Low-temperature plasma jet suppresses bacterial colonisation and affects wound healing through reactive species.

An argon-based low-temperature plasma jet (LTPJ) was used to treat chronically infected wounds in Staphylococcus aureus-laden mice. Based on physicochemical property analysis and in vitro antibacterial experiments, the effects of plasma parameters on the reactive nitrogen and oxygen species (RNOS) content and antibacterial capacity were determined, and the optimal treatment parameters were determined to be 4 standard litre per minute and 35 W. Additionally, the plasma-treated activation solution had a bactericidal effect. Although RNOS are related to the antimicrobial effect of plasma, excess RNOS may be detrimental to wound remodelling. In vivo studies demonstrated that medium-dose LTPJ promoted MMP-9 expression and inhibited bacterial growth during the early stages of healing. Moreover, LTPJ increased collagen deposition, reduced inflammation, and restored blood vessel density and TGF-ß levels to normal in the later stages of wound healing. Therefore, when treating chronically infected wounds with LTPJ, selecting the medium dose of plasma is more advantageous for wound recovery. Overall, our study demonstrated that low-temperature plasma jets may be a potential tool for the treatment of chronically infected wounds.

NS7a of SADS-CoV promotes viral infection via inducing apoptosis to suppress type III interferon production.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a newly discovered swine coronavirus with potential cross-species transmission risk. Although SADS-CoV-induced host cell apoptosis and innate immunity antagonization has been revealed, underlying signaling pathways remain obscure. Here, we demonstrated that infection of SADS-CoV induced apoptosis in vivo and in vitro, and that viral protein NS7a is mainly responsible for SADS-CoV-induced apoptosis in host cells. Furthermore, we found that NS7a interacted with apoptosis-inducing factor mitochondria associated 1 (AIFM1) to activate caspase-3 via caspase-6 in SADS-CoV-infected cells, and enhanced SADS-CoV replication. Importantly, NS7a suppressed poly(I:C)-induced expression of type III interferon (IFN-λ) via activating caspase-3 to cleave interferon regulatory factor 3 (IRF3), and caspase-3 inhibitor protects piglets against SADS-CoV infection in vivo. These findings reveal how SADS-CoV induced apoptosis to inhibit innate immunity and provide a valuable clue to the development of effective drugs for the clinical control of SADS-CoV infection.IMPORTANCEOver the last 20 years, multiple animal-originated coronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV), middle east respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2, have caused millions of deaths, seriously jeopardized human health, and hindered social development, indicating that the study of animal-originated coronaviruses with potential for cross-species transmission is particularly important. Bat-originated swine acute diarrhea syndrome coronavirus (SADS-CoV), discovered in 2017, can not only cause fatal diarrhea in piglets, but also infect multiple human cells, with a potential risk of cross-species transmission, but its pathogenesis is unclear. In this study, we demonstrated that NS7a of SADS-CoV suppresses IFN-λ production via apoptosis-inducing factor mitochondria associated 1 (AIFM1)-caspase-6-caspase-3-interferon regulatory factor 3 (IRF3) pathway, and caspase-3 inhibitor (Z-DEVD-FMK) can effectively inhibit SADS-CoV replication and protect infected piglets. Our findings in this study contribute to a better understanding of SADS-CoV-host interactions as a part of the coronaviruses pathogenesis and using apoptosis-inhibitor as a drug as potential therapeutic approaches for prevention and control of SADS-CoV infection.

Immunoassay System Based on the Technology of Time-Resolved Fluorescence Resonance Energy Transfer.

To enhance the specificity and sensitivity, cut the cost, and realize joint detection of multiple indicators, an immunoassay system based on the technology of time-resolved fluorescence resonance energy transfer (TR-FRET) was studied. Due to the FRET of the reagent, the donor probe and acceptor probe emitted specific fluorescence to enhance specificity. Long-lifetime specific fluorescence from the acceptor probe was combined with time-resolved technology to enhance sensitivity. A xenon flash lamp and a photomultiplier tube (PMT) were selected as the light source and detector, respectively. A filter-switching mechanism was placed in the light path, so the fluorescence signal from the donor and acceptor was measured alternately. The instrument's design is given, and some specificI parts are described in detail. Key technical specifications of the instrument and procalcitonin (PCT), C-reactive protein (CRP), and interleukin-6(IL-6) were tested, and the test results were presented subsequently. The CV value of the self-designed counting module is better than 0.01%, and the instrument noises for 620 nm and 665 nm are 41.44 and 10.59, respectively. When set at 37 °C, the temperature bias (B) is 0.06 °C, and the temperature fluctuation is 0.10 °C. The CV and bias are between ±3% and 5%, respectively, when pipetting volumes are between 10 µL and 100 µL. Within the concentration range of 0.01 nM to 10 nM, the luminescence values exhibit linear regression correlation coefficients greater than 0.999. For PCT detection, when the concentration ranges from 0.02 ng/mL to 50 ng/mL, the correlation coefficient of linear fitting exceeds 0.999, and the limit of quantification is 0.096 ng/mL. For CRP and IL-6, the detection concentration ranges from 0 ng/mL to 500 ng/mL and 0 ng/mL to 20 ng/mL, respectively, with limits of quantification of 2.70 ng/mL and 2.82 ng/mL, respectively. The experimental results confirm the feasibility of the technical and instrumental solutions.

Single-Ended Eddy Current Micro-Displacement Sensor with High Precision Based on Temperature Compensation.

To measure the micro-displacement reliably with high precision, a single-ended eddy current sensor based on temperature compensation was studied in detail. At first, the principle of the eddy current sensor was introduced, and the manufacturing method of the probe was given. The overall design plan for the processing circuit was induced by analyzing the characteristics of the probe output signal. The variation in the probe output signal was converted to pulses with different widths, and then it was introduced to the digital phase discriminator along with a reference signal. The output from the digital phase discriminator was processed by a low-pass filter to obtain the DC component. At last, the signal was amplified and compensated to reduce the influence of temperature. The selection criteria of the frequency of the exciting signal and the design of the signal conditioning circuit were described in detail, as well as the design of the temperature-compensating circuit based on the digital potentiometer with an embedded temperature sensor. Finally, an experimental setup was constructed to test the sensor, and the results were given. The results show that nonlinearity exists in the single-ended eddy current sensor with a large range. When the range is 500 µm, the resolution can reach 46 nm, and the repeatability error is ±0.70% FR. Within the temperature range from +2 °C to +58 °C, the voltage fluctuation in the sensor is reduced to 44 mV after temperature compensation compared to the value of 586 mV before compensation. The proposed plan is verified to be feasible, and the measuring range, precision, and target material should be considered in real-world applications.

High-Voltage Electrostatic Field Hydrogel Microsphere 3D Culture System Improves Viability and Liver-like Properties of HepG2 Cells.

Three-dimensional (3D) hepatocyte models have become a research hotspot for evaluating drug metabolism and hepatotoxicity. Compared to two-dimensional (2D) cultures, 3D cultures are better at mimicking the morphology and microenvironment of hepatocytes in vivo. However, commonly used 3D culture techniques are not suitable for high-throughput drug screening (HTS) due to their high cost, complex handling, and inability to simulate cell-extracellular matrix (ECM) interactions. This article describes a method for rapid and reproducible 3D cell cultures with ECM-cell interactions based on 3D culture instrumentation to provide more efficient HTS. We developed a microsphere preparation based on a high-voltage electrostatic (HVE) field and used sodium alginate- and collagen-based hydrogels as scaffolds for 3D cultures of HepG2 cells. The microsphere-generating device enables the rapid and reproducible preparation of bioactive hydrogel microspheres. This 3D culture system exhibited better cell viability, heterogeneity, and drug-metabolizing activity than 2D and other 3D culture models, and the long-term culture characteristics of this system make it suitable for predicting long-term liver toxicity. This system improves the overall applicability of HepG2 spheroids in safety assessment studies, and this simple and controllable high-throughput-compatible method shows potential for use in drug toxicity screening assays and mechanistic studies.

Low Temperature Plasma Jet Affects Acute Skin Wounds in Diabetic Mice Through Reactive Components.

As a common complication of diabetes, diabetic foot ulcers serious affect the life quality even lead to amputation if it's not properly treated. In this paper, we developed a Low Temperature Plasma Jet (LTPJ) system for treating diabetic foot ulcers on streptozotocin-induced diabetic mice. This system generates time-dependent reactive nitrogen and oxygen species (RNOS), which have temperature below 40°C. The wound area of normal mice was significantly reduced after LTPJ treatment. Histological and immunohistochemistry analysis showed faster deposition of collagen and more vessel formation both in plasma-treated normal and diabetic mice on Day 3. However, diabetic wounds showed poor collagen deposition and angiogenesis on Day 8, which might be the reason of slow wound healing. Reactive nitrogen species (RNS) that generated by LTPJ can promote endogenous nitric oxide (NO) production in diabetic wounds, thus promoting inflammation, stromal deposition, angiogenesis, cell proliferation and remodeling, while excess reactive oxygen species (ROS) will exacerbate oxidative stress in wound tissues of diabetic mice. In conclusion, LTPJ improved acute wound healing in normal mice, increased collagen deposition and angiogenesis in initial diabetic wound healing, but had no significant effect on diabetic wound healing rate.

A study on the detection of free and bound biotin based on TR-FRET technology.

Biotin is widely used in biological applications due to its highly selective and stable interaction with avidin, which highlights the great potential value of the quantitative determination of biotin concentration. However, the currently reported methods have many defects such as complicated operation processes and low sensitivity. Here, the time-resolved fluorescence resonance energy transfer (TR-FRET) assay is introduced to establish a convenient, rapid and sensitive biotin quantitative detection strategy. Europium cryptate (Eu3+) acts as an energy donor to label streptavidin, while APC acts as an energy acceptor to label biotin. Biotin in aqueous solution interacts with streptavidin in a competition mode. The obtained biotin detection range is 0.05-100 nM and the optimal limit of detection (LOD) of 0.03 nM biotin is obtained. Furthermore, an enzyme digestion test and a competition mode test were performed to analyze biotin in different states. The method used in this work has greatly improved the sensitivity of biotin quantitative detection and it's for the first time that a systematic study on the difference between free and bound biotin based on concentration results is conducted. It can be further extended to the detection of other biological molecules or multiplex detection of other small molecules.