Flexible iontronics with super stretchability, toughness and enhanced conductivity based on collaborative design of high-entropy topology and multivalent ion-dipole interactions.

All-solid-state ionic conductive elastomers (ASSICEs) are emerging as a promising alternative to hydrogels and ionogels in flexible electronics. Nevertheless, the synthesis of ASSICEs with concomitant mechanical robustness, superior ionic conductivity, and cost-effective recyclability poses a formidable challenge, primarily attributed to the inherent contradiction between mechanical strength and ionic conductivity. Herein, we present a collaborative design of high-entropy topological network and multivalent ion-dipole interaction for ASSICEs, and successfully mitigate the contradiction between mechanical robustness and ionic conductivity. Benefiting from the synergistic effect of this design, the coordination, de-coordination, and intrachain transfer of Li+ are effectively boomed. The resultant ASSICEs display exceptional mechanical robustness (breaking strength: 7.45 MPa, fracture elongation: 2621%, toughness: 107.19 MJ m-3) and impressive ionic conductivity (1.15 × 10-2 S m-1 at 25 °C). Furthermore, these ASSICEs exhibit excellent environmental stability (fracture elongation exceeding 1400% at 50 °C or -60 °C) and recyclability. Significantly, the application of these ASSICEs in a strain sensor highlights their potential in various fields, including human-interface communication, aerospace vacuum measurement, and medical balloon monitoring.

Magnetic Nanoparticle-Based Microfluidic Platform for Automated Enrichment of High-Purity Extracellular Vesicles.

Extracellular vesicles (EVs) are available in various biological fluids and have highly heterogeneous sizes, origins, contents, and functions. Rapid enrichment of high-purity EVs remains crucial for enhancing research on EVs in tumors. In this work, we present a magnetic nanoparticle-based microfluidic platform (ExoCPR) for on-chip isolation, purification, and mild recovery of EVs from cell culture supernatant and plasma within 29 min. The ExoCPR chip integrates bubble-driven micromixers and immiscible filtration assisted by surface tension (IFAST) technology. The bubble-driven micromixer enhances the mixing between immunomagnetic beads and EVs, eliminating the need for manual pipetting or off-chip oscillatory incubation. The high-purity EVs were obtained after passing through the immiscible phase interface where hydrophilic or hydrophobic impurities nonspecifically bound to SIMI were removed. The ExoCPR chip had a capture efficiency of 75.8% and a release efficiency of 62.7% for model EVs. We also demonstrated the powerful performance of the ExoCPR in isolating EVs from biological samples (>90% purity). This chip was further employed in clinical plasma samples and showed that the number of GPC3-positive EVs isolated from hepatocellular carcinoma patients was significantly higher than that of healthy individuals. This ExoCPR chip may provide a promising tool for EV-based liquid biopsy and other fundamental research.

Squeezed state in the hydrodynamic focusing regime for Escherichia coli bacteria detection.

Flow cytometry is an essential technique in single particle analysis and cell sorting for further downstream diagnosis, exhibiting high-throughput and multiplexing capabilities for many biological and biomedical applications. Although many hydrodynamic focusing-based microfluidic cytometers have been demonstrated with reduced size and cost to adapt to point-of-care settings, the operating conditions are not characterized systematically. This study presents the flow transition process in the hydrodynamic focusing mechanism when the flow rate or the Reynolds number increases. The characteristics of flow fields and mass transport were studied under various operating conditions, including flow rates and microchannel heights. A transition from the squeezed focusing state to the over-squeezed anti-focusing state in the hydrodynamic focusing regime was observed when the Reynolds number increased above 30. Parametric studies illustrated that the focusing width increased with the Reynolds number but decreased with the microchannel height in the over-squeezed state. The microfluidic cytometric analyses using microbeads and E. coli show that the recovery rate was maintained by limiting the Reynolds number to 30. The detailed analysis of the flow transition will provide new insight into microfluidic cytometric analyses with a broad range of applications in food safety, water monitoring and healthcare sectors.

A one-stage deep learning based method for automatic analysis of droplet-based digital PCR images.

Droplet-based dPCR offers many advantages over chip-based dPCR, such as lower processing cost, higher droplet density, higher throughput, while requiring less sample. However, the stochastic nature of droplet locations, uneven illuminations, and unclear droplet boundaries make automatic image analysis challenging. Most methods currently used to count a large amount of microdroplets rely on flow detection. Conventional machine vision algorithms cannot extract all information of the targets from complex backgrounds. Some two-stage methods, which first locate and then classify droplets according to their grayscale values, require high-quality imaging. In this study, we addressed these limitations by improving a one-stage deep learning algorithm named YOLOv5 and applying it to the detection task to realize one-stage detection. We introduced an attention mechanism module to increase the detection rate of small targets and used a new loss function to speed up the training process. Furthermore, we employed a network pruning method to facilitate the deployment of the model on mobile devices while preserving its performance. We validated the model with captured droplet-based dPCR images and found that the improved model accurately identified negative and positive droplets in complex backgrounds with an error rate of 0.65%. This method is characterized by its fast detection speed, high accuracy, and ability to be used on mobile devices or cloud platforms. Overall, the study presents a novel approach for detecting droplets in large-scale microdroplet images and provides a promising solution for accurate and efficient droplet counting in droplet-based dPCR.

Carboxymethylcellulose reinforced, double-network hydrogel-based strain sensor with superior sensing stability for long-term monitoring.

Hydrogel-based strain sensors have garnered significant attention for their potential for human health monitoring. However, its practical application has been hindered by water loss, freezing, and structural impairment during long-term motion monitoring. Here, a strain sensor based on double-network (DN) hydrogel of polyacrylamide (PAAm)/carboxymethylcellulose (CMC) was developed in a ternary solvent system of lithium chloride (LiCl)/ethylene glycol (EG)/H2O through a facile one-pot radical polymerization strategy. The incorporation of EG effectively mitigated the hydration of lithium salts by generating stable ion clusters with Li+ and stronger hydrogen bonds within the polymer matrix. The sensor demonstrated excellent mechanical properties, including a stretchability of 1858 %, toughness of 1.80 MJ/m3, and recoverability of 102 %. Furthermore, the LiCl/EG/H2O ternary system resulted in high conductivity, excellent anti-freezing performance, and superior sensing stability. In addition, the sensor exhibited remarkable sensitivity, enabling the monitoring of human movements ranging from subtle to significant deformations, including throat motion and bending of the elbow, wrist, finger, and lower limb. This study presents a viable approach for constructing hydrogel-based strain sensors with exceptional sensing stability for long-term tracking of human motions.

Immunomagnetic Separation Method Integrated with the Strep-Tag II System for Rapid Enrichment and Mild Release of Exosomes.

Exosomes are important participants in numerous pathophysiological processes and hold promising application value in cancer diagnosis, monitoring, and prognosis. However, the small size (40-160 nm) and high heterogeneity of exosomes make it still challenging to enrich exosomes efficiently from the complex biological fluid microenvironment, which has largely restricted their downstream analysis and clinical application. In this work, we introduced a novel method for rapid isolation and mild release of exosomes from the cell culture supernatant. A Strep-tag II-based immunomagnetic isolation (SIMI) system was constructed by modifying the capture antibodies onto magnetic nanoparticles through specific and reversible recognition between Strep-Tactin and Strep-tag II. Due to their high affinity and binding selectivity, exosomes could be isolated within 38 min with an isolation efficiency of 82.5% and a release efficiency of 62%. Compared with the gold-standard ultracentrifugation, the SIMI system could harvest nearly 59% more exosomes from the 293 T cell culture medium with shorter isolation time and higher purity. In addition, cellular uptake assay indicated that exosomes released from magnetic nanoparticles could maintain their high biological activity. These superior characteristics show that this novel method is a fast, efficient, and nondestructive exosome isolation tool and thus could potentially be further utilized in various exosome-related applications, e.g., disease diagnosis and drug delivery.

NOS2/miR-493-5p Signaling Regulates in the LPS-Induced Inflammatory Response in the RAW264.7 Cells.

Tuberculosis (TB) is a fatal infectious disease; however, the molecular mechanisms underlying the pathogenicity of TB remain elusive. The present study aims to identify potential biomarkers associated with Mycobacterium tuberculosis (M.tb) infection by using integrated bioinformatics and in vitro validation studies. GSE50050, GSE78706, and GSE108844 data from the gene expression omnibus (GEO) database were downloaded to identify differentially expressed genes (DEGs). The functions of DEGs were further subjected to gene ontology (GO) and KEGG pathway analysis. The hub genes from the DEGs were determined based on the protein-protein interaction (PPI) network analysis. Finally, the hub genes were experimentally validated using the in vitro functional studies. A total of 26 common DEGs were identified among GSE50050, GSE78706, and GSE108844. The functional enrichment analysis showed that the common DEGs were associated with cytokines response and TB pathways. The PPI network analysis identified nine hub genes. Further in vitro studies showed that nitric oxide synthase 2 (NOS2) was up-regulated in RAW264.7 cells upon lipopolysaccharides (LPS) stimulation, which was accompanied by increased inflammatory cytokines release. Furthermore, NOS2 was found to be a target of miR-493-5p, which was confirmed by luciferase reporter assay. NOS2 was repressed by miR-493-5p overexpression and was up-regulated after miR-493-5p inhibition in RAW264.7 cells. The rescue experiments showed that LPS-induced increase in the inflammatory cytokines of the RAW264.7 cells was significantly attenuated by NOS2 knockdown and miR-493-5p overexpression. Collectively, our results for the first time demonstrated that NOS2/miR-493-5p signaling pathway may potentially involve in the inflammatory response during bacterial infection such as M. tb infection.

A sample-to-answer DNA detection microfluidic system integrating sample pretreatment and smartphone-readable gradient plasmonic photothermal continuous-flow PCR.

As the gold standard for nucleic acid detection, full-process polymerase chain reaction (PCR) analysis often falls into the dilemma of complex workflow, time-consuming, and high equipment costs. Therefore, we designed and optimized a DNA quantification microfluidic system by strategically integrating sample pretreatment and a smartphone-readable gradient plasmonic photothermal (GPPT) continuous-flow PCR (CF-PCR). Through preloading and sequential injection of immiscible extraction reagents, combined with magnetic bead (MB) manipulation, the microfluidic chip successfully purified and concentrated 100 µL of HBV-DNA spiked plasma into a 20-µL purified sample within 14 minutes. With a digital PCR platform, the optimized experiments showed that the DNA extraction efficiency can reach 69% at an immiscible reagent configuration ratio of 10 : 10 : 1 : 12 : 2 (sample : lysis/binding buffer : MB : silicone oil : eluent) and a flow rate of 25 µL min-1. For the first time, we used gold nanorod (AuNR)-doped PDMS to prepare a CF-PCR submodule for the amplification of a 40 µL PCR mixture. Due to the plasmonic photothermal effect of AuNRs and the gradient intensity of an expanded laser spot, the PCR thermal gradient was formed on a coin-sized area. The compact annular thermal-microfluidic layout, optimized DNA dye concentration, and chip transmittance synergistically enable a rarely reported smartphone-based fluorescence CF-PCR, greatly simplifying thermal control and detection setup. Prototype construction and validation experiments show that the microsystem can complete the sample-to-answer quantification of HBV-DNA with a dynamic linear range from 1.2 × 101 to 1.2 × 106 copies per µL in ∼37 minutes. This novel microfluidic solution effectively bridges the technical gap between the CF-PCR, sample pretreatment and result characterization, making the workflow standardized and rapid and requiring <15% of the commercial instrument cost. The simplicity, rapidity and low cost of this work make it promising for applications in decentralized laboratories and low-resource settings.

A one-pot CRISPR/Cas13a-based contamination-free biosensor for low-cost and rapid nucleic acid diagnostics.

The pandemic due to the outbreak of 2019 coronavirus disease (COVID-19) caused by novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has raised significant public health concerns. Rapid, affordable, and accurate diagnostic testing not only paves the way for the effective treatment of diseases, but also plays a crucial role in preventing the spreading of infectious diseases. Herein, a one-pot CRISPR/Cas13a-based visual biosensor was proposed and developed for the rapid and low-cost nucleic acid detection. By combining Cas13a cleavage and Recombinase Polymerase Amplification (RPA) in a one-pot reaction in a disposable tube-in-tube vessel, amplicon contamination could be completely avoided. The RPA reaction is carried out in the inner tube containing two hydrophobic holes at the bottom. After the completion of amplification reaction, the reaction solution enters the outer tube containing pre-stored Cas13a reagent under the action of centrifugation or shaking. Inner and outer tubes are combined to form an independent reaction pot to complete the nucleic acid detection without opening the lid. This newly developed nucleic acid detection method not only meets the need of rapid nucleic acid detection at home without the need for any specialized equipment, but also fulfils the requirement of rapid on-site nucleic acid detection with the aid of small automated instruments. In this study, CRISPR/Cas13a and CRISPR/Cas12a were used to verify the reliability of the developed one-pot nucleic acid detection method. The performance of the system was verified by detecting the DNA virus, i.e., African swine fever virus (ASFV) and the RNA virus, i.e., SARS-Cov-2. The results indicate that the proposed method possesses a limit of detection of 3 copy/µL. The negative and positive test results are consistent with the results of real-time fluorescence quantitative polymerase chain reaction (PCR), but the time required is shorter and the cost is lower. Thus, this study makes this method available in resource-limited areas for the purpose of large-scale screening and in case of epidemic outbreak.

Anisotropic conductive reduced graphene oxide/silk matrices promote post-infarction myocardial function by restoring electrical integrity.

Myocardial infarction (MI) remains the leading cause of death globally, often leading to impaired cardiac function and pathological myocardial microenvironment. Electrical conduction abnormalities of the infarcted myocardium not only induce adverse myocardial remodeling but also prevent tissue repair. Restoring the myocardial electrical integrity, particularly the anisotropic electrical signal propagation within the injured area after infarction is crucial for an effective function recovery. Herein, optimized reduced graphene oxide (rGO) functionalized electrospun silk fibroin (rGO/silk) biomaterials presenting anisotropic conductivity and enhanced suturablity were developed and investigated as cardiac patches for their potential in improving the post-MI myocardial function of rat models. The results show that the anisotropic conductive rGO/silk patches exhibit remarkable therapeutic effect on repairing the infarcted myocardium compared to the nonconductive silk and isotropic conductive rGO/silk patches as determined by the enhanced pumping function, reduced susceptibility to arrhythmias, thickened left ventricular walls and improved survival of functional cardiomyocytes. Their notable effect on promoting the angiogenesis of capillaries in the infarcted myocardium has also been demonstrated. This study highlights an effective and biomimetic reconstruction of the electrical myocardial microenvironment based on the anisotropic conductive rGO/silk biomaterials as a promising option for promoting the repair of infarcted myocardium. STATEMENT OF SIGNIFICANCE: The dysfunctional electrical microenvironment in the infarcted myocardium not only aggravates the adverse myocardial remodeling but also limits the effect of cardiac regenerative medicine. Although various conductive biomaterials have been employed to restore the electrical network in the infarcted myocardium in vivo, the anisotropic nature of the myocardial electrical microenvironment which enables directional electrical signal propagation were neglected. In this study, an anisotropic conductive rGO/silk biomaterial system is developed to improve the myocardial function post infarction by restoring the anisotropic electrical microenvironment in the infarcted myocardium. The promoted effects of anisotropic conductive grafts on repairing infarcted hearts are demonstrated with improved pumping function, cardiomyocyte survival, resistance to ventricular fibrillation, and angiogenesis of capillary network.

Massive droplet generation for digital PCR via a smart step emulsification chip integrated in a reaction tube.

Step emulsification (SE) devices coupled with parallel generation nozzles are widely used in the production of large-scale monodisperse droplets, especially for droplet-based digital polymerase chain reaction (ddPCR) analysis. Although current ddPCR systems based on the SE method can provide a fully enclosed ddPCR scheme, high demands on chip fabrication and system control will increase testing costs and reduce its flexibility in ddPCR analysis. In this study, a compact SE device, integrating a smart SE chip into a reaction tube, was developed to prepare large-scale water-in-fluorinated-oil droplets for ddPCR analysis. The SE chip contained dozens of droplet-generation nozzles. By adjusting the nozzle height of the SE chip, monodisperse droplets in a picolitre to nanolitre vloume could be prepared at a production rate of tens to hundreds of microlitres per minute. Subsequently, we utilized such an integrated SE device to prepare monodisperse droplets for ddPCR experiments. The volume of PCR reagent and the number of droplets could be flexibly adjusted according to the requirements of the ddPCR analysis. The quantitative results showed that emulsions prepared by the SE device could achieve ddPCR detection with high accuracy, good repeatability, and an adaptive dynamic range, which also demonstrated the robustness and reliability of such devices in the droplet preparation. Thus, this compact SE device provides an inexpensive, flexible, and simplified droplet preparation method for digital PCR quantitative analysis.

Silencing KPNA2 inhibits IL-6-induced breast cancer exacerbation by blocking NF-κB signaling and c-Myc nuclear translocation in vitro.

AIMS: Recently, studies indicated that inflammation could exacerbate the development of BC. Karyopherin α-2 (KPNA2) is a molecule which modulates nucleocytoplasmic transport and is involved in malignant cellular behavior and carcinogenesis. Our study aims to elucidate the role of KPNA2 in BC pathogenesis and explore the mechanism of KPNA2 in regulating inflammation-induced BC exacerbations. MAIN METHODS: We measured the expression of KPNA2 in BC cells. Through loss-of-function experiments, the functional role of KPNA2 in MCF-7 and MDA-MB-468 cells was evaluated. SK-BR-3 cells were treated with IL-6 as an inflammatory in vitro model of BC. ELISA determination exhibited the contents of cytokines. RANKL and leptomycin B treatments activated NF-κB signaling and inhibited the nuclear translocation of c-Myc, respectively. KEY FINDINGS: The results showed that KPNA2 was significantly up-regulated in BC and silencing KPNA2 inhibited the proliferation, migration and invasion of BC cells, while the cycle arrest was induced, via blocking NF-κB signaling and c-Myc nuclear translocation. IL-6 stimulated the secretions of IL-8 and IL-17 in BC cells, and elevated KPNA2 expression. However, KPNA2 knockdown suppressed the inflammatory responses and malignant progression of BC induced by IL-6. SIGNIFICANCE: In conclusion, our study illustrated that KPNA2 regulated BC development, as well as IL-6-induced inflammation and exacerbation, via NF-κB signaling and c-Myc nuclear translocation. This may provide a novel target for BC therapy.

Down-regulation of lncRNA NEAT1 regulated by miR-194-5p/DNMT3A facilitates acute myeloid leukemia.

OBJECTIVE: miR-194-5p and NEAT1 have been reported to be associated with multiple malignancies, but their roles in acute myeloid leukemia (AML) remains not fully understood. METHODS: Bone marrow samples were collected for monocyte separation. qRT-PCR assay was performed to investigate the expression patterns of NEAT1 and miR-194-5p in AML. CCK-8, soft agar colony formation, flow cytometry and transwell assays were employed to explore the biological functions of NEAT1 or miR-194-5p. Methylation PCR was performed to monitor the methylation of NEAT1. Luciferase reporter assay was subjected to verify the relationship between miR-194-5p and DNMT3A. Immunofluorescence and western blotting were performed to detect the alterations of protein expression. RESULTS: NEAT1 and miR-194-5p were both down-regulated in AML. Overexpression of either NEAT1 or miR-194-5p repressed proliferation, induced apoptosis and restrained migration and invasion of AML cells. There was a negative correlation between NEAT1 and DNMT3A in AML. Knockdown of DNMT3A dramatically decreased the methylation of NEAT1. Moreover, DNMT3A was identified as a downstream target of miR-194-5p. Furthermore, down-regulation of DNMT3A rescued the impacts on the malignant phenotypes of NEAT1 inhibition by miR-194-5p inhibitor. CONCLUSION: Altogether, down-regulation of NEAT1 mediated by miR-194-5p/DNMT3A axis promotes AML progression, which might provide therapeutic targets in AML treatment.

An LED-Driven AuNPs-PDMS Microfluidic Chip and Integrated Device for the Detection of Digital Loop-Mediated Isothermal DNA Amplification.

The sensitive quantification of low-abundance nucleic acids holds importance for a range of clinical applications and biological studies. In this study, we describe a facile microfluidic chip for absolute DNA quantifications based on the digital loop-mediated isothermal amplification (digital LAMP) method. This microfluidic chip integrates a cross-flow channel for droplet generation with a micro-cavity for droplet tiling. DNA templates in the LAMP reagent were divided into ~20,000 water-in-oil droplets at the cross-flow channel. The droplets were then tiled in the micro-cavity for isothermal amplification and fluorescent detection. Different from the existing polydimethylsiloxane (PDMS) microfluidic chips, this study incorporates gold nanoparticles (AuNPs) into PDMS substrate through silica coating and dodecanol modification. The digital LAMP chip prepared by AuNPs-PDMS combines the benefits of the microstructure manufacturing performance of PDMS with the light-to-heat conversion advantages of AuNPs. Upon illumination with a near infrared (NIR) LED, the droplets were stably and efficiently heated by the AuNPs in PDMS. We further introduce an integrated device with a NIR heating unit and a fluorescent detection unit. The system could detect HBV (hepatitis B virus)-DNA at a concentration of 1 × 101 to 1 × 104 copies/µL. The LED-driven digital LAMP chip and the integrated device; therefore, demonstrate high accuracy and excellent performance for the absolute quantification of low-abundance nucleic acids, showing the advantages of integration, miniaturization, cost, and power consumption.

Integrated Single Microbead-Arrayed µ-Fluidic Platform for the Automated Detection of Multiplexed Biomarkers.

An automated, single microbead-arrayed µ-fluidic immunoassay (AMIA) device is innovatively devised in this study, which enables the highly sensitive and simultaneous detection of multiplex biomarkers with fully automatic operations. The AMIA platform not only achieves automated assay processing and multiplexed target detection by integrating single microbead manipulation, sample loading, multistep washing, and immunoreaction on a microfluidic chip but also confers high sensitivity due to the highly efficient signal enriching effect on a single microbead by the use of only a routine sandwich immunoreaction. As such, as low as the pg/mL level of multiplexed protein biomarkers can be simultaneously determined in a quite small volume of serum (∼20 µL is enough), which can well meet the clinical demand for disease screening and prognosis. What is more, the detection results of several clinically important biomarkers in clinical samples with the AMIA platform exhibit excellent consistency with those obtained by using a standard clinical test. Thus, in virtue of the excellent features in terms of high sensitivity, multiplexing capability, generality, and high degree of automation, the AMIA provides a practical and user-friendly platform for assaying different biomarkers in clinical diagnostics and point-of-care testing.

Smartphone-Based Droplet Digital LAMP Device with Rapid Nucleic Acid Isolation for Highly Sensitive Point-of-Care Detection.

While advances in microfluidics have enabled rapid and highly integrated detection of nucleic acid targets, the detection sensitivity is still unsatisfactory in the current POC (point-of-care) detection systems, especially for low abundance samples. In this study, a chip that integrates rapid nucleic acid extraction based on IFAST (immiscible phase filtration assisted by surface tension) and digital isothermal detection was developed to achieve highly sensitive POC detection within 60 min. Based on the interface theory, the factors influencing the interface stability of the IFAST process were studied, and the IFAST nucleic acid extraction conditions were optimized to increase the nucleic acid extraction recovery rate to 75%. Spiral mixing channel and flow-focusing droplet generation structure were designed to achieve the mixing and sample partitioning by applying negative pressure. A portable microdroplet fluorescence detection device was developed based on smartphone imaging. Validation tests were carried out for quantification of low-abundance cfDNA and detection of mutations.

Sample-to-Answer Hepatitis B Virus DNA Detection from Whole Blood on a Centrifugal Microfluidic Platform with Double Rotation Axes.

A point-of-care apparatus for hepatitis virus detection requires simple and easy-to-use processing steps and should have the same diagnostic capability as that in the central laboratory. However, no automated and efficient methods for hepatitis B virus (HBV) sample-to-answer detection include serum separation, and complete prestorage of reagents has been developed. We developed an automated sample-to-answer disc for rapid HBV detection from whole blood based on a double rotation axes centrifugal microfluidic platform. The disc with complete prestorage of reagents features fully automated and integrated serum separation from whole blood, magnetic bead-based DNA extraction, aliquoting of the nucleic acid, and real-time polymerase chain reaction. A laser diode for sequential release of prestored liquid reagents was used. Processing merely requires manual loading of the sample into the disc. We demonstrate successful sample-to-answer detection of HBV in a 500 µL whole blood sample with sample concentrations down to 102 copies/mL. The total time of the whole detection from sample-to-result is about 48 min. The disc provides a user-friendly molecular diagnostic system for rapid analysis of HBV without demanding a complicated laboratory instrument and major manual operation time. Overall, the results indicated that the developed disc could be used for HBV molecular diagnosis.

Knockdown of TRB3 improved the MPP+/MPTP-induced Parkinson's disease through the MAPK and AKT signaling pathways.

This study aims to investigate the function and molecular mechanisms of Tribbles homolog 3 (TRB3) on the MPP+/MPTP-induced Parkinson's disease (PD). In this study, MPP+-induced PD cellular model and MPTP-caused PD mice model were established. Following the transfection with TRB3-shRNA, cell viability, cell apoptosis, ROS level, and the ratio of p-p38/ p38, p-JNK/JNK, p-AKT/AKT were examined. At the same time, behavior assessment of wild type female C57BL/6 mice and whole-body TRB3 knockout mice PD models caused by MPTP were performed by Rotarod test and Open-field test. The results showed that TRB3 was markedly upregulated in MPP+-induced cellular model through ATF4/CHOP pathway. Knockdown of TRB3 significantly decreased the MPP+-induced reduction of cell viability, augment of cell apoptosis and accumulation of ROS, inhibited the phosphorylation of p38 and JNK, and promoted the phosphorylation of AKT, in vitro. Further, knockout of TRB3 improved the behavior impairment of PD mice induced by MPTP, in vivo. In conclusion, knockdown of TRB3 has a neuroprotective effect on MPTP/MPP+-induced PD cellular and mice models, through regulating MAPK and AKT signaling pathways.

MiR-140 modulates the inflammatory responses of Mycobacterium tuberculosis-infected macrophages by targeting TRAF6.

This study aimed to examine miR-140 expression in clinical samples from tuberculosis (TB) patients and to explore the molecular mechanisms of miR-140 in host-bacterial interactions during Mycobacterium tuberculosis (M tb) infections. The miR-140 expression and relevant mRNA expression were detected by quantitative real-time PCR (qRT-PCR); the protein expression levels were analysed by ELISA and western blot; M tb survival was measured by colony formation unit assay; potential interactions between miR-140 and the 3' untranslated region (UTR) of tumour necrosis factor receptor-associated factor 6 (TRAF6) was confirmed by luciferase reporter assay. MiR-140 was up-regulated in the human peripheral blood mononuclear cells (PBMCs) from TB patients and in THP-1 and U937 cells with M tb infection. Overexpression of miR-140 promoted M tb survival; on the other hand, miR-140 knockdown attenuated M tb survival. The pro-inflammatory cytokines including interleukin 6, tumour necrosis-α, interleukin-1ß and interferon-γ were enhanced by M tb infection in THP-1 and U937 cells. MiR-140 overexpression reduced these pro-inflammatory cytokines levels in THP-1 and U937 cells with M tb infection; while knockdown of miR-140 exerted the opposite actions. TRAF6 was identified to be a downstream target of miR-140 and was negatively modulated by miR-140. TRAF6 overexpression increased the pro-inflammatory cytokines levels and partially restored the suppressive effects of miR-140 overexpression on pro-inflammatory cytokines levels in THP-1 and U937 cells with M tb infection. In conclusion, our results implied that miR-140 promoted M tb survival and reduced the pro-inflammatory cytokines levels in macrophages with M tb infection partially via modulating TRAF6 expression.

Rapid isolation of cfDNA from large-volume whole blood on a centrifugal microfluidic chip based on immiscible phase filtration.

Cell-free (cf) nucleic acids are considered important and have been used as selective biomarkers. Conventional techniques for cf nucleic acid biomarker isolation from blood are generally time-consuming, complicated, and expensive. This study describes a lab-on-a-disk system equipped with newly developed immiscible filtration assisted by surface tension (IFAST), which can achieve the rapid isolation of cfDNA from whole blood. The principle of centrifugal IFAST (C-IFAST) is introduced. An arch-like channel for magnetic bead transfer in the immiscible phase is designed, which builds both a virtual water-air "wall" and an air-oil "wall" to prevent the blending of water and oil. The entire process requires less than 15 min and achieves the recovery of 65% of cfDNA from plasma and 30% from whole blood. Experiments were performed to test the validity of the chip, showing that this technique takes less time to obtain results of identical quality compared to commercial kits. The proposed C-IFAST method enables rapid and reliable cfDNA isolation from large whole blood volume (4 ml) and can potentially be used in "liquid biopsy" point-of-care diagnosis.