Multi-omics study reveals different pathogenesis of the generation of skin lesions in SLE and IDLE patients.

Lupus erythematosus (LE) is a heterogeneous, antibody-mediated autoimmune disease. Isolate discoid LE (IDLE) and systematic LE (SLE) are traditionally regarded as the two ends of the spectrum, ranging from skin-limited damage to life-threatening multi-organ involvement. Both belong to LE, but IDLE and SLE differ in appearance of skin lesions, autoantibody panels, pathological changes, treatments, and immunopathogenesis. Is discoid lupus truly a form of LE or is it a completely separate entity? This question has not been fully elucidated. We compared the clinical data of IDLE and SLE from our center, applied multi-omics technology, such as immune repertoire sequencing, high-resolution HLA alleles sequencing and multi-spectrum pathological system to explore cellular and molecular phenotypes in skin and peripheral blood from LE patients. Based on the data from 136 LE patients from 8 hospitals in China, we observed higher damage scores and fewer LE specific autoantibodies in IDLE than SLE patients, more uCDR3 sharing between PBMCs and skin lesion from SLE than IDLE patients, elevated diversity of V-J recombination in IDLE skin lesion and SLE PBMCs, increased SHM frequency and class switch ratio in IDLE skin lesion, decreased SHM frequency but increased class switch ratio in SLE PBMCs, HLA-DRB1*03:01:01:01, HLA-B*58:01:01:01, HLA-C*03:02:02:01, and HLA-DQB1*02:01:01:01 positively associated with SLE patients, and expanded Tfh-like cells with ectopic germinal center structures in IDLE skin lesions. These findings suggest a significant difference in the immunopathogenesis of skin lesions between SLE and IDLE patients. SLE is a B cell-predominate systemic immune disorder, while IDLE appears limited to the skin. Our findings provide novel insights into the pathogenesis of IDLE and other types of LE, which may direct more accurate diagnosis and novel therapeutic strategies.

Gravity-driven flow control in a fully integrated microfluidic cartridge for molecular point-of-care testing.

Molecular point-of-care testing (POCT) system is crucial for the timely prevention and control of infectious diseases. We recently proposed a gravity-driven microfluidic cartridge for molecular POCT detection, without the need for external sources or actuators, demonstrating the advantages in terms of the reduced cartridge size and low development costs. How to achieve precise control of liquid flow behavior is challenging for the gravity-driven cartridge. In this work, we explored the underlying mechanism of flow control in the cartridge and offered optimized solutions for our cartridge design to achieve precise control of dynamic flow rates and enhance pumping efficiency significantly. Through the computational fluid dynamics simulations, we demonstrated that adopting an asymptotic contraction chamber geometry design and a closed-loop air flow channel design with the cartridge inlet can facilitate stable laminar flow of the liquid in our microfluidic cartridge, enabling precise control of flow velocity. We further optimized the microchannel diameter and the contact angle of the liquid with the microchannel wall. The effectiveness of the optimized cartridge for POCT detection was well validated by the accurate detection of the human papillomavirus type 16 virus in the 120 clinical swab samples.

Novel aerosol detection platform for SARS­CoV­2: Based on specific magnetic nanoparticles adsorption sampling and digital droplet PCR detection.

The SARS­CoV­2 virus is released from an infectious source (such as a sick person) and adsorbed on aerosols, which can form pathogenic microorganism aerosols, which can affect human health through airborne transmission. Efficient sampling and accurate detection of microorganisms in aerosols are the premise and basis for studying their properties and evaluating their hazard. In this study, we built a set of sub-micron aerosol detection platform, and carried out a simulation experiment on the SARS­CoV­2 aerosol in the air by wet-wall cyclone combined with immunomagnetic nanoparticle adsorption sampling and ddPCR. The feasibility of the system in aerosol detection was verified, and the influencing factors in the detection process were experimentally tested. As a result, the sampling efficiency was 29.77%, and extraction efficiency was 98.57%. The minimum detection limit per unit volume of aerosols was 250 copies (102 copies/mL, concentration factor 2.5).

Programmable Biosensors Based on RNA-Guided CRISPR/Cas Endonuclease.

Highly infectious illnesses caused by pathogens constitute severe threats to public health and lead to global economic loss. The use of robust and programmable clustered regularly interspaced short palindromic repeat and CRISPR-associated protein (CRISPR-Cas) systems, repurposed from genome-engineering applications has markedly improved traditional nucleic acid detection for precise identification, independently enabling rapid diagnostics of multiplex biomarker with genetic and mutation related to tumors, and microbial pathogens. In this review, we delineate the utility of the current CRISPR-Cas enzyme as biosensors by which these effector toolkits achieve recognition, signaling amplification, and finally, accurate detection. Additionally, we discuss the details of the dominance and hurdles related to expanding this revolutionary technology into an effective and convenient contraption crucial for improving the rational redesign to CRISPR/Cas biosensing. Overall, this review provides an insight into the current status of rapid and POC diagnostic systems by CRISPR/Cas tools.

Encapsulation of glucose oxidase in Fe(III)/tannic acid nanocomposites for effective tumor ablation via Fenton reaction.

Increasing the content of reactive oxygen species (ROS) with the assistance of nanoformulations in cancer cells via the Fenton reaction is considered an effective method to treat cancer. However, the efficiency of the Fenton reaction is affected by the level of H2O2, the selection of iron ions in different nanoformulations, etc. Herein, we use FeIII-tannic acid (FeIIITA) nanocomposites as the carrier to deliver glucose oxidase (GOD) which can solve the problem of insufficient endogenous H2O2 by catalytically converting the glucose. In comparison with traditional Fe2+/Fe3+, FeIIITA nanocomposites perform higher catalytic activity in converting H2O2 to high toxic hydroxyl radicals (·OH) due to the TA-mediated reduction of Fe3+. So, the integration of GOD and TA in the construction of nanocomposites significantly enhances the efficiency of the Fenton reaction. In vitro experiments show that ·OH produced by GOD-FeIIITA nanocomposites can not only achieve a good anticancer effect at a low concentration but also promote degradability of the nanocomposites. When it is only 1.08 µg · ml-1, the cell apoptosis rate has reached 76.91%. In vivo experiments further demonstrate that GOD-FeIIITA nanocomposites can significantly inhibit tumor growth. So this work lays a good foundation for Fenton reaction-based cancer treatment.

Application of magnetic nanoparticles in nucleic acid detection.

Nucleic acid is the main material for storing, copying, and transmitting genetic information. Gene sequencing is of great significance in DNA damage research, gene therapy, mutation analysis, bacterial infection, drug development, and clinical diagnosis. Gene detection has a wide range of applications, such as environmental, biomedical, pharmaceutical, agriculture and forensic medicine to name a few. Compared with Sanger sequencing, high-throughput sequencing technology has the advantages of larger output, high resolution, and low cost which greatly promotes the application of sequencing technology in life science research. Magnetic nanoparticles, as an important part of nanomaterials, have been widely used in various applications because of their good dispersion, high surface area, low cost, easy separation in buffer systems and signal detection. Based on the above, the application of magnetic nanoparticles in nucleic acid detection was reviewed.

A Sensitive Aptasensor Based on a Hemin/G-Quadruplex-Assisted Signal Amplification Strategy for Electrochemical Detection of Gastric Cancer Exosomes.

Emerging evidence indicates that exosomes derived from gastric cancer cells enhance tumor migration and invasion through the modulation of the tumor microenvironment. However, it remains a major problem to detect cancer-specific exosomes due to technical and biological challenges. Most of the methods reported could not achieve efficient detection of tumor-derived exosomes in the background of normal exosomes. Herein, a label-free electrochemical aptasensor is presented for specific detection of gastric cancer exosomes. This platform contains an anti-CD63 antibody modified gold electrode and a gastric cancer exosome specific aptamer. The aptamer is linked to a primer sequence that is complementary to a G-quadruplex circular template. The presence of target exosomes could trigger rolling circle amplification and produce multiple G-quadruplex units. This horseradish peroxidase mimicking DNAzyme could catalyze the reduction of H2 O2 and generate electrochemical signals. This aptasensor exhibits high selectivity and sensitivity toward gastric cancer exosomes with a detection limit of 9.54 × 102 mL-1 and a linear response range from 4.8 × 103 to 4.8 × 106 exosomes per milliliter. Therefore, this electrochemical aptasensor is expected to become a useful tool for the early diagnosis of gastric cancer.

Importance of Polyacrylamide Hydrogel Diverse Chains and Cross-Linking Density for Cell Proliferation, Aging, and Death.

Diverse chains and cross-linking density of polymers are important for cell proliferation, aging, and death. In this study, by controlling the component ratio of N,N'-methylenebisacrylamide (Bis)/acrylamide (Acr), we prepared polyacrylamide (PAM) hydrogels with three different polymer structures using ultraviolet irradiation. Moreover, we quantified their Flory's cross-linking densities, gel concentrations, and mechanical properties and evaluated their influence to HL-7702 liver cell behavior and metabolism. Results showed that PAM hydrogel at a ratio of Bis/Acr = 1:50 (Acr-50) owned the highest cross-linking density (0.04), which provided abundant binding sites for cell adhesion and allowed for rapid cell proliferation. On the basis of the binding sites, cells had strong traction interaction from fibrillate adhesion with the polymers, allowed easy cell migration, and induced the living cell aggregations with a diameter of 800 µm. Cells in aggregation exhibited healthy cell phenotypes and functions, and also the mitosis of the cells in aggregation is exactly the same with the cells in tissue. It is concluded that cell proliferation, aging, and death can be controlled by adjusting the cross-linking density and diverse chains of matrix hydrogels. This work will be helpful to design new functional soft biomaterials for tissue regeneration in the future.

QuickAnalysis: A Software Designed and Developed for a Portable On-Site Pathogen Detection System.

To develop a software program used for controlling the portable on-site pathogen detection system which is based on real-time PCR, this software is developed by C# over the Microsoft Visual Studio 2013 and used on local computers which are equipped with Windows systems. Taking the actual demand as the guidance, it constructs according to the framework design and unifies the barcode technology, the cloud service, and the Bluetooth technology. Based on the above methods, this software can meet the demands of the system such as free experiment design, auto-control of the hardware and efficient data management. During the operation of this program, the serial port and network communication between it and the device and web server remains stable, no matter it is processing the data collection or data analysis and display. It is feasible and practical to apply this software to the portable on-site pathogen detection system in clinical practices and research work.

The Liquid Level Detection System Based on Pressure Sensor.

Micro-liquid distribution operation has been a core part of the fully automatic liquid handling platform in clinical testing. Accurate and fast automatic detection of liquid level with disposable tip is the precondition to ensure the stability of the whole operation of liquid handling platform. By further research on the liquid level sensing technology of pressure sensor, in this paper we designed a new type of accurate and stable micro-liquid level detection system and verified its stability, instantaneity and accuracy by setting up a small reagent distribution platform. It was shown that the liquid level detection system designed in this paper has reliable stability, good instantaneity and high accuracy, with the liquid level detecting accuracy up to 100%, the response speed after detecting the liquid level reaching millisecond and the depth in the liquid level less than 0.3 mm. Besides, there is no limit on the physical and chemical properties of the liquids to be tested, which is consistent with the technical indicators of the liquid handling platform.

CRISPR-Cas13a mediated nanosystem for attomolar detection of canine parvovirus type 2.

Canine parvovirus type 2 (CPV-2) infection is the most lethal disease of dogs with higher mortality in puppies worldwide. In today's world, dogs are an integral part of our communities as well as dogs breeding and rearing has become a lucrative business. Therefore, a fast, accurate, portable, and cost-effective CPV-2 detection method with the ability for on-site detection is highly desired. In this study, we for the first time proposed a nanosystem for CPV-2 DNA detection with RNA-guided RNA endonuclease Cas13a, which upon activation results in collateral RNA degradation. We expressed LwCas13a in prokaryotic expression system and purified it through nickel column. Activity of Cas13a was verified by RNA-bound fluorescent group while using a quenched fluorescent probe as signals. Further Cas13a was combined with Recombinase polymerase amplification (RPA) and T7 transcription to establish molecular detection system termed specific high-sensitivity enzymatic reporter un-locking (SHERLOCK) for sensitive detection of CPV-2 DNA. This nanosystem can detect 100 amol/L CPV-2 DNA within 30 min. The proposed nanosystem exhibited high specificity when tested for CPV-2 and other dog viruses. This CRISPR-Cas13a mediated sensitive detection approach can be of formidable advantage during CPV-2 outbreaks because it is time-efficient, less laborious and does not involve the use of sophisticated instruments.

Molecular Engineering-Based Aptamer-Drug Conjugates with Accurate Tunability of Drug Ratios for Drug Combination Targeted Cancer Therapy.

Polytherapy (or drug combination cancer therapy (DCCT)), targeting multiple mechanisms associated with tumor proliferation, can efficiently maximize therapeutic efficacy, decrease drug dosage, and reduce drug resistance. However, most DCCT strategies cannot coordinate the specific delivery of a drug combination in an accurately tuned ratio into cancer cells. To address these limitations, the present work reports the engineering of circular bivalent aptamer-drug conjugates (cb-ApDCs). The cb-ApDCs exhibit high stability, specific recognition, excellent cellular uptake, and esterase-triggered release. Furthermore, the drug ratios in cb-ApDCs can be tuned for an enhanced synergistic effect without the need for complex chemistry. Therefore, cb-ApDCs provide a promising platform for the development of DCCT strategies for different drug combinations and ratios.

A sample-in-digital-answer-out system for rapid detection and quantitation of infectious pathogens in bodily fluids.

A variety of automated sample-in-answer-out systems for in vitro molecular diagnostics have been presented and even commercialized. Although efficient in operation, they are incapable of quantifying targets, since quantitation based on analog analytical methods (via standard curve analysis) is complex, expensive, and challenging. To address this issue, herein, we describe an integrated sample-in-digital-answer-out (SIDAO) diagnostic system incorporating DNA extraction and digital recombinase polymerase amplification, which enables rapid and quantitative nucleic acid analysis from bodily fluids within a disposable cartridge. Inside the cartridge, reagents are pre-stored in sterilized tubes, with an automated pipetting module allowing facile liquid transfer. For digital analysis, we fabricate a simple, single-layer polydimethylsiloxane microfluidic device and develop a novel and simple sample compartmentalization strategy. Sample solution is partitioned into an array of 40,044 fL-volume microwells by sealing the microfluidic device through the application of mechanical pressure. The entire analysis is performed in a portable, fully automated instrument. We evaluate the quantitative capabilities of the system by analyzing Mycobacterium tuberculosis genomic DNA from both spiked saliva and serum samples, and demonstrate excellent analytical accuracy and specificity. This SIDAO system provides a promising diagnostic platform for quantitative nucleic acid testing at the point-of-care. Graphical abstract ᅟ.

Comparison of the Off-Target Effects Among One-Base to Three-Base Mismatched Targets of gRNA Using a Blue to White Assay.

The present study was designed to test a new strategy for comparing the off-target effects of CRISPR/cas9 employing a blue/white colony based assay. Eight types of AmpR plasmids with matched, one base mismatched, two bases mismatched and three bases mismatched targets were constructed. The wild typed alpha peptide of the beta-glactosidase gene and some with mutations, chosen as the targets, were successfully subcloned into the plasmids in an inframe pattern. The relevant gRNA and cas9 were subcloned into the ChloR plasmid. Transformation of the plasmid harboring the target gene yielded blue colonies, while white colonies formed in the plates following the co-transformation of target gene plasmid and the CRISPR/cas9 plasmid. Colonies in different levels of blue color were formed from the plasmids having completely matched target and mismatched targets. The appearance of different blue levels following the co-transformation of the different target plasmid and CRISPR/cas9 plasmid qualitatively indicated the effectiveness of the gRNA, while the ratio of the AMP/CL colonies can be used for relatively quantitative evaluation. The target and off-target effects among different targets of gRNA were differentially identifiable and these effects in turn demonstrated the effectiveness of the blue to white assay. Either the qualitative or the quantitative analysis of the blue to white depends on the final interactions among the target, the cas9 and the gRNA. Clearly, when co-transformation is replaced by single transformation using a specialized nanoparticle containing these three components, the target and off-target effect will be more accurately evaluated. Thus, this study provided a model for application of nanoparticle in gene editing studies.

A Novel Assay Coupling Dephosphorylation and Blue/White Colony Screening for the G > A Hotspot Mutation at Codon 13 of KRAS Gene.

Development of sensitive assay for detection of hotspot mutations of cancer driving gene is crucial for circulating tumor DNA analysis. This study tested the possibilities of applying restriction enzyme digestion and dephosphorylation coupled with blue/white screening technology for analyzing a hotspot point mutation in codon 13 of KRAS gene. The present study has documented that the combination of PCR with restriction digestion, dephosphorylation, blue/white screening and Sanger's sequencing can identify rare mutations with sensitivities at 0.003%. This novel assay with high sensitivity may have application in the diagnosis of early cancer targeting ctDNAs.

Wet Chemical Synthesis of Silica Nanosheets via Ethyl Acetate-Mediated Hydrolysis of Silica Precursors and Their Applications.

Freestanding and transferable silica nanosheets with thicknesses of ≈5-7 nm are prepared via ethyl acetate-mediated hydrolysis of silica precursors in aqueous solution. The resulting silica nanosheets have shown many potential applications. For example, they can be used as the support film on the finer mesh grids for transmission electron microscopy imaging and as the precursor for the synthesis of silicon nanosheets.

Aptamer selection and applications for breast cancer diagnostics and therapy.

Aptamers are short non-coding, single-stranded oligonucleotides (RNA or DNA) developed through Systematic Evolution of Ligands by Exponential enrichment (SELEX) in vitro. Similar to antibodies, aptamers can bind to specific targets with high affinity, and are considered promising therapeutic agents as they have several advantages over antibodies, including high specificity, stability, and non-immunogenicity. Furthermore, aptamers can be produced at a low cost and easily modified, and are, therefore, called "chemical antibodies". In the past years, a variety of aptamers specifically bound to both breast cancer biomarkers and cells had been selected. Besides, taking advantage of nanomaterials, there were a number of aptamer-nanomaterial conjugates been developed and widely investigated for diagnostics and targeted therapy of breast cancer. In this short review, we first present a systematical review of various aptamer selection methods. Then, various aptamer-based diagnostic and therapeutic strategies of breast cancer were provided. Finally, the current problems, challenges, and future perspectives in the field were thoroughly discussed.

Comparative Cytotoxicity induced by Zinc Oxide Nanoparticles in Human Prostate Cells.

Despite increasing biomedical applications of zinc oxide nanoparticles (ZnO NPs), there is a lack of information concerning the biological effects of ZnO NPs on human cells. The purpose of this study was to assess the comparative cytotoxicity in human prostate cells (PC-3 and RWPE-1) exposure to 20-nm ZnO NPs. Exposure to concentrations from 0 to 50 µg/mL of ZnO NPs reduced cell viability of PC-3 cells in a dose- and time-dependent manner; whereas it did not affect RWPE-1 cells. A dose-dependent increase in LDH leakage and intracellular reactive oxygen species was observed in PC-3 cells but not in RWPE-1 cells exposure to ZnO NPs at concentrations of 8 ˜ 50 µg/mL for 24 h (P < 0.05). That the percentage of apoptotic cells increased significantly was observed in PC-3 cells induced by ZnO NPs at 10 µg/mL exposure for 8 h. Our results showed that ZnO NPs induced in vitro preferential cytotoxicity in the human prostate cancer cells. We indicated that the different cytotoxicity of ZnO NPs is likely due to the different cell-nanoparticle interaction and response behavior rather than to hydrodynamic sizes of particles. It is suggested that ZnO NPs are expected to find a very promising targeting therapeutic application for human prostate cancer.

Porous Magnetic Pseudo-Carbon Paste Electrode Electrochemical Biosensor for DNA Detection.

A novel and sensitive electrochemical sensor based on magnetic porous pseudo-carbon paste electrode (MPPCPE) for DNA detection was described in this study. The MPPCPE was fabricated by mixing polymethyl methacrylate (PMMA) microspheres, as template, graphite powders, as filler, pyrrole, as precursor of polymer and magnetic powder. It was demonstrated that the fabricated MPPCPE had more sensitivity for detecting DNA when compared with magnetic carbon paste electrode (MPCPE). Results from anodic stripping voltammetry (ASV) experiments showed that the Ag peak current versus concentration of complementary DNA at MPPCPE had two linear regions. The first region demonstrated linearity over a concentration range from 0.5 to 20 nM, with correlation coefficient of 0.992, while the second linear region had concentration of complementary DNA at 0.005 to 0.1 nM range, with correlation coefficient of 0.957. The detection limit was as low as 0.005 nM and the electrochemical sensor was easy to preserve for a long time, and had good reusability and stability.

Full-Automated Thermal Cycler in Nucleic Acid Testing Workstation.

Integrated automation in laboratory has recruited many traditional standalone devices provided by various vendors, however, because of semi-automatization or lack of standard control interfaces, current available devices are difficult to be integrated into workstation. This paper reported a full-automated thermal cycler system (AutoTCS) equipped with a typical 96-well thermo-cycler module, and especially with a well-designed sliding hot lid for polymerase chain reaction (PCR), which makes it very suitable for using in an automatic nucleic acid testing (NAT) workstation. By a PC software or the touch screen on AutoTCS with user-friendly graphical user interface (GUI), the presented system can not only serve as a PCR module of a NAT workstation, but also can be treated as a single PCR system. Experiments have demonstrated that the AutoTCS achieved a temperature accuracy of ±0.1 °C, a heating rate of approximately 2.5 °C/sec, and cooling rate of approximately 1.6 °C/sec. The AutoTCS was tested with PLCE1 gene and hepatitis B virus (HBV) DNA, and this had successfully achieved an excellent PCR performance.