS AND CONCLUSION: By constructing the regulated ceRNA network, six genes (FAM126B, SORL1, PRLR, CPEB2, RAP2C, and YOD1) and 16 miRNAs (hsa-miR-338-5p, hsa-miR-650, hsa-miR-9-5p, hsa-miR-1290, hsa-miR-544a, hsa-miR-3179, hsa-miR-1269a, hsa-miR-3679-5p, hsa-miR-149-5p, hsa-miR-615-3p, hsa-miR-33b-5p, hsa-miR-31-5p, hsa-miR-4639-5p, hsa-miR-204-5p, hsa-miR-5588-5p, and hsa-mir-196a-5p) were detected. Five long non-coding RNAs (lnc-CORO2B-1, lnc-MBL2-7, lnc-TRIM45-1, lnc-CHST10-2, and lnc-TNP1-6) were found to target these miRNAs in this ceRNA network. The ceRNA network based on transcriptome data revealed that FAM126B, SORL1, PRLR, CPEB2, RAP2C, and YOD1 were crucial proteins of differential effects in periodontitis and periimplantitis. The lncRNA-miRNA-mRNA interaction involved the regulation of the Hippo signaling pathway, Wnt signaling pathway, Toll-like receptor signaling pathway, NOD signaling pathway, oxidative stress, and innate immune process. These regulated pathways and biological processes may be factors contributing to the pathogenesis of periimplantitis being distinct from that of periodontitis.

Long Noncoding RNA DLX6-AS1 Promotes the Progression in Cervical Cancer by Targeting miR-16-5p/ARPP19 Axis.

Background: Cervical cancer (CC) is regarded as one of the most common gynecological malignancies. LncRNA DLX6-AS1 has been proven vital in various cancers, whereas its exact function is still largely unestablished in CC. Materials and Methods: The expression pattern of DLX6-AS1 and miR-16-5p in CC cells was determined by real-time quantitative polymerase chain reaction (RT-qPCR). ARPP19 expression was assessed by RT-qPCR and Western blot assays in CC cells. The precise function of DLX6-AS1 in CC was detected by Cell-Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), caspase-3 activity, transwell, and Western blot experiments. RNA immunoprecipitation (RIP) and luciferase reporter assays were employed to certify the combination between miR-16-5p and DLX6-AS1 (or ARPP19). Nuclear cytoplasmic segmentation determined the localization of DLX6-AS1 in CC cells. A xenograft mouse model assay studied the influences of DLX6-AS1 silencing on CC progression in vivo. Results: Elevated DLX6-AS1 expression was disclosed in CC cells. DLX6-AS1 silence attenuated proliferation, migration, and epithelial-mesenchymal transition program as well as enhanced CC cell apoptosis. DLX6-AS1 was uncovered to sponge and negatively modulate miR-16-5p in CC. Besides, ARPP19 was uncovered as a downstream target gene of miR-16-5p in CC. Rescue experiments indicated that DLX6-AS1 enhanced the cellular process of CC cells through upregulating ARPP19. Moreover, in vivo assay confirmed that DLX6-AS1 promoted CC growth. Conclusions: DLX6-AS1 accelerates the progression of CC through sponging miR-16-5p and upregulates ARPP19, which offers a novel insight into prognosis and remedy of CC.

Development of a horseradish peroxidase-nanobody fusion protein for visual detection of ochratoxin A by dot immunoassay.

Ochratoxin A (OTA) is a common cereal mycotoxin that seriously threatens food safety and public health. Herein a horseradish peroxidase-nanobody fusion protein (HRP-Nb) retaining antibody and enzyme activity was obtained after inclusion body denaturation and renaturation and enzyme reconstitution, which served both as the primary antibody and reporter enzyme and was applied to develop a membrane-based dot immunoassay (HN-DIA) for OTA visual detection. Based on the optimal experimental conditions, the HN-DIA could be finished in 10 min with a cut-off limit of 50 µg kg-1 in rice and oat samples by eye. The HN-DIA showed high selectivity for OTA and had good accuracy and reproducibility in the recovery experiments. Spiked sample analysis results of the HN-DIA and high performance liquid chromatography (HPLC) correlated well with each other. Therefore, the proposed HN-DIA has the potential for rapid screening of OTA and other small molecule pollutants in food and the environment by naked eye.

Dual blocking of PI3K and mTOR signaling by NVP-BEZ235 inhibits proliferation in cervical carcinoma cells and enhances therapeutic response.

NVP-BEZ235 is a novel dual PI3K/mTOR inhibitor that shows dramatic effects on many tumors, but its effects on cervical carcinoma cells are largely unknown. In the present study, we investigated the effects of NVP-BEZ235 on the proliferation and invasion of cervical carcinoma cells in vitro and clarified its mechanism of action. In cellular settings with human cervical carcinoma cell lines, this molecule effectively and specifically blocked dysfunctional PI3K/mTOR pathway activation, suppressed cell growth in a time- and concentration-dependent manner, led to G1 cell cycle arrest, and induced apoptosis. NVP-BEZ235 suppressed HeLa cell invasiveness and metastasis by inhibiting the PI3K/Akt/MMP-2 pathway. We further demonstrated that NVP-BEZ235 treatment in combination with cisplatin or carboplatin induced a synergistic anti-tumoral response in cervical carcinoma cells. These findings suggested that NVP-BEZ235 could regulate growth and invasion of cervical carcinoma cells; thus it may provide a potential therapy for cervical carcinoma.

Temozolomide in combination with metformin act synergistically to inhibit proliferation and expansion of glioma stem-like cells.

Glioblastoma is the most common and most aggressive brain tumor in adults. The introduction of temozolomide (TMZ) has advanced chemotherapy for malignant gliomas, but it is not curative. The difficulties in treating glioblastoma may be as a result of the presence of glioma stem cells (GSCs), which are a source of relapse and chemoresistance. Another reason may be that endogenous Akt kinase activity may be activated in response to clinically relevant concentrations of TMZ. Akt activation is correlated with the increased tumorigenicity, invasiveness and stemness of cancer cells and overexpression of an active form of Akt increases glioma cell resistance to TMZ. Mounting evidence has demonstrated that cancer stem cells are preferentially sensitive to an inhibitor of Akt and down-regulation of the PI3K/Akt pathway may enhance the cytotoxicity of TMZ. Metformin (MET), the first-line drug for treating diabetes, it has been proved that it reduces AKT activation and selectively kills cancer stem cells, but whether it can potentiate the cytotoxicity of TMZ for GSCs remains unknown. In the present study, the GSCs isolated from human glioma cell line U87 and Rat glioma cell line C6, in vitro treatment with TMZ either alone or with MET. The present study demonstrates that MET acts synergistically with TMZ in inhibiting GSCs proliferation and generating the highest apoptotic rates when compared to either drug alone. These findings implicate that GSCs cytotoxicity mediated by TMZ may be stimulated by MET, have a synergistic effect, but the definite mechanisms remain elusive.

Giant recurrent dumbbell-shaped hypoglossal schwannoma in an elderly male: A case report.

Dumbbell-shaped hypoglossal Schwannomas of the 12th cranial nerve are extremely rare, and complete removal of these tumors is difficult, particularly in elderly patients with recurrent tumors. The present study reports the case of a 61-year-old male with a giant recurrent dumbbell-shaped hypoglossal schwannoma that arose extracranially. The recurrent tumor was completely removed in a one-stage surgical procedure via the far lateral suboccipital approach in combination with the transcervical approach. To the best of our knowledge, such a lesion has not been reported previously. The life expectancy and natural course of the disease are important factors to take into account when considering the individual end-point of surgery in patients. More studies on hypoglossal schwannomas are required, particularly cases in which the hypoglossal schwannoma was not totally resected, not only in order to develop more definitive and secure surgical treatments, but also to reduce the resultant unnecessary suffering of patients.

Efficacy and safety of bevacizumab for the treatment of glioblastoma.

Glioblastoma (GBM) is the most common and devastating primary malignant intracranial tumor in adults. The current first-line treatment for patients with newly diagnosed GBM is surgical resection followed by radiotherapy plus concomitant and adjuvant temozolomide. This treatment protocol may prolong the survival period of the patient, however it is not curative and more effective therapeutic strategies are required. GBM is a type of highly vascularized tumor with increased expression levels of vascular endothelial growth factor (VEGF), which is a significant mediator of angiogenesis. Since angiogenesis is essential for tumor growth, anti-angiogenic therapies hold potential for the treatment of GBM, and targeting VEGF has demonstrated promising results in previous studies. Bevacizumab (BEV) is a recombinant humanized monoclonal antibody that inhibits VEGF and is approved by the US Food and Drug Administration as a monotherapy treatment for patients with recurrent GBM and is associated with manageable toxicity. Previous studies have demonstrated that BEV may be an effective treatment for recurrent GBM, with prolonged progression-free survival and overall survival, and maintained patient quality of life and functional status. The present review article briefly outlines the mechanism of action of BEV and summarizes the current literature and clinical trial research on the role of BEV for the treatment of patients with recurrent and newly diagnosed GBM.

Metformin and temozolomide act synergistically to inhibit growth of glioma cells and glioma stem cells in vitro and in vivo.

Glioblastoma (GBM) is the most frequent and aggressive brain tumor in adults. In spite of advances in diagnosis and therapy, the prognosis of patients with GBM has remained dismal. The fast recurrence and multi-drug resistance are some of the key challenges in combating brain tumors. Glioma stem cells (GSCs) which are considered the source of relapse and chemoresistance, the need for more effective therapeutic options is overwhelming. In our present work, we found that combined treatment with temozolomide (TMZ) and metformin (MET) synergistically inhibited proliferation and induced apoptosis in both glioma cells and GSCs. Combination of TMZ and MET significantly reduced the secondary gliosphere formation and expansion of GSCs. We first demonstrated that MET effectively inhibited the AKT activation induced by TMZ, and a combination of both drugs led to enhanced reduction of mTOR, 4EBP1 and S6K phosphorylation. In addition, the combination of the two drugs was accompanied with a powerful AMP-activated protein kinase (AMPK) activation, while this pathway is not determinant. Xenografts performed in nude mice demonstrate in vivo demonstrated that combined treatment significantly reduced tumor growth rates and prolonged median survival of tumor-bearing mice. In conclusion, TMZ in combination with MET synergistically inhibits the GSCs proliferation through downregulation of AKT-mTOR signaling pathway. The combined treatment of two drugs inhibits GSCs self-renewal capability and partly eliminates GSCs in vitro and in vivo. This combined treatment could be a promising option for patients with advanced GBM.

NVP-BEZ235, a novel dual PI3K-mTOR inhibitor displays anti-glioma activity and reduces chemoresistance to temozolomide in human glioma cells.

Glioblastoma multiforme (GBM) is the most frequent and most aggressive brain tumor in adults. The introduction of temozolomide (TMZ) has advanced chemotherapy for malignant gliomas. However, a considerable number of GBM cases are refractory to TMZ, the need for more effective therapeutic options is overwhelming. Mounting evidence shows that endogenous AKT (protein kinase B) activity can be activated in response to clinically relevant concentrations of TMZ. AKT activation correlated with the increased tumorigenicity, invasiveness and stemness and overexpression of an active form of AKT increases glioma cell resistance to TMZ. Previous studies also show that TMZ contributes to glioma cell apoptosis by inhibiting mTOR signaling. Thus, we hypothesized that the dual PI3K-mTOR inhibitor NVP-BEZ235 may act as antitumor agent against gliomas and potentiate the cytotoxicity of TMZ. In the present study, we found that NVP-BEZ235 treatment of glioma cell lines led to G1 cell cycle arrest, and induced apoptosis. Combination treatment with both TMZ and NVP-BEZ235 resulted in synergistically inhibited glioma cell growth and induced apoptosis (combination index CI<1) in a subset of glioma cell lines, as shown in the increased levels of Bax, and active Caspase-3, and decreased level of Bcl-2. Furthermore, NVP-BEZ235 treatment reversed p-AKT levels enhanced by TMZ. Inhibition of mTOR (p70S6K) signaling with the combination of TMZ and NVP-BEZ235 can be augmented beyond that achieved using each agent individually. In vivo xenograft models in mice, the combinatorial treatment with TMZ and NVP-BEZ235 significantly reduced tumor growth rates and prolonged median survival of tumor-bearing mice. These findings exhibit that TMZ in combination with NVP-BEZ235 act synergistically to inhibit proliferation of glioma cells by down-regulating of the PI3K-AKT-mTOR pathway, suggesting TMZ and NVP-BEZ235 combination therapy may be an option for GBM treatment.