Spherical nucleic acid enzyme programmed network to accelerate CRISPR assays for electrochemiluminescence biosensing applications.

Given the targeted binding ability and cleavage activity of the emerging CRISPR/Cas12a assay which transduces the target into its cleavage activity exhibited broadly prospective applications in integrated sensing and actuating system. Here, we elaborated a universal approach to quickly activate CRISPR/Cas12a for low-abundance biomarker detection based on the amplification strategy of a target-induced spherical nucleic acid enzyme (SNAzyme) network that could accelerate the output of activators. Specifically, multifunctional Y-shaped probes and hairpin probes (HPs, which contained the specific sequence of the activators of CRISPR/Cas12a and the substrate chain of DNAzyme) were rationally designed to construct SNAzyme. Target recognition induced disassembly of the Y-shaped probes, which released DNAzyme strands to active DNAzyme and accompanied by SNAzyme self-assembly into SNAzyme network. Interestingly, compared with randomly dispersed SNAzyme, the reaction kinetics of the SNAzyme network enhanced 1.6 times in response to Α-methyl acyl-CoA racemase (AMACR, a biomarker for prostate cancer), which was attributed to the promoted catalytic efficiency of DNAzyme by the confined SNAzyme network. Benefiting from these, the prepared biosensor based on electrochemiluminescence (ECL) platform by loading AuAg nanoclusters (AuAgNCs) into metal-organic framework-5 (MOF-5) exhibited satisfying detection performance for AMACR with a wide linear range (0.001 µg/mL to 100 µg/mL) and a low detection limit (1.0 × 10-4 µg/mL, which exhibited significant potential in clinical diagnoses.

Peritumoral Radiomics Strategy Based on Ensemble Learning for the Prediction of Gleason Grade Group of Prostate Cancer.

RATIONALE AND OBJECTIVES: To develop and evaluate a peritumoral radiomic-based machine learning model to differentiate low-Gleason grade group (L-GGG) and high-GGG (H-GGG) prostate lesions. MATERIALS AND METHODS: In this retrospective study, a total of 175 patients with prostate cancer (PCa) confirmed by puncture biopsy were recruited and included 59 patients with L-GGG and 116 patients with H-GGG. The original PCa regions of interest (ROIs) were delineated on T2-weighted (T2WI), diffusion-weighted imaging (DWI), and apparent diffusion coefficient (ADC) maps, and then centra-tumoral and peritumoral ROIs were defined. Features were meticulously extracted from each ROI to establish radiomics models, employing distinct sequence datasets. Peritumoral radiomics models were specifically developed for both the peripheral zone (PZ) and transitional zone (TZ), utilizing dedicated PZ and TZ datasets, respectively. The performances of the models were evaluated by using the receiver operating characteristic (ROC) curve and precision-recall curve. RESULTS: The classification model with combined peritumoral features based on T2 + DWI + ADC sequence dataset demonstrated superior performance compared to the original tumor and centra-tumoral classification models. It achieved an area under the ROC curve (AUC) of 0.850 [95% confidence interval, 0.849, 0.860] and an average accuracy of 0.950. The combined peritumoral model outperformed the regional peritumoral models with AUC of 0.85 versus 0.75 for PZ lesions and 0.88 versus 0.69 for TZ lesions, respectively. The peritumoral classification models exhibit greater efficacy in predicting PZ lesions as opposed to TZ lesions. CONCLUSION: The peritumoral radiomics features showed excellent performance in predicting GGG in PCa patients and might be a valuable addition to the non-invasive assessment of PCa aggressiveness.

Radiomics-Based Machine Learning Models for Predicting P504s/P63 Immunohistochemical Expression: A Noninvasive Diagnostic Tool for Prostate Cancer.

Objective: To develop and validate a noninvasive radiomic-based machine learning (ML) model to identify P504s/P63 status and further achieve the diagnosis of prostate cancer (PCa). Methods: A retrospective dataset of patients with preoperative prostate MRI examination and P504s/P63 pathological immunohistochemical results between June 2016 and February 2021 was conducted. As indicated by P504s/P63 expression, the patients were divided into label 0 (atypical prostatic hyperplasia), label 1 (benign prostatic hyperplasia, BPH) and label 2 (PCa) groups. This study employed T2WI, DWI and ADC sequences to assess prostate diseases and manually segmented regions of interest (ROIs) with Artificial Intelligence Kit software for radiomics feature acquisition. Feature dimensionality reduction and selection were performed by using a mutual information algorithm. Based on screened features, P504s/P63 prediction models were established by random forest (RF), gradient boosting decision tree (GBDT), logistic regression (LR), adaptive boosting (AdaBoost) and k-nearest neighbor (KNN) algorithms. The performance was evaluated by the area under the ROC curve (AUC) and accuracy. Results: A total of 315 patients were enrolled. Among the 851 radiomic features, the 32 top features were derived from T2WI, in which the gray-level run length matrix (GLRLM) and gray-level cooccurrence matrix (GLCM) features accounted for the largest proportion. Among the five models, the RF algorithm performed best in general evaluations (microaverage AUC=0.920, macroaverage AUC=0.870) and provided the most accurate result in further sublabel prediction (the accuracies of label 0, 1, and 2 were 0.831, 0.831, and 0.932, respectively). In comparative sequence analyses, T2WI was the best single-sequence candidate (microaverage AUC=0.94 and macroaverage AUC=0.78). The merged datasets of T2WI, DWI, and ADC yielded optimal AUCs (microaverage AUC=0.930 and macroaverage AUC=0.900). Conclusions: The radiomic-based RF classifier has the potential to be used to evaluate the presurgical P504s/P63 status and further diagnose PCa noninvasively and accurately.

Inhibition of Endoplasmic Reticulum Stress Apoptosis by Estrogen Protects Human Umbilical Vein Endothelial Cells Through the PI3 Kinase-Akt Signaling Pathway.

We aimed to investigate whether the cardioprotective effect of estrogen is mediated by inhibiting the apoptosis induced by endoplasmic reticulum stress (ERS) and to explore the underlying signaling pathway responsible for this effect. The effect of estrogen on ERS apoptosis, the mechanism responsible for that effect, and the ERS signaling pathways were examined in human umbilical vein endothelial cells (HUVECs) and measured using Western blot, Hoechst stains and caspase-3 activity assay. In vitro, 10-8 mol/l estrogen directly inhibited the up-regulation of the ERS marker glucose-regulated protein 78 (GRP78) and ERS apoptosis marker C/EBP homologous protein (CHOP). ERS was induced using the ERS inducer tunicamycin (TM, 10 µmol/l) or dithiothreitol (DTT, 2 mmol/l) in HUVECs. Estrogen can also decrease the apoptosis cells mediated by ERS, based on the results of Hoechst stains. Protein expression in the three main ERS signaling pathways was upregulated in TM- or DTT-induced HUVEC ERS. Increases in p-PERK/PERK were the most obvious, and estrogen significantly inhibited the upregulation of p-PERK/PERK, p-IRE1/IRE1, and ATF6. These inhibitory effects were abolished by specific estrogen receptor antagonists (ICI182, 780, and G15) and inhibitors of the E2 post-receptor signaling pathway, including phosphoinositide 3-kinase (PI3K) inhibitor LY294002, p38-mitogen activated protein kinase (p38-MAPK) inhibitor SB203580, c-Jun N-terminal kinase (JNK) inhibitor SP600125 and extracellular signal-regulated kinases1/2 (ERK1/2) inhibitor U0126; of these inhibitors, LY294002 was the most effective. Further experiments showed that when the PI3K pathway was blocked, the inhibitory effect of estrogen on ERS apoptosis was reduced. Estrogen can prevent HUVEC apoptosis by inhibiting the ERS apoptosis triggered by the PERK pathway, which may protect vascular endothelial cells and the cardiovascular system. The main mechanism responsible for ERS inhibition is the activation of the PI3K-Akt pathway for the activated estrogen receptor. J. Cell. Biochem. 118: 4568-4574, 2017. © 2017 Wiley Periodicals, Inc.

Electrochemiluminescence biosensor for determination of organophosphorous pesticides based on bimetallic Pt-Au/multi-walled carbon nanotubes modified electrode.

A novel and highly sensitive electrochemiluminescence (ECL) biosensing system was designed and developed for individual detection of different organophosphorous pesticides (OPs) in food samples. Bimetallic Pt-Au nanoparticles were electrodeposited on multi-walled carbon nanotubes (MWNTs)-modified glass carbon electrode (GCE) to increase the surface area of electrode and ECL signals of luminol. Biocomposites of enzymes from acetylcholinesterase and choline oxidase (AChE and ChOx) were immobilized onto the electrode surface to produce massive hydrogen peroxides (H2O2), thus amplifying ECL signals. Based on the dual-amplification effects of nanoparticles and H2O2 produced by enzymatic reactions, the proposed biosensor exhibits highly sensitivity. The proposed biosensing approach was then used for detecting OPs by inhibition of OPs on AChE. Under optimized experimental conditions, the ECL intensity decreased accordingly with the increase in concentration of OPs, and the inhibition rates of OPs were proportional to their concentrations in the range of 0.1-50nmolL(-1) for malathion, methyl parathion and chlorpyrifos, with detection limit of 0.16nmolL(-1), 0.09nmolL(-1) and 0.08nmolL(-1), respectively. The linearity range of the biosensor for pesticide dufulin varied from 50 to 500nmolL(-1), with the detection limit of 29.7nmolL(-1). The resulting biosensor was further validated by assessment of OPs residues in cabbage, which showed a fine applicability for the detection of OPs in the realistic sample.

Association of ABCB1, CYP3A4, EPHX1, FAS, SCN1A, MICA, and BAG6 polymorphisms with the risk of carbamazepine-induced Stevens-Johnson syndrome/toxic epidermal necrolysis in Chinese Han patients with epilepsy.

OBJECTIVE: This study explored the association between the risk of carbamazepine (CBZ)-induced Stevens-Johnson syndrome (SJS)/toxic epidermal necrolysis (TEN) and CBZ dose, dose-adjusted concentration, and ABCB1, CYP3A4, EPHX1, FAS, SCN1A, MICA, and BAG6 polymorphisms in patients of Han ethnicity with epilepsy who were living in northeastern China. MATERIALS AND METHODS: We determined the genotypes of patients with CBZ-SJS/TEN and CBZ-tolerant patients, who were used as controls, for ABCB1, CYP3A4, EPHX1, FAS, SCN1A, MICA, and BAG6 polymorphisms by polymerase chain reaction (PCR) amplification and direct sequencing. We measured the steady-state serum CBZ concentrations using fluorescence polarization immunoassay for the control patients. RESULTS: We observed statistically significant differences in EPHX1 c.337T>C polymorphisms between patients with CBZ-SJS/TEN and CBZ-tolerant controls in terms of allelic and genotypic frequencies (p = 0.011 and p = 0.007, respectively). The C allele and the C-G diplotype of EPHX1 may play important roles in increasing the risk of CBZ-SJS/TEN development (odds ratio [OR] 0.478, 95% confidence interval [CI] = 0.267-0.855, p = 0.011; OR = 0.213, 95% CI = 0.049-0.930, p = 0.025, respectively). We did not observe any significant associations between ABCB1, CYP3A4, EPHX1, FAS, SCN1A, MICA or BAG6 genes and CBZ dose or dose-adjusted concentration in CBZ-tolerant patients. SIGNIFICANCE: We found a significant association between EPHX1 c.337T>C polymorphisms and the development of CBZ-SJS/TEN in patients of Han ethnicity living in northeastern China. EPHX1 c.337T>C polymorphisms may contribute to the risk of severe CBZ-SJS/TEN by increasing the concentration of a CBZ metabolite, CBZ-10,11-epoxide, in patients with epilepsy.

Influence of ABCB1, CYP3A4*18B and CYP3A5*3 polymorphisms on cyclosporine A pharmacokinetics in bone marrow transplant recipients.

The aim of this study was to retrospectively evaluate the effect of polymorphisms in the CYP3A4, CYP3A5 and ABCB1 genes on the dose-adjusted concentration and dose requirement of cyclosporine A(CsA) in Chinese recipients during the early period after bone marrow or hematopoietic stem cell transplantation. Ninety-one bone marrow transplant recipients were genotyped by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay or by direct sequencing for the C1236T, G2677T/A and C3435T polymorphisms in CYP3A4*18B, CYP3A5*3, and ABCB1, respectively. The concentration at zero before administration (C0) and concentration at 2 h after administration (C2) of whole blood CsA were measured by fluorescence polarization immunoassay. Dose-adjusted C0 and C2 were determined and compared among groups with different genotypes. Compared with CYP3A5*3/*3 individuals, CYP3A5*1/*1 subjects have a significantly lower dose-adjusted C0 and C2 at days 1-10 and a higher dose requirement for CsA at days 16-30 (p < 0.05). In addition, homozygotes for the ABCB1 3435T mutant have a significantly higher dose-adjusted C0 and C2 and a lower dose requirement compared with wildtype (p < 0.05). Similar results were also derived for carriers of the T-G-C haplotype in CYP3A5 producers compared with non-carriers (p < 0.05 and p < 0.01, respectively). In summary, the ABCB1 3435T SNP, T-G-C haplotype in CYP3A5 producers, and CYP3A5*3 SNP are all associated with differences in CsA pharmacokinetics and dose requirements during the first month after bone marrow or hematopoietic stem cell transplantation. Genetic testing can therefore help to determine initial dosage and individualize immunosuppressive therapy.

Methotrexate ameliorates pristane-induced arthritis by decreasing IFN-γ and IL-17A expressions.

OBJECTIVE: This study was carried out to test the effects of methotrexate (MTX) and black seed oil (BSO) on pristane-induced arthritis (PIA) in rats. METHODS: Inbred dark agouti (DA) rats were induced by a single subcutaneous injection of pristane, and then treated with MTX or BSO. Arthritis severity was evaluated macroscopically and microscopically. Plasma nitric oxide (NO) concentration was determined by the Griess method and cytokine mRNA expression in the spleen was detected by the real-time reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The clinical arthritis severity was decreased after MTX treatment, while the BSO groups did not show significant changes compared with the disease group. The plasma NO level of the MTX group was significantly decreased compared with the disease group, but the BSO groups showed no difference from the disease group in plasma NO levels. The interferon-γ (IFN-γ) and interleukin-17A (IL-17A) mRNA expressions in the spleens were significantly decreased in the MTX group, but only showed a declining trend in the BSO groups compared with the disease group. Neither MTX nor BSO had an effect on the mRNA expressions of IL-4, transforming growth factor ß (TGF-ß), and tumor necrosis factor-α (TNF-α) in the spleen. CONCLUSIONS: MTX, but not BSO, can reduce the arthritis severity and decrease the mRNA expressions of IFN-γ and IL-17A in pristane-induced arthritis of rats.

[Changes of cell adhesion molecules and proinflammatory cytokines during cardiopulmonary bypass and the effect of intervention].

OBJECTIVE: To investigate the changes of the cell adhesion molecules and proinflammatory cytokines during cardiopulmonary bypass, and to observe the effect of intervention with ulinastatin. METHODS: Twenty-two ASA II-III patients (9 males, 13 females), aged 20-60 years, undergoing cardiac operation with CPB were randomly divided into 2 groups: the control group (Group C, n=11) and the ulinastatin group (Group W, n=11). In Group W, patient received ulinastatin 1.2 x 10(4) U/kg, and half of the dose was given intravenously after the induction of anesthesia, while the same amount of ulinastatin added into the primary solution. And in Group C, normal saline was given instead of ulinastatin. Blood samples were taken from radial artery before the operation (T1), 20 min after the initiation of CPB (T2), 1 h (T3), 6 h (T4 ), 24 h (T5) after the CPB for the determination of plasma TNF-alpha, IL-6, sICAM-1 and sP-Selectin concentrations. RESULTS: The concentrations of TNF-alpha, IL-6 increased significantly at T2-T4 in both groups compared with T1 (P < 0.05), and returned to the baseline level at Ts in Group W. The concentrations of TNF-alpha, IL-6 in Group C at T2-T5 were higher than that in Group W (P < 0.01). The concentrations of sICAM-1, sP-Selectin increased significantly at T3, T4 in both groups compared with that at T1 (P < 0.05). But at T5, the concentrations of sICAM-1, sP-Selectin decreased, especially in Group W the concentrations of sICAM-1, sP-Selectin returned to the baseline level. The sI-CAM-1, sP-Selectin concentrations in group C at T4, T5 were higher than that in group W (P < 0.05). CONCLUSION: Ulinastatin can reduce the increase of the cell adhesion molecules and proinflammatory cytokines during cardiopulmonary bypass and effectively weaken the inflammatory response to CPB.

[Effects of ulinastatin on cerebral inflammatory response during cardiopulmonary bypass].

OBJECTIVE: To investigate the effects of ulinastatin (UTI) on cerebral inflammatory response during cardiopulmonary bypass (CPB). METHODS: Twenty-four NYHA II-III patients (13 males and 11 females) aged 23-45 years, undergoing elective cardiac valve replacement under hypothermic CPB were randomly divided into 2 groups: ulinastatin group (Group U, n=12) and control group (Group C, n=12). In group U, UTI (1.2 x 10(4) U/kg) was given intravenously after the induction of anesthesia, 0.6 x 10(4) U/kg UTI was added to the priming solution, and 0.6 x 10(4) U/kg UTI was given about 5 min before the aortic decamping. In Group C, normal saline was given instead of UTI. Internal jugular vein was cannulated and the catheter was advanced retrogradely till jugular bulb. Blood samples were taken simultaneously from artery and jugular bulb after induction of anesthesia (T1), 60 min (T2) and 6 h (T3) after discontinuation of CPB for determination of TNFalpha, IL-6, IL-8 and IL-10. The juguloarterial gradients of these cytokines (deltaTNFalpha, deltaIL-6, deltaIL-8, and deltaIL-10) were calculated. RESULTS: In Group C, arterial levels of TNFalpha, IL-6, IL-8, IL-10 at T2 and T3, deltaTNFalpha, deltaIL-8 and deltaIL-10 at T2, deltaTNFalpha, deltaIL-6 and deltaIL-10 at T3 significantly increased (P < 0.01). deltaIL-8 increased at T3 (P < 0.05). In Group U, arterial levels of IL-6, IL-8, IL-10 at T2, arterial levels of IL-6, IL-8,IL-L-10 and deltaTNFalpha, deltaIL-8 at T3 significantly increased (P < 0.01). Arterial levels of TNFalpha at T2 and T3, deltaTNFalpha, deltaIL-10 at T2, deltaIL-6 at T3 increased (P < 0.05). Arterial levels of TNFalpha, IL-6 and deltaTNFalpha, deltaIL-8 at T2, arterial levels of TNFalpha and deltaIL-6 at T3 in Group U were lower than those in Group C (P < 0.05). Arterial levels of IL-6 at T3, IL-8 at T2 and T3 in Group U were significantly lower than those in Group C (P < 0.01). Arterial levels of IL-10 and deltaIL-10 at T3 in Group U were higher than those in Group C (P < 0.05). CONCLUSION: Systemic and cerebral activation of inflammatory response during CPB can be alleviated by ulinastatin.

[Effects of ulinastatin on erythrocyte lipid peroxidation in patients undergoing open heart surgery].

OBJECTIVE: To investigate the effects of ulinastatin on erythrocyte lipid peroxidation in patients undergoing open heart surgery. METHODS: Twenty adult patients with rheumatic heart disease undergoing elective value replacement were divided randomly into 2 groups of 10 patients each: a control group (group C) and an ulinastatin group (group W). The patients were premedicated with intramuscular morphine 0.08 mg/kg and scopolamine 0.06 mg/kg. Anesthesia was induced with midazolam 0.1 mg/kg, fentanyl 5 microg/kg and vecuronium 0.1 mg/kg. After the tracheal intubation, the patients were mechanically ventilated. Anesthesia was maintained with midazolam, fentanyl and isoflurane. Blood samples were taken from radial artery before the operation (T1), 30 min after the initiation of CPB (T2), at the end of the CPB (T3), 30 min after the aorta declamping (T4) and 24 h after the operation (T5) for the determination of plasma and erythrocyte MDA (P-MDA and E-MDA) and erythrocyte SOD (E-SOD). RESULTS: The levels of P-MDA and E-MDA increased significantly after the initiation of CPB and the level of E-SOD was higher than the baseline level at T2, and then decreased from T3 to T5 in group C (P <0.001). The levels of P-MDA and E-MDA didn't increased until T4 (P <0.001) and returned to the baseline level at T5 in group W. The levels of P-MDA and E-MDA were significantly higher in group C than those in group W and the level of E-SOD was markedly lower than that in group W (P <0.05). CONCLUSION: Ulinastatin can alleviate erythrocyte lipid peroxidation in patients undergoing open heart surgery.