Hapten synthesis, monoclonal antibody production and immunoassay development for analyzing spiropidion residues.

Spiropidion developed by Syngenta shows high insecticidal and acaricidal activity against a wide range of sucking pests. In this study, according to the structure of spiropidion, two haptens were synthesized by introducing carboxyl groups from the ester group. After cell fusion, a monoclonal antibody (mAb 8B5) of spiropidion was obtained. The IC50 of the established heterologous indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was 7.36 ng/mL, and its working range was 1.75-34.92 ng/mL. The average recoveries were 76.05-124.78% in the Yangtze River and citrus samples. Moreover, the ic-ELISA results of 15 citrus samples agreed well with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Overall, the established ic-ELISA could be applied for the spiropidion residue monitor in food and agricultural samples.

Preparation of limonin monoclonal antibody and establishment of a sensitive icELISA for analyzing limonin in citrus and herbal samples.

Limonin is an intensely bitter and highly oxidized tetracyclic triterpenoid secondary metabolite, which is abundant in the Rutaceae and Meliaceae, especially in Citrus. In order to detect limonin content in complex substrates such as citrus and traditional Chinese medicine, monoclonal antibodies specifically recognizing limonin were prepared and an indirect competitive enzyme-linked immunosorbent assay (icELISA) was established. The median inhibition concentration (IC50) was 5.40 ng/mL and the linear range was 1.25-23.84 ng/mL. The average recoveries from citrus peel and pulp samples were 95.9%-118.8% and 77.5%-113.1%, respectively. Moreover, the contents of limonin in 6 citrus samples and 4 herbal samples were analyzed by icELISA and UPLC-MS, and the results of the two methods were consistent. This validation is sufficient to demonstrate that the developed immunoassay is applicable for the detection of limonin in citrus and herbal samples and has the advantage of high efficiency, sensitivity, and convenience.

First experimental evaluation of a high-resolution deep silicon photon-counting sensor.

Purpose: Current photon-counting computed tomography detectors are limited to a pixel size of around 0.3 to 0.5 mm due to excessive charge sharing degrading the dose efficiency and energy resolution as the pixels become smaller. In this work, we present measurements of a prototype photon-counting detector that leverages the charge sharing to reach a theoretical sub-pixel resolution in the order of 1 µm. The goal of the study is to validate our Monte-Carlo simulation using measurements, enabling further development. Approach: We measure the channel response at the MAX IV Lab, in the DanMAX beamline, with a 35 keV photon beam, and compare the measurements with a 2D Monte Carlo simulation combined with a charge transport model. Only a few channels on the prototype are connected to keep the number of wire bonds low. Results: The measurements agree generally well with the simulations with the beam close to the electrodes but diverge as the beam is moved further away. The induced charge cloud signals also seem to increase linearly as the beam is moved away from the electrodes. Conclusions: The agreement between measurements and simulations indicates that the Monte-Carlo simulation can accurately model the channel response of the detector with the photon interactions close to the electrodes, which indicates that the unconnected electrodes introduce unwanted effects that need to be further explored. With the same Monte-Carlo simulation previously indicating a resolution of around 1 µm with similar geometry, the results are promising that an ultra-high resolution detector is not far in the future.

First experimental evaluation of count-rate performance for micrometre resolution deep silicon detector.

Objective.An ultra-fine-pitch deep silicon detector has been developed for clinical photon-counting computed tomography (CT). With a small pixel size of 14 × 650µm2, it has shown potential to reach micrometre spatial resolution in previous simulation studies. A detector prototype with such geometry has been manufactured, and we report on the first experimental evaluation of its count-rate performance.Approach.The measurement was carried out at MAX IV synchrotron laboratory with 35 keV monochromatic x-rays. By inserting tungsten attenuators of 50, 75, 100, 150, 200, 225, 325µm-thicknesses into the beam, the response of the detector to fluence rates from 3.3 × 107to 1.3 × 1011mm-2s-1was characterized.Main results.The measurement result showed that the detector exhibited count rate linearity up to 6.66 × 108mm-2s-1with 13% count loss and was still functional at count rate up to 2.9 × 1010mm-2s-1. A semi-nonparalyzable dead-time model was fitted to the count-rate behaviour of the detector, showing great agreement with the measured data, with an estimated nonparalyzable dead time of 2.9 ns.Significance.This is the first experimental evaluation of the count-rate performance for a deep silicon detector with such small pixel geometry. The results suggest that this type of detector shows the potential to be used at fluence rates encountered in clinical CT with little count loss due to pile-up.

CRTAC1 enhances the chemosensitivity of non-small cell lung cancer to cisplatin by eliciting RyR-mediated calcium release and inhibiting Akt1 expression.

Sensitivity to platinum-based combination chemotherapy is associated with a favorable prognosis in patients with non-small cell lung cancer (NSCLC). Here, our results obtained from analyses of the Gene Expression Omnibus database of NSCLC patients showed that cartilage acidic protein 1 (CRTAC1) plays a role in the response to platinum-based chemotherapy. Overexpression of CRTAC1 increased sensitivity to cisplatin in vitro, whereas knockdown of CRTAC1 decreased chemosensitivity of NSCLC cells. In vivo mouse experiments showed that CRTAC1 overexpression increased the antitumor effects of cisplatin. CRTAC1 overexpression promoted NFAT transcriptional activation by increasing intracellular Ca2+ levels, thereby inducing its regulated STUB1 mRNA transcription and protein expression, accelerating Akt1 protein degradation and, in turn, enhancing cisplatin-induced apoptosis. Taken together, the present results indicate that CRTAC1 overexpression increases the chemosensitivity of NSCLC to cisplatin treatment by inducing Ca2+-dependent Akt1 degradation and apoptosis, suggesting the potential of CRTAC1 as a biomarker for predicting cisplatin chemosensitivity. Our results further reveal that modulating the expression of CRTAC1 could be a new strategy for increasing the efficacy of cisplatin in chemotherapy of NSCLC patients.

Preparation of spirodiclofen monoclonal antibody and establishment of indirect competitive enzyme-linked immunosorbent assay.

Spirodiclofen, a spirocyclic tetronic acid derivative, has excellent acaricidal effect and is used worldwide to control the majority of important mite species. For monitoring its residue in food and environmental samples, two haptens containing different spacer arms were synthesized, a monoclonal antibody (mAb 5A4) against spirodiclofen was prepared, and a heterologous indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was established. The 50% inhibition concentration (IC50) of ic-ELISA was 25.46 ng/mL, and the working range was 5.59-133.85 ng/mL. The ic-ELISA showed no cross-reactivity with structural analogs of spirodiclofen and other commonly-used acaricides. The average recoveries from Shiranui citrus samples and Yangtze River water were 85.62%-97.74% and 85.95%-99.30%, respectively. In the analysis of 12 citrus samples, the results of the ic-ELISA were quite similar to those of ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Hence, the new immunosorbent assay provides a substitute method for the qualitative and quantitative of spirodiclofen in food and environmental samples.

Downregulation of microRNA-6125 promotes colorectal cancer growth through YTHDF2-dependent recognition of N6-methyladenosine-modified GSK3ß.

BACKGROUND: MicroRNAs (miRNAs), the key regulator of gene expression, and N6-methyladenosine (m6A) RNA modification play a significant role in tumour progression. However, regulation of m6A-modified mRNAs by miRNAs in colorectal cancer (CRC), and its effect on progression of CRC, remains to be investigated. METHODS: Expression of miR-6125 and YTH Domain-Containing Family Protein 2 (YTHDF2) was detected by western blotting and immunohistochemistry. The effects of miR-6125 and YTHDF2 on proliferative capacity of CRC cells were analysed using soft agar, ATP, CCK8 and EdU assays, and in animal experiments. RESULTS: MiR-6125 expression was downregulated markedly in CRC, and expression correlated negatively with tumour size and prognosis. MiR-6125 targeted the 3'-UTR of YTHDF2 and downregulated the YTHDF2 protein, thereby increasing the stability of m6A-modified glycogen synthase kinase 3 beta (GSK3ß) mRNA. Increased GSK3ß protein levels inhibited the expression of Wnt/ß-catenin/Cyclin D1 pathway-related proteins, leading to G0-G1 phase arrest and ultimately inhibiting the proliferation of CRC cells. CONCLUSIONS: MiR-6125 regulates YTHDF2 and thus plays a critical role in regulating the Wnt/ß-catenin pathway, thereby affecting the growth of CRC. Collectively, these results suggest that miR-6125 and YTHDF2 are potential targets for treatment of CRC.

Coiled-Coil Domain-Containing 68 Downregulation Promotes Colorectal Cancer Cell Growth by Inhibiting ITCH-Mediated CDK4 Degradation.

Coiled-coil domain-containing 68 (CCDC68) plays different roles in cancer and is predicted as a tumor suppressor in human colorectal cancer (CRC). However, the specific role of CCDC68 in CRC and the underlying mechanisms remain unknown. Here, we showed that CCDC68 expression was lower in CRC than that in corresponding normal tissues, and CCDC68 level was positively correlated with disease-free survival. Ectopic expression of CCDC68 decreased CRC cell proliferation in vitro and suppressed the growth of CRC xenograft tumors in vivo. CCDC68 caused G0/G1 cell cycle arrest, downregulated CDK4, and upregulated ITCH, the E3 ubiquitin ligase responsible for CDK4 protein degradation. This increased CDK4 degradation, which decreased CDK4 protein levels and inhibited CRC tumor growth. Collectively, the present results identify a novel CDK4 regulatory axis consisting of CCDC68 and ITCH, which suggest that CCDC68 is a promising target for the treatment of CRC.