Establishment of genomic RNA reference materials for BCR-ABL1 P210 measurement.

Quantitation of BCR-ABL1 with the quantitative reverse transcriptase polymerase chain reaction (RT-PCR) is very important in monitoring chronic myeloid leukemia (CML), which relies on an RNA reference material. A genomic RNA reference material (RM) containing the BCR-ABL1 P210 fusion mutation was developed, and an absolute quantitative method based on one-step reverse transcription digital PCR (RT-dPCR) was established for characterizing the RM. The proposed dPCR method demonstrates high accuracy and excellent analytical sensitivity, as shown by the linear relationship (0.94 < slope < 1.04, R2≧0.99) between the measured and nominal values of b2a2, b3a2, and ABL1-ref within the dynamic range (104-101 copies/reaction). Homogeneity and stability assessment based on dPCR indicated that the RM was homogeneous and stable for 24 months at -80 °C. The RM was used to evaluate inter-laboratory reproducibility in eight different laboratories, demonstrating that participating laboratories could consistently produce copy concentrations of b3a2 and ABL1-ref, as well as the BCR-ABL1/ABL1 ratio (CV < 2.0%). This work suggests that the RM can be employed in establishing metrological traceability for detecting mutations in the BCR-ABL1 fusion gene, as well as in quality control for testing laboratories.

Rapid detection of trace Salmonella in milk and chicken by immunomagnetic separation in combination with a chemiluminescence microparticle immunoassay.

Rapid detection of trace Salmonella is urgently needed to ensure food safety. We present an innovative pretreatment strategy, based on a two-step enrichment culture and immunomagnetic separation, combined with a chemiluminescence microparticle immunoassay to detect at least one proliferative Salmonella cell in 25 mL (25 g) food. The capture performance of immunomagnetic beads (IMBs) of sizes for Salmonella was investigated, and the IMBs of size 2.8 µm showed a high capture efficiency of 60.7% in 25 mL milk and 74.5% in 25 mL chicken culture filtrate, which ensured the successful capture of trace Salmonella after 2.5 h in situ enrichment even from only one Salmonella cell. The separated Salmonella cells, reaching an amount of 103 colony-forming units (CFU) by a secondary enrichment for 3 h, were detected by a horseradish peroxidase chemiluminescence reaction with 4-(1-imidazolyl)phenol as an enhancer, which evidenced a linear response for Salmonella concentrations ranging from 2.3 × 102 to 7.8 × 104 CFU/mL. The entire detection process was completed within 8 h, with a very low detection limit of 1 CFU/25 mL (25 g), which was verified by colony counting, and a small degree of interference of 0.17-1.06%. Trace Salmonella from five different serovars in milk and chicken was successfully detected without false negative or false positive results. Furthermore, this study provides a basis to develop a fully automated instrument based on IMBs that includes all steps from sample preparation to chemiluminescence microparticle immunoassay for high-throughput screening of foodborne pathogens. Graphical abstract.

The development of methods for the detection of Salmonella in chickens by a combination of immunomagnetic separation and PCRs.

Micro- and nanoimmunomagnetic beads (MIMBs and NIMBs) used for immunomagnetic separation (IMS) with PCR were studied for the rapid detection of Salmonella. The capture efficiency of the two different IMBs was evaluated by a conventional plate counting method, and the binding pattern was studied using scanning electron microscopy. The specificity of the IMBs was tested with Salmonella, Shigella flexneri, enterohemorrhagic Escherichia coli O157:H7, and Listeria monocytogenes. By comparing the pre-enrichment IMS and the IMS enrichment steps with a 5.5-H enrichment time, this study developed a rapid and sensitive method for the detection of Salmonella in chicken. The method was implemented by IMS enrichment and PCR with MIMBs and NIMBs, with a total analysis time of 8 H. We showed that the method was sensitive based on NIMBs with a detection limit of 10° CFU for Salmonella in 25 g of chicken.

In vivo anti-tumor effect of DC-CIK cells on human lymphoma cell line Raji.

To research on the effect of DC-CIK cells on human lymphoma cell line Raji the immunophenotype of DC-CIK cells was analyzed using flow cytometry, and its proliferation inhibition effect was detected using MTT assay. 24 nude mice (4-6 weeks old) were employed and inoculated Raji cells on right axillaries for constructing human Burkitt lymphoma model. MTT results showed that DC-CIK cells had a significant inhibitory effect on Raji cells with obvious dose- and time- dependent effect. Western Blot results confirmed that DC-CIK cells could significantly down regulate the expression of BCL-2 (P<0.05). DC-CIK cells possesses significant anti-tumor effect on human Burkitt lymphoma bearing nude mice, and down regulation of Raji induced BCL-2 cell apoptosis may be one of the inhibitory mechanisms of DC-CIK cells.

Accelerating microfluidic immunoassays on filter membranes by applying vacuum.

This paper describes a vacuum-accelerated microfluidic immunoassay (we abbreviate it as VAMI) by sandwiching a filter membrane between a two-layer chip. A direct assay of IgG demonstrated that VAMI could simultaneously achieve higher sensitivity and require less time compared with conventional microfluidic immunoassays. We further applied VAMI to carry out a 3-step competitive assay (including antigen immobilization, competitive reaction and 2(nd) antibody reaction) for detecting the illegal food additive Sudan Red. A total assay time of 15 min with a limit of detection (LOD) of 1 ng ml(-1) is achieved.

pH-Regulated Strategy and Mechanism of Antibody Orientation on Magnetic Beads for Improving Capture Performance of Staphylococcus Species.

Immunomagnetic beads (IMBs) have been widely used to capture and isolate target pathogens from complex food samples. The orientation of the antibody immobilized on the surface of magnetic beads (MBs) is closely related to the effective recognition with an antigen. We put forward an available strategy to orient the antibody on the surface of MBs by changing the charged amino group ratio of the reactive amino groups at optimal pH value. Quantum dots labeling antigen assay, antigen-binding fragment (Fab) accessibility assay and lysine mimicking were used for the first time to skillfully illustrate the antibody orientation mechanism. This revealed that the positively charged ε-NH2 group of lysine on the Fc relative to the uncharged amino terminus on Fab was preferentially adsorbed on the surface of MBs with a negatively charged group at pH 8.0, resulting in antigen binding sites of antibody fully exposed. This study contributes to the understanding of the antibody orientation on the surface of MBs and the potential application of IMBs in the separation and detection of pathogenic bacteria in food samples.

Hapten designs based on aldicarb for the development of a colloidal gold immunochromatographic quantitative test strip.

The common carbamate insecticide aldicarb is considered one of the most acutely toxic pesticides. Herein, rational design was used to synthesize two haptens with spacers of different carbon chain lengths. The haptens were then used to immunize mice. The antibodies obtained were evaluated systematically, and a colloidal gold immunochromatographic strip was developed based on an anti-aldicarb monoclonal antibody. The 50% inhibition concentration and linear range of anti-aldicarb monoclonal antibody immunized with Hapten 1 were 0.432 ng/mL and 0.106-1.757 ng/mL, respectively. The cross-reactivities for analogs of aldicarb were all <1%. The limit of detection of the colloidal gold immunochromatographic strip was 30 µg/kg, and the average recoveries of aldicarb ranged from 80.4 to 110.5% in spiked samples. In the analysis of spiked samples, the test strip could accurately identify positive samples detected by the instrumental method in the GB 23200.112-2018 standard but produced some false positives for negative samples. This assay provides a rapid and accurate preliminary screening method for the determination of aldicarb in agricultural products and environments.

Rapid detection of trace Salmonella in milk using an effective pretreatment combined with droplet digital polymerase chain reaction.

Salmonella is one of the most dangerous food-borne pathogens around the world to cause a threat to humans and it is urgent to develop the rapid detection method of trace Salmonella in food. Although many advanced techniques have been widely applied to shorten the detection time, the pretreatment method usually used of traditional enrichment and plate culturing to separate Salmonella are complicated and time-consuming. Herein, we developed an effective pretreatment method based on in situ enrichment culture with an immunomagnetic separation step, combined with droplet digital polymerase chain reaction (ddPCR) technology to achieve rapid detection of trace Salmonella in milk, which allowed detecting as low as 10-1 CFU/mL level of Salmonella. It took 8 h to perform the entire testing process from pretreatment to ddPCR detection and analysis. The pretreatment method could be a suitable platform integrating with many detection techniques for the rapid detection of trace Salmonella.

Establishment of primary reference measurement procedures and reference materials for EGFR variant detection in non-small cell lung cancer.

Circulating tumor DNA (ctDNA)-based mutation detection is promising to change the clinical practice of genotype-directed therapy for cancer. A growing number of non-invasive tests for cancer screening and monitoring that involve the detection of ctDNA have been commercialized. Primary reference measurement procedures (PRMPs) and reference materials (RMs) are urgently needed to assess the non-invasive tests. In this study, a PRMP based on digital PCR (dPCR) and ctDNA RMs for quantification of the frequently occurring variant in epidermal growth factor receptor (EGFR L858R, T790M, and 19Del) in non-small cell lung cancer (NSCLC) were established. The candidate dPCR PRMP showed high specificity (false positive rate 0-0.003%), good repeatability (coefficient of variance (CV), 2-3% for 104 copies/reaction), and high interlaboratory reproducibility (3-10%). A good linearity (0.97 < slope < 1.03, R2 ≥ 0.9999) between the measured mutant (MU) value and prepared value was observed for all assays over the fractional abundance (FA) range, between 25% and 0.05%. The limit of quantification (LoQ) was determined to be 34 L858R, 23 T790M, and 34 19Del copies/reaction, corresponding to a FA of 0.2%. An inter-laboratory study of using the EGFR ctDNA RMs and dPCR assays demonstrated that the participating laboratories produced consistent concentrations of MU and wild-type (WT), as well as FA. This study demonstrates that dPCR can act as a potential PRMP for EGFR mutation for validation of NSCLC genotyping tests and ctDNA quantitative tests. The PRMP and RMs established here could improve interlaboratory repeatability and reproducibility, which supports rapid translation and application of non-invasive tests into clinical practice.

Monodispersed magnetic polystyrene beads with excellent colloidal stability and strong magnetic response.

Monodispersed polystyrene beads incorporated with Fe(3) O(4) nanoparticles are prepared via dispersion polymerization. The resultant magnetic beads present well-defined composite structures, excellent colloidal stability, and strong magnetic response. The formation mechanism for the monodispersed composite beads, incorporated with preformed Fe(3) O(4) nanocrystals, was investigated. The potential applications of the monodispersed magnetic beads in bacteria capturing were demonstrated. After being coated with anti-Salmonella CSA-1 antibody, the magnetic beads show capturing efficiencies of >99.4% in isolating Salmonella sp.

Interlaboratory assessment of droplet digital PCR for quantification of BRAF V600E mutation using a novel DNA reference material.

Droplet digital PCR (ddPCR) has attracted much attention in the detection of genetic signatures of cancer present at low levels in circulating tumor DNA (ctDNA) in blood. A growing number of laboratory-developed liquid biopsy tests based on such technology have become commercially available for clinical settings. To obtain consistent and comparable results, an international standard is necessary for validation of the analytical performance. In this study, a novel and SI-traceable "ctDNA" reference material (RM) carrying BRAF V600E was prepared by gravimetrically mixing a 152 bp PCR amplicon and sonicated wild-type genomic DNA. The ddPCR performance was evaluated by analyzing serial "ctDNA" dilutions using a competitive MGB assay. The mutant frequency concordance (k) between ddPCR and the gravimetrical value was 1.03 in the range from 53.9% to 0.1%. The limit of blank (LoB), detection (LoD) and quantification (LoQ) of ddPCR assay were determined to be 0.01%, 0.02% and 0.1%, respectively. Results from the interlaboratory study, using challenging low levels of BRAF V600E ctDNA RMs, demonstrated that the participating laboratories had the appropriate technical competency to perform accurate ddPCR-based low level of ratio measurements. However, a systematic error caused by uncorrected droplet volume in Naica Crystal ddPCR platform was found by using the ctDNA RM. Between-laboratory consistency in copy number measurement was greatly improved when a correct droplet volume was applied for the ddPCR measurement by using the ctDNA RM. This confirms that the "ctDNA" RM is fit for the validation of ddPCR systems for ctDNA quantification. This would also support translation of tests for circulating tumor DNA by ddPCR into routine use.

A rheumatoid arthritis magnetic resonance imaging contrast agent based on folic acid conjugated PEG-b-PAA@SPION.

Superparamagnetic iron oxide nanoparticles (SPIONs) offer unique properties for magnetic resonance imaging (MRI). Targeting imaging of rheumatoid arthritis in vivo requires a specific, magnetic sensitive and ultra-stable MRI contrast agent. In this study, SPIONs with a preferable colloid stability and optimized size were obtained by using an in situ polyol method with the diblock copolymer PEG-b-PAA acting as a surface ligand. Increasing the degree of polymerization (DP) of PAA from 18 to 36 to 57 led to the decreasing size of the iron oxide nanoparticles from 52 nm to 17 nm to 9 nm, respectively. Folic acid was conjugated onto PEG-PAAx@SPION as a specific targeting molecule for activated macrophages in a rheumatoid arthritis joint. To evaluate the stability and magnetic properties of the particle, a series of tests were conducted to evaluate and optimize the nanoparticles. In vitro endocytosis experiments confirmed the better performance of the folic acid conjugated SPIONs than the non-folic acid modified SPIONs. In vivo MRI clearly demonstrated the significant signal diminishment of the arthritis joint in antigen induced arthritis (AIA) rats by intravenous injection of the optimized nanoparticles FA-PEG-b-PAA36@SPION. These results indicated that FA-PEG-b-PAA36@SPION could serve as a promising candidate for the MRI of rheumatoid arthritis.

Folic acid-conjugated glucose and dextran coated iron oxide nanoparticles as MRI contrast agents for diagnosis and treatment response of rheumatoid arthritis.

Coating superparamagnetic iron oxide (SPIO) with dextran increases the stability of the magnetic nanoparticles during blood circulation, yet this is accompanied by an increase in the particle size and the vascular permeability efficiency of the SPIO nanoparticles into the joints decreases. In our study, the thickness of the dextran coated onto SPIO (dex-SPIO) was optimized without affecting the magnetic quality of iron oxide by adding a suitable amount of glucose into the crystal growth process. To further improve the signal enhancement effect of this glucose and dextran coated SPIO (glu-dex-SPIO) for the detection of the inflammatory site of arthritis, folic acid (FA) was conjugated to glu-dex-SPIO. This FA glu-dex-SPIO was used as a negative contrast agent for MRI to visualize the antigen induce arthritis (AIA) model in rats using a 7 T MR scans. MR imaging revealed more significant differences between the synovium and surrounding tissues with FA glu-dex SPIO than when using the non-targeting glu-dex-SPIO over a long period of time (24 h) after intravenous injection. Moreover, the therapeutic efficacy of the cyclooxygenase 2 (COX-2) inhibitor treatment of the inflamed joints also could be confirmed by using FA glu-dex SPIO enhanced MRI, indicating that this type of nanoparticles could also have potential as a contrast agent for measuring the treatment response of rheumatoid arthritis.

Theranostic self-assembly structure of gold nanoparticles for NIR photothermal therapy and X-Ray computed tomography imaging.

The controllable self-assembly of amphiphilic mixed polymers grafted gold nanoparitcles (AuNPs) leads to strong interparticle plasmonic coupling, which can be tuned to the near-infrared (NIR) region for enhanced photothermal therapy (PTT). In this study, an improved thiolation method was adopted for ATRP and ROP polymer to obtain amphiphilic brushes of PMEO2MA-SH and PCL-SH. By anchoring PCL-SH and PMEO2MA-SH onto the 14 nm AuNPs, a smart hybrid building block for self-assembly was obtained. Increasing the PCL/PMEO2MA chain ratio from 0.8:1, 2:1 and 3:1 to 7:1, the structure of gold assemblies (GAs) was observed to transfer from vesicle to large compound micelle (LCM). Contributed to the special dense packed structure of gold nanoparticles in LCM, the absorption spectrometry of gold nanoparticles drastically red-shifted from 520 nm to 830 nm, which endowed the GAs remarkable NIR photothermal conversion ability. In addition, gold has high X-ray absorption coefficient which qualifies gold nanomaterial a potential CT contrast agent Herein, we obtain a novel gold assembly structure which can be utilized as potential photothermal therapeutic and CT contrast agents. In vitro and In vivo studies testified the excellent treatment efficacy of optimum GAs as a PTT and CT contrast agent. In vitro degradation test, MTT assay and histology study indicated that GAs was a safe, low toxic reagent with good biodegradability. Therefore, the optimum GAs with strong NIR absorption and high X-ray absorption coefficient could be used as a theranostic agent and the formation of novel gold large compound micelle might offers a new theory foundation for engineering design and synthesis of polymer grafted AuNPs for biomedical applications.

miR-375 affects the proliferation and invasion of hepatoma cells via targetingYAP / 中国肿瘤生物治疗杂志

@# Objective: To investigate the mechanisms of miR-375 affecting the proliferation and invasion of hepatoma cells via targeting YAP (Yes-associated protein). Methods: The cancerous tissues and corresponding para-cancerous tissues of 70 patients with hepatocellular carcinoma (HCC) who underwent surgical resection at the Second Affiliated Hospital of Henan University of Traditional Chinese Medicine from January 2015 to December 2016, as well as the hepatoma cell lines SMMC-7721, Hb611, HepG2 and BEL-7405 were collected for this study. qPCR method was used to detect the expression level of miR-375 in collected HCC tissues and different hepatoma cell lines; Dual luciferase reporter gene assay was used to verify the interaction between miR-375 and YAP; The relationship between miR-375 and clinicopathological features of HCC patients was also analyzed; MTT assay was used to detect the effect of miR375 on the proliferation of hepatoma cells; Transwell invasion assay was used to detect the invasive ability of hepatoma cells after inhibiting the expression of miR-375; Western blotting was used to detect the expression of YAP in HepG2 cells. The nude mouse model of subcutaneously transplanted xenograft was established, and the tumor volume and mass of transplanted hepatoma cells were detected after inhibiting the expression of miR-375. The expression of YAP in xenograft of nude mice was detected by immunohistochemistry and Western blotting. Results: The expression of miR-375 and YAP in HCC tissues was significantly higher than that in para-cancerous tissues (all P<0.05). The expression of miR-375 in HepG2 cells was the highest (P<0.05). miR-375 could specifically bind to the 3' UTR of YAP and regulate the expression activity of YAP. After inhibiting the expression of miR-375, the proliferation and invasion abilities of HepG2 cells were reduced (all P<0.05); The tumor volume and mass of transplanted xenografts were significantly reduced (both P<0.05); The expression of YAP protein in the transplanted xenografts was down-regulated (P<0.05). Conclusion: miR-375 plays an important role in the occurrence and development of liver cancer, and can influence the malignant biological behaviors of hepatoma cells by targeting and regulating the expression ofYAP.

An efficient isolation method for domestic hen (Gallus domesticus) ovarian primary follicles.

An enzymatic method of isolating primary follicles in the turkey has been described previously, but no similar work has been done in the hen. In this study, primary follicles from domestic hens (Gallus domesticus) were isolated using an enzymatic method, and the isolated follicular quantity, quality, and development in vitro were assessed. About 400 primary follicles ranging from 60 to 1125 microm in diameter were recovered with trypsin and collagenase from hen ovaries (per ovary). Of these, 76.5% were intact follicles with a complete single layer of granulose cells surrounded by the basement membrane, and their ultrastructures appeared this way in situ. Follicles (351 to 500 microm in diameter) were cultured in vitro, and 46.67% of them survived after 5 days. Ultrastructural examination showed that elongated mitochondria forming a ring were distributed to the periphery of the oocyte, the Golgi was oriented with the maturing face toward the granulosa cell layer, and the oocyte plasma membrane presented a few short microvilli lying on the oocyte surface, which confirmed that the surviving follicles were developmental. These results suggest that a simple, rapid, effective enzymatic method can be used to isolate a great number of intact primary follicles from the hen ovary.