Enzyme Method-Based Microfluidic Chip for the Rapid Detection of Copper Ions

Metal ions in high concentrations can pollute the marine environment. Human activities and industrial pollution are the causes of Cu2+ contamination. Here, we report our discovery of an enzyme method-based microfluidic that can be used to rapidly detect Cu2+ in seawater. In this method, Cu2+ is reduced to Cu+ to inhibit horseradish peroxidase (HRP) activity, which then results in the color distortion of the reaction solution. The chip provides both naked eye and spectrophotometer modalities. Cu2+ concentrations have an ideal linear relationship, with absorbance values ranging from 3.91 nM to 256 μM. The proposed enzyme method-based microfluidic chip detects Cu2+ with a limit of detection (LOD) of 0.87 nM. Other common metal ions do not affect the operation of the chip. The successful detection of Cu2+ was achieved using three real seawater samples, verifying the ability of the chip in practical applications. Furthermore, the chip realizes the functions of two AND gates in series and has potential practical implementations in biochemical detection and biological computing.

Hydroxychloroquine inhibits proteolytic processing of endogenous TLR7 protein in human primary plasmacytoid dendritic cells

Toll-like receptor 7 (TLR7) triggers antiviral immune responses through its capacity to recognize single-stranded RNA. Proteolytic cleavage of TLR7 protein is required for its functional maturation in the endosomal compartment. Structural studies demonstrated that the N- and C-terminal domains of TLR7 are connected and involved in ligand binding after cleavage. Hydroxychloroquine (HCQ), an antimalarial drug, has been studied for its antiviral effects. HCQ increases pH in acidic organelles and has been reported to potently inhibit endosomal TLR activation. Whether HCQ can prevent endogenous TLR7 cleavage in primary immune cells, such as plasmacytoid dendritic cells (pDCs), had never been examined. Here, using a validated anti-TLR7 antibody suitable for biochemical detection of native TLR7 protein, we show that HCQ-treatment of fresh PBMCs, CAL-1 leukemic and primary human pDCs inhibits TLR7 cleavage and results in accumulation of full-length protein. As a consequence, we observe an inhibition of pDC activation in response to TLR7 stimulation with synthetic ligands and viruses including inactivated SARS-CoV2, which we show herein activates pDCs through TLR7-signaling. Together, our finding suggests that the major pathway by which HCQ inhibits ssRNA-sensing by pDCs may rely on its capacity to inhibit endosomal acidification and the functional maturation of TLR7 protein.

MODERN MICROBIOLOGICAL TECHNOLOGIES IN DIAGNOSIS OF MYCOSES IN OTORHINOLARYNGOLOGICAL PRACTICE

The review article highlights the latest approaches to solve one of the important problems of modern otorhinolaryngology, the diagnosis of opportunistic mycoses of the upper respiratory tract and ear. Opportunistic mycoses of the ENT organs in recent decades have posed a significant challenge for modern clinical medicine not only in Ukraine but also throughout the world. According to the scientific literature, the share of fungal lesions of the ear and upper respiratory tract in the structure of chronic inflammation of these organs makes up 22.1%. The main causative agents of mycotic lesions of the ENT organs are opportunistic fungi of the genera Aspergillus, Рenicillium, Mucor and yeast-like fungi of the genus Сandida, which are characterized by a low level of pathogenicity and are a part of the resident microflora of the macroorganism. Classical methods of microbiological diagnosis have certain limitations in the identification of micromycetes. Therefore, the use of more reliable, fast and accurate methods in clinical practice will contribute to the timely and effective treatment of fungal diseases of the ENT organs. This review presents an analysis of modern microbiological diagnostic technologies, such as biochemical detection of microorganisms using identification plate test systems (ARI RapID, CrystalTM), semi-automatic and automatic microbiological identification using analyzers VITEK, VITEK 2, Walk Away. Identification of fungal pathogens can also be performed by performing direct protein profiling using mass spectrometry, polymerase chain reaction and sequencing. These methods with the highest level of reliability enable to identify the pathogen, as well as to assess its sensitivity to chemotherapeutic drugs. The combination of classical and modern microbiological technologies should become a standard for the diagnosis of fungal diseases, including the upper respiratory tract and ear.

Chloroplast proteotoxic stress-induced autophagy is involved in the degradation of chloroplast proteins in Chlamydomonas reinhardtii

Intraorganellar proteases and cytoplasmic proteolytic systems such as autophagy orchestrate the degradation of organellar proteins to ensure organelle homeostasis in eukaryotic cells. The green alga Chlamydomonas reinhardtii is an ideal unicellular model organism for elucidating the mechanisms maintaining proteostasis in chloroplasts. However, the autophagic pathways targeting the photosynthetic organelles of these algae have not been clearly elucidated. Here, we explored the role of autophagy in chloroplast protein degradation in Chlamydomonas cells. We labeled the chloroplast protein Rubisco small subunit (RBCS) with the yellow fluorescent protein Venus in a Chlamydomonas strain in which expression of the chloroplast gene clpP1, encoding a major catalytic subunit of the chloroplast Clp protease, can be conditionally repressed to selectively perturb chloroplast protein homeostasis. We observed transport of both nucleus-encoded RBCS-Venus fusion protein and chloroplast-encoded Rubisco large subunit (rbcL) from the chloroplast to the vacuoles in response to chloroplast proteotoxic stress induced by clpP1 inhibition. This process was retarded by the addition of autophagy inhibitors. Biochemical detection of lytic cleavage of RBCS-Venus supported the notion that Rubisco is degraded in the vacuoles via autophagy. Electron microscopy revealed vacuolar accumulation of autophagic vesicles and exposed their ultrastructure during repression of clpP1 expression. Treatment with an autophagy activator also induced chloroplast autophagy. These results indicate that autophagy contributes to chloroplast protein degradation in Chlamydomonas cells.

Hong-Hui-Xiang Alleviates Pain Hypersensitivity in a Mouse Model of Monoarthritis

Background. Hong-Hui-Xiang (HHX) is a sterilized aqueous solution extracted from Illicium lanceolatum A.C. Smith widely used for pain relief in China. Despite its history, it is not well understood. In the present study, we used a mouse model of arthritic knee pain to investigate the antinociceptive effects of HHX and its potential side effects on weight and respiratory function, as well as on the liver, kidney, and heart. Methods. Mice were randomly assigned to four groups: saline and HHX at three doses (1 μl, 10 μl, and 50 μl). Each group was randomly divided to two subgroups: saline and CFA. After the first injection of HHX or saline on day 7, mechanical hyperalgesia was tested via the hind paw. Only after the tests had established that the analgesic effect had subsided was the next injection administered. A total of five injections were administered. Blood, knee joints, and other organs were collected for histopathological observation and biochemical detection. Objectives. We found that mechanical threshold of hind paw increased 2 h after of the initial injection HHX (10 μl and 50 μl), which lasted for at least 3 h. The analgesic effect lasted for three days after the second injection on day 8 and was approximately maintained for five days each time after the third injection. We also found a reduction in the diameter of the knee joint and suppression of synovial inflammation in response to treatment of HHX (10 μl and 50 μl). Meanwhile, HHX had no toxic effects on the liver, kidneys, and heart via histological and biochemical assays in all groups. Conclusion. HHX exerts antinociceptive and anti-inflammatory effects in a mouse model of arthritic knee pain. There were no obvious side effects on the liver, kidneys, or heart.

ROC Curve Analysis of the Sensitivity and Specificity of Biochemical Detection of Intrahepatic Cholestasis during Pregnancy

Introduction Intrahepatic cholestasis of pregnancy (ICP) is associated with an increased risk of adverse pregnancy outcomes in mothers and infants. The aim was to evaluate the sensitivity and specificity of biochemical detection of ICP by ROC curve and to determine the threshold of more reliable experimental indicators. Materials and Methods 305 patients and 305 healthy pregnant women were enrolled in the study. Results The average levels of TBA, ALT, and AST in the ICP group were much higher than those in the control group (P<0. 001); the area of both CG and TBA under ROC curve was up to 0.99, the sensitivity was 97.7%, and the specificity was 99.3%. Conclusions This study did not find any single specificity and sensitivity markers that could be used to reliably diagnose ICP. In the future, we will pay more attention to the correlation between sensitive biochemical indicators and adverse pregnancy outcomes.