Long non-coding FAM201A accelerates the tumorigenesis and progression of colorectal cancer through miR-3163/MACC1 axis.

Colorectal cancer (CRC) is the leading contributor to cancer-relevant deaths worldwide with severe incidence and mortality. An extensive body of evidence has demonstrated that lncRNA plays a critical role in the oncogenicity of CRC. Despite the oncogenic function of FAM201A in esophageal squamous cell cancer and non-small-cell lung cancer, the potential of FAM201A in CRC progression remains unknown. FAM201A expression level was significantly enhanced in CRC cells compared with normal cells. Further, functional experiments illustrated that knockdown of FAM201A restrained cell growth, stemness and promoted chemoresistance of CRC cells. By exploring molecular mechanism of FAM201A, we found that FAM201A acted as a sponge of miR-3163. More importantly, oncogene MACC1 was confirmed to be a direct target of miR-3163 and FAM201A modulated MACC1 expression level via competing for miR-3163. Subsequently, we testified that FAM201A exerted its role in the tumorigenesis and development of CRC through targeting miR-3163/MACC1. Animal assay certified that FAM201A expedited CRC cell growth in vivo. In conclusion, our study was the first to unveil that FAM201A promoted cell proliferation and CSC characteristics in CRC via regulation of the miR-3163/MACC1 axis, which provided clues for the clinical treatment of patients with this disease.

Low-sample-consumption and ultrasensitive detection of procalcitonin by boronate affinity recognition-enhanced dynamic light scattering biosensor.

Accurate determination of procalcitonin (PCT) is highly crucial in bacterial infection diagnosis. Many biosensors previously developed suffer from large sample consumption or lengthy waiting time, which raise difficulties for more vulnerable patients, such as infants, old people, and other critically ill patients. To address this dilemma, we present an innovative boronate affinity recognition (BAR)-enhanced dynamic light scattering (DLS) biosensor to achieve ultrasensitive PCT detection. In this biosensing system, monoclonal antibody-modified magnetic nanoparticles (MNP@mAb) are designed as probes to capture PCT from serum samples and generate DLS signal transduction. Polyvalent phenylboronic acid-labeled bovine serum albumin (BSA@PBA) is used as scaffold to aggregate MNP@mAb and PCT (MNP@mAb-PCT) complex because of the specific interaction of cis-diol-containing PCT with boronic acid ligands on the surface of BSA@PBA. The BAR-enhanced DLS biosensor shows ultrahigh sensitivity to PCT determination due to high binding affinity, with the limit of detection of 0.03 pg/mL. The total detection time of PCT in whole blood or serum is less than 15 min with small sample consumption (about 1 µL) due to the rapid magnetic separation and aggregation of MNP@mAb-PCT triggered by BSA@PBA. In addition, the proposed DLS biosensor exhibits a high specificity for PCT quantitative detection. Therefore, this work provides a promising and versatile strategy for extending DLS biosensor to rapid and ultrasensitive detection of trace PCT for broader patients and more urgent cases.

CD38 deficiency up-regulated IL-1ß and MCP-1 through TLR4/ERK/NF-κB pathway in sepsis pulmonary injury.

As a disease with high mortality, many cytokines and signaling pathways are associated with sepsis. The pro-inflammatory cytokines and chemokines are participating in the pathogenesis of sepsis, especially in early stage. Moreover, the releases and expressions of cytokines are regulated by numerous signaling pathways, including TLR4/ERK pathway. But despite many studies have expounded the pathogenesis of sepsis and the regulation of cytokines in sepsis, how CD38 influence the expressions of related molecules in sepsis are still unknown. The aim of this study is illuminating the alteration of cytokines and signaling pathways in CD38-/- mice injected with Escherichia coli. Compared with WT mice, E. coli infection results in more severe pulmonary injuries and higher mRNA expressions of cytokines. Compared with E. coli infected WT mice, CD38 knockout leads to aggravated pulmonary injury, increased phosphorylated ERK1/2, p38 and NF-κB p65, and enhanced levels of IL-1ß, iNOS and MCP-1. While compared with E. coli infected CD38-/- mice, TLR4 mutation results in alleviated pulmonary injury, down-regulated phosphorylated ERK1/2 and NF-κB p65, and decreased expressions of IL-1ß and MCP-1. CD38 deficiency increased the expressions of IL-1ß andMCP-1 and aggravated pulmonary injury through TLR4/ERK/NF-κB pathway in sepsis.

TLR4-NLRP3-GSDMD-Mediated Pyroptosis Plays an Important Role in Aggravated Liver Injury of CD38-/- Sepsis Mice.

Clinically, severe bacterial infection can cause septicemia and multiple organ dysfunction syndrome, especially liver injury. CD38 is closely related to many inflammatory pathways, but its role in liver injury caused by bacterial infection remains unclear. The purpose of this study is to discuss the specific role of CD38 in bacterial liver injury. Eight-week-old male C57BL/6 mice (WT, CD38-/- and CD38-/-TLR4mut) were used and stimulated with Escherichia coli (ATCC25922) or PBS, intraperitoneally. After 3 hours of bacterial stimulation, serum was collected to detect ALT and AST concentration, and liver tissue was harvested for hematoxylin and eosin staining and bacterial culture. The mRNA expressions of TLR4, NLRP3, IL-1ß, IL-18, and GSDMD were quantitatively determined by RT-qPCR. The expressions of TLR4, MyD88, TRIF, NF-κB p65, NLRP3, GSDMD, and cytokines were detected by Western blot. The expression and localization of ERK1/2 were detected by immunohistochemistry and Western blot. The results showed that bacterial stimulation could upregulate the expression of inflammatory cytokines, leading to hepatic dysfunction. Moreover, bacterial stimulation of CD38-deficient mice can aggravate the inflammatory response, the expressions of TLR4, NF-κB, and ERK1/2 were significantly increased, and the biomarkers related to pyroptosis also manifested more obvious pyroptosis. However, TLR4 mutation significantly alleviated inflammation and pyroptosis in the liver caused by bacteria, on the basis of CD38 deficiency. Overall, CD38 knockout exacerbates bacteria-induced liver damage through TLR4-NLRP3-GSDMD-mediated pyroptosis.

Synthesis of PDA-Mediated Magnetic Bimetallic Nanozyme and Its Application in Immunochromatographic Assay.

Immunochromatographic assay (ICA) is widely applied in various fields. However, severe matrix interference and weak signal output present major challenges in achieving accurate and ultrasensitive detection in ICA. Here, a polydopamine (PDA)-mediated magnetic bimetallic nanozyme (Fe3O4@PDA@Pd/Pt) with peroxidase-like activity was synthesized and used as a probe in ICA. The magnetic property of Fe3O4@PDA@Pd/Pt enabled effective magnetic enrichment of targets, thereby reducing the matrix interference in the sample. PDA coating on the magnetic bimetallic nanozyme was employed as a mediator and a stabilizer. It improved the catalytic ability and stability of the magnetic bimetallic nanozyme by providing more coordination sites for Pd/Pt growth and functional groups (-NH and -OH). In addition, the Pd/Pt bimetallic synergistic effect could further enhance the catalytic ability of the nanozyme. A method was developed by integrating Fe3O4, PDA, and Pd/Pt into Fe3O4@PDA@Pd/Pt as a probe in ICA. With the proposed method, human chorionic gonadotropin and Escherichia coli O157:H7 were successfully detected to be as low as 0.0094 mIU/mL in human blood serum and 9 × 101 CFU/mL in the milk sample, respectively. This method may be readily adapted for accurate and ultrasensitive detection of other biomolecules in various fields.

Hydrazide mediated oriented coupling of antibodies on quantum dot beads for enhancing detection performance of immunochromatographic assay.

Immunochromatographic assays (ICAs) are one of the most popular on-site diagnostic tools. The bio-activity of the probe holds great promise for the detection performance of ICA. The orientation of antibodies on nanoparticle surface plays a vital role in improving the bio-activity of the probe. Hydrazide mediated oriented coupling (HDZ-O) strategy can erect the antibodies on the particle surface via the nucleophilic addition reaction between the hydrazide group of particles and the aldehyde group of the Fc region of antibodies. In this work, we synthesized the hydrazide and carboxylic group modified quantum dot beads (QBs-NH-NH2, and QBs-COOH) and compared two coupling strategies for preparing QB probes systematically. Results showed the reaction time for producing QB probe by using HDZ-O method was 20 min, and the optimal labeled antibody content was 80 µg per mg of QBs. Those for carbodiimide method were 180 min and 320 µg per mg of QBs. Moreover, the QB probe by HDZ-O showed a higher bio-activity than that by carbodiimide method under their optimal labeled antibody concentrations. Subsequently, the QB probe by HDZ-O strategy was applied further in sandwich ICA for hepatitis B surface antigen (HBsAg) detection. The results indicated that the strip has a widely dynamic linearity with HBsAg concentration ranging from 0.05 ng/mL to 3200 ng/mL. The limit of detection of the strip is 0.05 ng/mL, which is approximately fourfold lower than that of the probe prepared by carbodiimide method. In addition, the ICA strip also showed acceptable accuracy and precision for HBsAg quantitative detection in real serum samples, which have average recoveries in intra- and inter-assays that vary from 89.16% to 97.63% with the variable coefficients less than 6.98%. The proposed ICA strip showed good agreement (R2 > 0.94) with a commercial chemiluminescence kit for 40 of clinical serum samples. All in all, this study proposed a robust strategy to immobilize the antibodies with proper orientation on the surface of QBs, and the resultant QB probe can improve the detection performance of sandwich LFIA platform remarkably.

Core-Shell-Heterostructured Magnetic-Plasmonic Nanoassemblies with Highly Retained Magnetic-Plasmonic Activities for Ultrasensitive Bioanalysis in Complex Matrix.

Herein, a facile self-assembly strategy for coassembling oleic acid-coated iron oxide nanoparticles (OC-IONPs) with oleylamine-coated gold nanoparticles (OA-AuNPs) to form colloidal magnetic-plasmonic nanoassemblies (MPNAs) is reported. The resultant MPNAs exhibit a typical core-shell heterostructure comprising aggregated OA-AuNPs as a plasmonic core surrounded by an assembled magnetic shell of OC-IONPs. Owing to the high loading of OA-AuNPs and reasonable spatial distribution of OC-IONPs, the resultant MPNAs exhibit highly retained magnetic-plasmonic activities simultaneously. Using the intrinsic dual functionality of MPNAs as a magnetic separator and a plasmonic signal transducer, it is demonstrated that the assembled MPNAs can achieve the simultaneous magnetic manipulation and optical detection on the lateral flow immunoassay platform after surface functionalization with recognition molecules. In conclusion, the core-shell-heterostructured MPNAs can serve as a nanoanalytical platform for the separation and concentration of target compounds from complex biological samples using magnetic properties and simultaneous optical sensing using plasmonic properties.

Novel Multiple miRNA-Based Signatures for Predicting Overall Survival and Recurrence-Free Survival of Colorectal Cancer Patients.

BACKGROUND Colorectal cancer (CRC) has become a heavy health burden around the world, accounting for about 10% of newly diagnosed cancer cases. In the present study, we aimed to establish the miRNA-based prediction signature to assess the prognosis of CRC patients. MATERIAL AND METHODS A total of 451 CRC patients' expression profiles and clinical information were download from the TCGA database. LASSO Cox regression was conducted to construct the overall survival (OS)- and recurrence-free survival (RFS)-associated prediction signatures, by which CRC patients were divided into low- and high-risk groups. Kaplan-Meier (K-M) curve and receiver operating characteristic (ROC) curves were used to explore the discriminatory ability and stability of the signatures. Functional enrichment analyses were performed to identify the probable mechanisms. RESULTS miRNA-216a, miRNA-887, miRNA-376b, and miRNA-891a were used to build the prediction formula associated with OS, while miR-1343, miR-149, miR-181a-1, miR-217, miR-3130-1, miR-378a, miR-542, miR-6716, miR-7-3, miR-7702, miR-677, and miR-891a were obtained to construct the formula related to RFS. K-M curve and ROC curve revealed the good discrimination and efficiency of OS in the training (P<0.001, AUC=0.712) and validation cohorts (P=0.019, AUC=0.657), as well as the results of RFS in the training (P<0.001, AUC=0.714) and validation cohorts (P=0.042, AUC=0.651). The function annotations for the targeted genes of these miRNAs show the potential mechanisms of CRC. CONCLUSIONS We established 2 novel miRNA-based prediction signatures of OS and RFS, which are reliable tools to assess the prognosis of CRC patients.

Effect of different-sized gold nanoflowers on the detection performance of immunochromatographic assay for human chorionic gonadotropin detection.

Traditional colloidal gold based immunochromatographic assays (ICAs) that use 20-40 nm gold nanospheres (AuNSs) as signal reporters have low sensitivity given the insufficient brightness of AuNSs. Gold nanoflowers (AuNFs) possess high optical brightness and strong target binding affinity because of their multibranched structures and large specific surface areas. Thus, ICAs with AuNF reporters have better sensitivity than ICAs with conventional AuNSs reporters. In addition, large AuNFs exhibit higher optical absorbance than small AuNFs. To elucidate the effect of AuNF size on the sensitivity of sandwich ICA, AuNFs with five different sizes of 33, 47, 79, 152, and 195 nm were synthesized and used as reporters in ICA strips for pregnancy diagnosis based on the detection of human chorionic gonadotropin (HCG). Medium-sized AuNFs with diameters of 47-79 nm showed the highest sensitivity of 9 mIU/mL. When used to test actual HCG-spiked urine samples, the 47 nm AuNF-based ICA showed two independent linear correlations in low (9 mIU/mL ~ 288 mIU/mL) and high (288 mIU/mL ~ 2304 mIU/mL) HCG concentrations for HCG quantitative detection. The intra- and interassay recovery rates for HCG spiked urine samples ranged from 81.19% to 114.44% with the coefficient of variation (CV) from 3.29% to 8.08%. These results collectively indicate that the AuNF-based ICA has high accuracy. In addition, the proposed AuNF-based ICA method exhibited excellent selectivity for HCG determination. In summary, medium-sized AuNFs can serve as optimal ICA labels in the high-sensitivity detection of disease-related protein biomarkers.

Dual-mode fluorescent and colorimetric immunoassay for the ultrasensitive detection of alpha-fetoprotein in serum samples.

We present a novel dual-mode fluorescent and colorimetric immunosensor based on conventional immunoassay platforms by utilizing a gold nanoflower (AuNF)-loaded fluorescein molecule (AuNF@Fluorescein) as signal output. The AuNFs were modified with thiolated carboxyl ligand, which consisted of a hydrophobic alkane chain as hydrophobic wallet for fluorescein encapsulation, a tetra (ethylene glycol) unit for biocompatibility and solubility, and a functional carboxyl group for the conjugation of biorecognition molecules for biosensing. The resultant AuNFs showed a high loading capacity of 3.74 × 106 fluorescein molecules per AuNF because of its flower-like shape with many complex branches. By adjusting the solution pH to 8.0, the fluorescein molecules can almost entirely be released from the hydrophobic wallet of AuNF@Fluorescein, which led to strong fluorescent-signal amplification. Under the optimal detection conditions, the proposed immunoassay based on fluorescent signal exhibited ultrahigh sensitivity for alpha-fetoprotein (AFP) detection, with a limit of detection (LOD) of 29 fg/mL. This value is approximately 9.3 × 103-fold lower than that of corresponding horseradish peroxidase (HRP)-based immunoassay (LOD = 270 pg/mL). The fluorescein molecule also had intrinsic peroxidase-like activity to catalyze 3,3',5,5'-tetramethylbenzidine oxidation with hydrogen peroxide for colorimetric signal. The proposed method with colorimetric mode further exhibited a sensitivity with a LOD of 17.7 pg/mL, which is about 15-fold lower than that of conventional HRP-based immunoassay. The recoveries of the proposed dual-mode immunoassay for AFP spiked serum samples ranged within 89.85%-100.0%, with the coefficient of variations ranging from 0.5% to 2.4%, indicating acceptable accuracy and precision for AFP quantitative detection. The reliability of the developed dual-mode immunoassay was further compared with a commercial chemiluminescence immunoassay kit by analyzing 20 clinical serum samples, showing that the two methods well agreed with each other, with high correlation coefficients of 0.997 and 0.986 based on recorded fluorescence and colorimetric signals, respectively. In summary, the proposed method was highly suitable for the ultrasensitive analysis of biomarkers or infectious diseases by fluorescence mode and can be used for routine clinical diagnosis by colorimetric mode.

CHSY1 promoted proliferation and suppressed apoptosis in colorectal cancer through regulation of the NFκB and/or caspase-3/7 signaling pathway.

Colorectal cancer is a commonly observed malignant cancer. However, the limited therapies for colorectal cancer do not bring much benefit for patients. Chondroitin synthase-1 (CHSY1) is an enzyme responsible for the biosynthesis of chondroitin sulfate and has been implicated in the tumorigenesis of several cancer types; however, there is limited information regarding the role of CHSY1 in colorectal cancer. In the present study, CHSY1 was demonstrated to be highly expressed in colorectal cancer tissues and in cell lines, and the CHSY1 expression level was associated with the 5-year survival rate of patients with colorectal cancer. Following CHSY1 knockdown, the proliferation of colorectal cancer cells was significantly decreased. The number of RKO cells decreased by 50% following CHSY1 knockdown compared with that in the control after culture for 5 days. However, the apoptosis rate of RKO cells increased to 14.15% after CHSY1 knockdown. In addition, the activity of caspase-3/7 was also enhanced. Furthermore, the expression of B-cell lymphoma 2 (Bcl-2) was reduced, whereas the levels of Bcl-2-associated X protein (Bax) and truncated caspase-3/7 were increased following CHSY1 knockdown. Additionally, the phosphorylation level of IκB and the expression of nuclear factor (NF)κB also decreased. In contrast, forced expression of CHSY1 increased the level of Bcl-2, NFκB, and phosphorylated IκB, whereas the level of bax and truncated caspase-3/7 decreased. Therefore, the data of the present study suggest that CHSY1 promoted cell proliferation by regulating NFκB signaling and suppressed cell apoptosis by regulating/caspase-3/7 signaling in colorectal cancer. The present study also suggests that CHSY1 may be a potential target for colorectal cancer therapy.

Fluorescence ELISA based on glucose oxidase-mediated fluorescence quenching of quantum dots for highly sensitive detection of Hepatitis B.

Herein, we present a novel sandwich fluorescence enzyme linked immunosorbent assay (ELISA) for highly sensitive detection of Hepatitis B virus surface antigen (HBsAg) based on glucose oxidase (GOx)-induced fluorescence quenching of mercaptopropionic acid-modified CdTe quantum dots (MPA-QDs). In this system, hydrogen peroxide (H2O2) sensitive MPA-QDs was used as a signal output, and glucose oxidase (GOx) was used as label which can generate H2O2 via catalytic oxidation of glucose. The proposed method showed dynamic linear detection of HBsAg both in the range of 47pgmL-1 ~ 380pgmL-1 and 0.75ngmL-1 ~ 12.12ngmL-1. The detection limit of the proposed fluorescence ELISA was 1.16pgmL-1, which was approximately 430-fold lower than that of horseradish peroxidase (HRP)-based conventional ELISA. The average recoveries for HBsAg-spiked serum samples ranged from 98.0% to 126.8% with the relative standard derivation below 10%, thus indicating acceptable precision and high reproducibility of the proposed fluorescence ELISA for HBsAg detection. Additionally, the developed method showed no false positive results analyzing 35 real HBsAg-negative serum samples, and exhibited excellent agreement (R2=0.9907) with a commercial time-resolved fluorescence immunoassay (TRFIA) kit for detecting 31 HBsAg-positive serum samples. In summary, the proposed method based on fluorescence quenching of H2O2 sensitive QDs is considerably to be an excellent biodetection platform with ultrahigh sensitivity, good accuracy and excellent reliability.

Effect of cold stress on immunity in rats.

An increase in the morbidity of upper respiratory tract infections and the attack and exacerbation of autoimmune diseases has been observed to occur in the few days following sudden environmental temperature decreases, but the mechanisms for these phenomena are not well understood. To determine the effect of a sudden ambient temperature drop on the levels of stress hormones and T-lymphocyte cytokines in the plasma, the Toll-like receptor 4 (TLR4) expression of immunocompetent cells in rat spleens and the levels of regulatory T (Treg) cells in the peripheral blood, Sprague Dawley rats were divided into three groups of different ambient temperatures (20, 4 and -12°C). In each group, there were four observation time-points (1, 12, 24 and 48 h). Each ambient temperature group was subdivided into non-stimulation, lipopolysaccharide-stimulation and concanavalin A-stimulation groups. The levels of adrenocorticotropin (ACTH), epinephrine (EPI), angiotensin-II (ANG-II), interleukin-2 (IL-2), interferon-γ (IFN-γ), IL-4 and IL-10 in the plasma were determined using ELISA. The cellular expression levels of TLR4 and the presence of cluster of differentiation (CD)4+CD25+ and CD4+CD25+Forkhead box P3 (Foxp3)+ cells were determined using flow cytometry. The experiments demonstrated that the ACTH, EPI, ANG-II and IL-10 levels in the plasma were significantly increased at 4 and -12°C compared with those at 20°C, while the plasma levels of IFN-γ, IL-2 and IL-4, the TLR4 expression rates of immunocompetent cells in the rat spleen and the percentage of CD4+CD25+Foxp3+ Treg cells among the CD4+CD25+ Treg cells in the peripheral blood were decreased at 4 and -12°C compared with those at 20°C. These data indicate that cold stress affects the stress hormones and the innate and adaptive immunity functions in rats.