Two-dimensional MoS2 as a nano-binder for ssDNA: Ultrasensitive aptamer based amperometric detection of Ochratoxin A.

Two-dimensional (2D) MoS2 is found to possess different affinities for ssDNA and dsDNA. This finding is exploited in an amperometric aptamer-based method for the determination of the mycotoxin ochratoxin A (OTA). Initially, a dsDNA probe (formatted through the hybridization of OTA-aptamer with an auxiliary DNA) is self-assembled on a gold electrode. Upon introduction of OTA, it will bind to the aptamer and cause the unwinding of dsDNA, while the auxiliary DNA (with single-stranded structure) remains on the electrode. Since the affinity of 2D MoS2 for ssDNA is considerably larger than that for dsDNA, it will be adsorbed on the electrode by binding to the auxiliary DNA. Notably, 2D MoS2 possesses peroxidase-like activity. Hence, it can catalyze the amplification of electrochemical signal of the hydroquinone/benzoquinone redox system. Under optimal conditions, the amperometric signal (best measured at -0.2 V vs. SCE) increases with increasing OTA concentration in the range from 0.5 pg·mL-1 to 1.0 ng·mL-1, with a lower detection limit of 0.23 pg·mL-1. The method was applied to the determination of OTA in spiked red wine. Graphical abstract Herein we construct a convenient electrochemical aptasensor for sensitive monitor of ochratoxin A by using 2D MoS2 as a nano-binder to catalyze the amplification of electrochemical signal from hydroquinone/benzoquinone system.

Homogeneous electrochemical immunoassay of aflatoxin B1 in foodstuff using proximity-hybridization-induced omega-like DNA junctions and exonuclease III-triggered isothermal cycling signal amplification.

A new homogeneous electrochemical immunosensing platform was designed for sensitive detection of aflatoxin B1 (AFB1) in foodstuff. The system consisted of anti-AFB1 antibody labeled DNA1 (Ab-DNA1), AFB1-bovine serum albumin (BSA)-conjugated DNA2 (AFB1-DNA2), and methylene blue functionalized hairpin DNA. Owing to a specific antigen-antibody reaction between anti-AFB1 and AFB1-BSA, the immunocomplex formed assisted the proximity hybridization of DNA1 with DNA2, thus resulting in the formation of an omega-like DNA junction. Thereafter, the junction opened the hairpin DNA to construct a new double-stranded DNA, which could be readily cleaved by exonuclease III to release the omega-like DNA junction and methylene blue. The dissociated DNA junction could repeatedly hybridize with residual hairpin DNA molecules with exonuclease III-based isothermal cycling amplification, thereby releasing numerous free methylene blue molecules into the detection solution. The as-produced free methylene blue molecules could be captured by a negatively charged indium tin oxide electrode, each of which could produce an electronic signal within the applied potentials. On introduction of target AFB1, the analyte competed with AFB1-DNA2 for the conjugated anti-AFB1 on the Ab-DNA1, subsequently decreasing the amount of omega-like DNA junctions formed, hence causing methylene blue labeled hairpin DNA to move far away from the electrode surface. Under optimal conditions the detectable electrochemical signal decreased with increasing amount of target AFB1 in a dynamic working range of 0.01-30 ng mL-1 with a detection limit of 4.8 pg mL-1. In addition, the precision and reproducibility of this system were acceptable. Finally, the method was further evaluated for analysis of naturally contaminated or AFB1-spiked peanut samples, giving results that matched well with those obtained with a commercial AFB1 ELISA kit.

[Dysmenorrhea: a study of affected factors and approaches to relief among female students at a college in southern Taiwan].

BACKGROUND: Dysmenorrhea is a major problem in the general gynecology clinic. It causes discomfort among healthcare staffs and significant losses in terms of time and finances. PURPOSE: The purpose of this study was to determine the affected factors of dysmenorrhea and evaluate the self-perceived efficacy of relief methods. METHODS: A cross-sectional research design was used to collect data. Participants included 586 female students enrolled at a college in southern Taiwan. Data was analyzed using a t-test, one-way ANOVA, and Scheffe test. RESULTS: Traditional Chinese medicine pattern identifications related significantly to dysmenorrhea frequency perception. Lifestyle characteristics related significantly to dysmenorrhea level perception. Using shenghua decoction, siwu and pig blood decoction, Angelica drink, ginger, ziziphus jujube, brown sugar tea, and analgesics all related significantly to dysmenorrhea relief efficacy. CONCLUSIONS/IMPLICATIONS FOR PRACTICE: Strategies found to help relieve dysmenorrhea level perception include increasing the duration and regularity of sleep and avoiding the consumption of pungent foods. Seeking and adhering to physician recommendations can also increase dysmenorrhea self-care efficacy. Dysmenorrhea-relief courses should be improved in hospitals and schools to assist women to self-manage dysmenorrhea more effectively.

Evidence of premature lymphocyte aging in people with low anti-spike antibody levels after BNT162b2 vaccination.

SARS-CoV-2 vaccines have unquestionably blunted the overall impact of the COVID-19 pandemic, but host factors such as age, sex, obesity, and other co-morbidities can affect vaccine efficacy. We identified individuals in a relatively healthy population of healthcare workers (CORALE study cohort) who had unexpectedly low peak anti-spike receptor binding domain (S-RBD) antibody levels after receiving the BNT162b2 vaccine. Compared to matched controls, "low responders" had fewer spike-specific antibody-producing B cells after the second and third/booster doses. Moreover, their spike-specific T cell receptor (TCR) repertoire had less depth and their CD4+ and CD8+T cell responses to spike peptide stimulation were less robust. Single cell transcriptomic evaluation of peripheral blood mononuclear cells revealed activation of aging pathways in low responder B and CD4+T cells that could underlie their attenuated anti-S-RBD antibody production. Premature lymphocyte aging may therefore contribute to a less effective humoral response and could reduce vaccination efficacy.

TGF-ß1-induced miR-424 promotes pulmonary myofibroblast differentiation by targeting Slit2 protein expression.

Idiopathic pulmonary fibrosis (IPF) is a devastating interstitial lung disease with irreversible loss of lung tissue and function. Myofibroblasts in the lung are key cellular mediators of IPF progression. Transforming growth factor (TGF)-ß1, a major profibrogenic cytokine, induces pulmonary myofibroblast differentiation, and emerging evidence has established the importance of microRNAs (miRs) in the development of IPF. The objective of this study was to define the pro-fibrotic roles and mechanisms of miRs in TGF-ß1-induced pulmonary myofibroblast differentiation. Using RNA sequencing, we identified miR-424 as an important TGF-ß1-induced miR in human lung fibroblasts (HLFs). Quantitative RT-PCR confirmed that miR-424 expression was increased by 2.6-fold in HLFs in response to TGF-ß1 and was 1.7-fold higher in human fibrotic lung tissues as compared to non-fibrotic lung tissues. TGF-ß1-induced upregulation of miR-424 was blocked by the Smad3 inhibitor SIS3, suggesting the involvement of this canonical TGF-ß1 signaling pathway. Transfection of a miR-424 hairpin inhibitor into HLFs reduced TGF-ß1-induced expression of classic myofibroblast differentiation markers including ɑ-smooth muscle actin (ɑ-SMA) and connective tissue growth factor (CTGF), whereas a miR-424 mimic significantly enhanced TGF-ß1-induced myofibroblast differentiation. In addition, TGF-ß1 induced Smad3 phosphorylation in HLFs, and this response was reduced by the miR-424 inhibitor. In silico analysis identified Slit2, a protein that inhibits TGF-ß1 profibrogenic signaling, as a putative target of regulation by miR-424. Slit2 is less highly expressed in human fibrotic lung tissues than in non-fibrotic lung tissues, and knockdown of Slit2 by its siRNA enhanced TGF-ß1-induced HLF differentiation. Overexpression of a miR-424 mimic down-regulated expression of Slit2 but not the Slit2 major receptor ROBO1 in HLFs. Luciferase reporter assays showed that the miR-424 mimic represses Slit2 3' untranslated region (3'-UTR) reporter activity, and mutations at the seeding regions in the 3'-UTR of Slit2 abolish this inhibition. Together, these data demonstrate a pro-fibrotic role of miR-424 in TGF-ß1-induced HLF differentiation. It functions as a positive feed-back regulator of the TGF-ß1 signaling pathway by reducing expression of the negative regulator Slit2. Thus, targeting miR-424 may provide a new therapeutic strategy to prevent myofibroblast differentiation and IPF progression.

Transforming growth factor (TGF)-ß1-induced miR-133a inhibits myofibroblast differentiation and pulmonary fibrosis.

Transforming growth factor (TGF)-ß1, a main profibrogenic cytokine in the progression of idiopathic pulmonary fibrosis (IPF), induces differentiation of pulmonary fibroblasts to myofibroblasts that produce high levels of collagen, leading to concomitantly loss of lung elasticity and function. Recent studies implicate the importance of microRNAs (miRNAs) in IPF but their regulation and individual pathological roles remain largely unknown. We used both RNA sequencing and quantitative RT-PCR strategies to systematically study TGF-ß1-induced alternations of miRNAs in human lung fibroblasts (HFL). Our data show that miR-133a was significantly upregulated by TGF-ß1 in a time- and concentration-dependent manner. Surprisingly, miR-133a inhibits TGF-ß1-induced myofibroblast differentiation whereas miR-133a inhibitor enhances TGF-ß1-induced myofibroblast differentiation. Interestingly, quantitative proteomics analysis indicates that miR-133a attenuates myofibroblast differentiation via targeting multiple components of TGF-ß1 profibrogenic pathways. Western blot analysis confirmed that miR-133a down-regulates TGF-ß1-induced expression of classic myofibroblast differentiation markers such as ɑ-smooth muscle actin (ɑ-SMA), connective tissue growth factor (CTGF) and collagens. miRNA Target Searcher analysis and luciferase reporter assays indicate that TGF-ß receptor 1, CTGF and collagen type 1-alpha1 (Col1a1) are direct targets of miR-133a. More importantly, miR-133a gene transferred into lung tissues ameliorated bleomycin-induced pulmonary fibrosis in mice. Together, our study identified TGF-ß1-induced miR-133a as an anti-fibrotic factor. It functions as a feed-back negative regulator of TGF-ß1 profibrogenic pathways. Thus, manipulations of miR-133a expression may provide a new therapeutic strategy to halt and perhaps even partially reverse the progression of IPF.

Up-regulated miR-133a orchestrates epithelial-mesenchymal transition of airway epithelial cells.

Dysregulation of microRNAs (miRNAs) contributes to epithelial-mesenchymal transition (EMT) of cancer, but the pathological roles of miRNAs in airway EMT of lung diseases remains largely unknown. We performed sequencing and real-time PCR analysis of the miRNA expression profile of human airway epithelial cells undergoing EMT, and revealed miR-133a to be one of the most common up-regulated miRNAs. MiR-133a was previously reported to be persistently up-regulated in airway epithelial cells of smokers. We found that mice exposed to cigarette smoke (CS) showed airway hyper-responsiveness, a typical symptom occurring in CS-related lung diseases, up-regulation of miR-133a and EMT marker protein N-cadherin in airway epithelium. Importantly, miR-133a overexpression induces airway epithelial cells to undergo spontaneous EMT via down-regulation of grainyhead-like 2 (GRHL2), an epithelial specific transcriptional factor. Loss of GRHL2 causes down-regulation of epithelial splicing regulatory protein 1 (ESRP1), a central coordinator of alternative splicing processes that are critical in the regulation of EMT. Down-regulation of ESRP1 induces isoform switching of adherens junction-associated protein p120-catenin, and leads to the loss of E-cadherin. Our study is the first to demonstrate that up-regulated miR-133a orchestrates airway EMT via alternative splicing processes, which points to novel therapeutic possibilities for the treatment of CS-related lung disease.

Amplified impedimetric immunosensor based on instant catalyst for sensitive determination of ochratoxin A.

A new impedimetric immunosensor for the fast determination of ochratoxin A (OTA) in food samples was developed based on the instant catalyst as enhancer. Initially, the signal tags were prepared via co-immobilization of anti-OTA antibody and amine-terminated dendrimer (PAMAM) on the graphene oxide nanosheets through the covalent interaction, which were utilized as a good platform for combining manganese ion (anti-OTA-GO-PAMAM-Mn(2+)). Upon target OTA introduction, a competitive-type immunoreaction was implemented between the analyte and the immobilized OTA-BSA on the electrode for the anti-OTA antibody on the graphene oxide nanosheets labels. After a competitive immunoassay format, the anti-OTA-GO-PAMAM-Mn(2+) were captured onto the electrode surface, which could induce the in situ formation of MnO2via classical redox reaction between Mn(2+) and KMnO4 on the immunesensing platform. Moreover, the generated MnO2 nanoparticles act as efficient catalyst could catalyze the 4-chloro-1-naphthol (4-CN) oxidation without H2O2 to generate an insoluble precipitation on the platform. Under the optimal conditions, the instant catalyst based impedimetric immunosensor displayed a wide dynamic working range between 0.1pgmL(-1) and 30ngmL(-1). The detection limit (LOD) of the assay was 0.055pgmL(-1). The developed method exhibited high selectivity and can be used for the determination of OTA in real red wine samples.

Novel glucometer-based immunosensing strategy suitable for complex systems with signal amplification using surfactant-responsive cargo release from glucose-encapsulated liposome nanocarriers.

Methods based on surfactant-responsive controlled release systems of cargoes from nanocontainers have been developed for bioanalytical applications, but most were utilized for drug delivery and a few reports were focused on immunoassays. Herein we design an in situ amplified immunoassay protocol for high-efficient detection of aflatoxins (aflatoxin B1, AFB1 used in this case) based on surfactant-responsive cargo release from glucose-encapsulated liposome nanocarriers with sensitivity enhancement. Initially, biotinylated liposome nanocarrier encapsulated with glucose was synthesized using a reverse-phase evaporation method. Thereafter, the nanocarrier was utilized as the signal-generation tag on capture antibody-coating microplate through classical biotin-avidin linkage after reaction with biotinylated detection antibody. Upon addition of buffered surfactant (1X PBS-Tween 20 buffer) into the medium, the surfactant immediately hydrolyzed the conjugated liposome, and released the encapsulated glucose from the nanocarriers, which could be quantitatively determined by using a low-cost personal glucometer (PGM). The detectable signal increased with the increment of target analyte. Under the optimal conditions, the assay could allow PGM detection toward target AFB1 as low as 0.6 pg mL(-1) (0.6 ppt). Moreover, the methodology also showed good reproducibility and high specificity toward target AFB1 against other mycotoxins and proteins, and was applicable for quantitatively monitoring target AFB1 in the complex systems, e.g., naturally contaminated/spiked peanut samples and serum specimens, with the acceptable results. Taking these advantages of simplification, low cost, universality and sensitivity, our design provides a new horizon for development of advanced immunoassays in future point-of-care testing.