Dexmedetomidine protects against sepsis-induced lung injury through autophagy and Smad2/3 signaling pathway.

Objectives: Dexmedetomidine (Dex) is a potent α2-adrenergic receptor(α2-AR) agonist that has been shown to protect against sepsis-induced lung injury, however, the underlying mechanisms of this protection are not fully understood. Autophagy and the Smad2/3 signaling pathway play important roles in sepsis-induced lung injury, but the relationship between Dex and Smad2/3 is not clear. This study aimed to investigate the role of autophagy and the Smad2/3 signaling pathway in Dex-mediated treatment of sepsis-induced lung injury. Sepsis was performed using cecal ligation and puncture (CLP) in C57BL/6J mice. Materials and Methods: Mice were randomly assigned to four groups (n=6 per group): sham, CLP, CLP-Dex, and CLP-Dex-YOH, Yohimbine hydrochloride (YOH) is an α2-AR blocker. The cecum was carefully separated to avoid blood vessel damage and was identified and punctured twice with an 18-gauge needle. The pathological changes, inflammatory factor levels, oxidative stress, autophagy, Smad2/3 signaling pathway-related protein levels in lung tissues, and the activity of superoxide dismutase (SOD) and malonaldehyde (MDA) in the serum were measured. Results: CLP-induced lung injury was reflected by increased levels of inflammatory cytokines, apoptosis, and oxidative stress, along with an increase in the expression of autophagy and Smad2/3 signaling pathway-related proteins. Dex could reverse these changes and confer a protective effect on the lung during sepsis. However, the administration of YOH significantly reduced the positive effects of Dex in mice with sepsis. Conclusion: Dex exerts its beneficial effects against sepsis-induced lung injury through the regulation of autophagy and the Smad2/3 signaling pathway.

Probing the communication patterns of different chondrocyte subtypes in osteoarthritis at the single cell level using pattern recognition and manifold learning.

The patterns of communication among different chondrocyte subtypes in human cartilage degeneration and regeneration help us understand the microenvironment of osteoarthritis and optimize cell-targeted therapies. Here, a single-cell transcriptome dataset of chondrocytes is used to explore the synergistic and communicative patterns of different chondrocyte subtypes. We collected 1600 chondrocytes from 10 patients with osteoarthritis and analyzed the active communication patterns for the first time based on network analysis and pattern recognition at the single-cell level. Manifold learning and quantitative contrasts were performed to analyze conserved and specific communication pathways. We found that ProCs (Proliferative chondrocytes), ECs (Effector chondrocytes), preHTCs (Prehypertrophic chondrocytes), HTCs (Hypertrophic chondrocytes), and FCs (Fibrocartilage chondrocytes) are more active in incoming and outgoing signaling patterns, which is consistent with studies on their close functional cooperation. Among them, preHTCs play multiple roles in chondrocyte communication, and ProCs and preHTCs have many overlapping pathways. These two subtypes are the most active among all chondrocyte subtypes. Interestingly, ECs and FCs are a pair of "mutually exclusive" subtypes, of which ECs are predominant in incoming patterns and FCs in outgoing patterns. The active signaling pathways of ECs and FCs largely do not overlap. COLLAGEN and LAMININ are the main pivotal pathways, which means they are very important in the repair and expansion of joint homeostasis. Notably, only preHTCs assume multiple roles (including sender, receiver, mediator, and influencer) and are involved in multiple communication pathways. We have examined their communication patterns from the perspective of cellular interactions, revealed the relationships among different chondrocyte subtypes, and, in particular, identified a number of active subtypes and pathways that are important for targeted therapy in the osteoarthritic microenvironment. Our findings provide a new research paradigm and new insights into understanding chondrocyte activity patterns in the osteoarthritic microenvironment.

Xanthine oxidase, a therapeutic target of realgar for non-small cell lung cancer.

Background: The effects of realgar against non-small cell lung cancer (NSCLC) have been massively studied, but the direct therapeutic targets of realgar remain unclear. This study aimed to identify the molecular targets of realgar against NSCLC and explore their therapeutic mechanisms based on a network pharmacology approach and experimental validations. Methods: The BATMAN-TCM and Digsee databases were used to predict realgar targets and NSCLC-related genes, respectively. A protein-protein interaction network was constructed for each gene set, and the overlapping genes were identified as potential targets of realgar against NSCLC. The correlation between potential targets and NSCLC was analyzed using The Cancer Genome Atlas and International Cancer Genome Consortium databases, and the key target was validated by in-silico and in-vitro experiments. Results: Twenty-three overlapping genes, including xanthine oxidase (XO), were identified as potential targets of realgar against NSCLC. XO was selected as the key target for validation, as it was found to be upregulated in NSCLC tumor tissue, which correlated with poor overall survival. A possible interaction between realgar and XO was revealed by molecular docking which was further validated experimentally. Realgar treatment suppressed the activity of XO in NSCLC cells, as demonstrated by the unchanged XO protein levels. Finally, the mechanism of action of XO as a target against NSCLC through the cell-cell junction organization pathway was investigated. Conclusions: Overall, this study proposes a potential molecular mechanism illustrating that XO is a target of realgar against NSCLC and highlights the usefulness of XO as a therapeutic target for NSCLC.

CD 4+, CD 8+ and CD 19+cell surface antigen and abnormal mitochondria ultrastructure of peripheral blood P-type atypical lymphocytes in patients with schizophrenia.

OBJECTIVE: P-type atypical lymphocytes may play important roles in the aetiology and therapy of schizophrenia. However, there is merely a direct immunological characterisation of it. The aim of this study is to explore the surface antigens of these cells and their comparative ultrastructure in schizophrenia. METHODS: We recruited 25 age-and gender-matched patients with unmedicated schizophrenia, other mental diseases and healthy individuals. Peripheral venous blood was smeared and stained. CD4+, CD8+ and CD19+ cell surface antigen- positive lymphocytes were purified using magnetic beads and prepared for light microscopy and electron microscopy. RESULTS: The percentages of P-type atypical lymphocytes (34.53% ± 9.92%) were significantly higher (p < 0.0001) in schizophrenia than that of other mental diseases (9.79% ± 3.45%). These cells could present CD4+, CD8+ and CD19+ surface antigens. Their relative ultrastructure differed from that of normal lymphocytes, especially in mitochondria, which showed abundant, aggregated and quite irregular mitochondria; for example, slight dilation of the foci, swelling, degeneration, and even cavity. CONCLUSIONS: P-type atypical lymphocytes could be found among CD4+, CD8+, and CD19 + lymphocytes with schizophrenia. Their abnormal ultrastructure of mitochondria implied that energy metabolism might play an important role in the aetiology of schizophrenia.

Knockout of ABC transporters by CRISPR/Cas9 contributes to reliable and accurate transporter substrate identification for drug discovery.

It is essential to explore the relationship between drugs and transporters in the process of drug development. Strong background signals in nonhuman MDCK or LLC-PK1 cells and overlapping interference of inhibitors or RNAi in human Caco-2 cells mean that an ideal alternative could be to knock out specific transporter genes in Caco-2 cells. However, the application of gene knockout (KO) to Caco-2 cells is challenging because it is still inefficient to obtain rapidly growing Caco-2 subclones with double-allele KO through long-term monoclonal cultivation. Herein, CRISPR/Cas9, a low cost but more efficient and precise gene editing technology, was utilized to singly or doubly knockout the P-gp, BCRP, and MRP2 genes in Caco-2 cells. By combining this with single cell expansion, rapidly growing transporter-deficient subclones were successfully screened and established. Bidirectional transport assays with probe substrates and three protease inhibitors indicated that more reliable and detailed data could be drawn easily with these KO Caco-2 models. The six robust KO Caco-2 subclones could contribute to efficient in vitro drug transport research.

Efficient generation of a CYP3A4-T2A-luciferase knock-in HepaRG subclone and its optimized differentiation.

CRISPR/Cas9 has allowed development of better and easier-to-use ADME models than traditional methods by complete knockout or knock-in of genes. However, gene editing in HepaRG cells remains challenging because long-term monoclonal cultivation may alter their differentiation capacity to a large extent. Here, CRISPR/Cas9 was used to generate a CYP3A4-T2A-luciferase knock-in HepaRG subclone by Cas9-mediated homologous recombination and monoclonal cultivation. The knock-in HepaRG-#9 subclone retained a similar differentiation potential to wildtype HepaRG cells (HepaRG-WT). To further improve differentiation and expand the applications of knock-in HepaRG cells, two optimized differentiation procedures were evaluated by comparison with the standard differentiation procedure using the knock-in HepaRG-#9 subclone and HepaRG-WT. The results indicated that addition of forskolin (an adenylate cyclase activator) and SB431542 (a TGF-ß pathway inhibitor) to the first optimized differentiation procedure led to better differentiation consequence in terms of not only the initiation time for differentiation and morphological characterization, but also the mRNA levels of hepatocyte-specific genes. These data may contribute to more extensive applications of genetically modified HepaRG cells in ADME studies.

Validation Study to Determine the Accuracy of Widespread Promoterless EGFP Reporter at Assessing CRISPR/Cas9-Mediated Homology Directed Repair.

An accurate visual reporter system to assess homology-directed repair (HDR) is a key prerequisite for evaluating the efficiency of Cas9-mediated precise gene editing. Herein, we tested the utility of the widespread promoterless EGFP reporter to assess the efficiency of CRISPR/Cas9-mediated homologous recombination by fluorescence expression. We firstly established a promoterless EGFP reporter donor targeting the porcine GAPDH locus to study CRISPR/Cas9-mediated homologous recombination in porcine cells. Curiously, EGFP was expressed at unexpectedly high levels from the promoterless donor in porcine cells, with or without Cas9/sgRNA. Even higher EGFP expression was detected in human cells and those of other species when the porcine donor was transfected alone. Therefore, EGFP could be expressed at certain level in various cells transfected with the promoterless EGFP reporter alone, making it a low-resolution reporter for measuring Cas9-mediated HDR events. In summary, the widespread promoterless EGFP reporter could not be an ideal measurement for HDR screening and there is an urgent need to develop a more reliable, high-resolution HDR screening system to better explore strategies of increasing the efficiency of Cas9-mediated HDR in mammalian cells.

Rapid and accurate detection of SARS-CoV-2 mutations using a Cas12a-based sensing platform.

The increasing prevalence of SARS-CoV-2 variants with spike mutations has raised concerns owing to higher transmission rates, disease severity, and escape from neutralizing antibodies. Rapid and accurate detection of SARS-CoV-2 variants provides crucial information concerning the outbreaks of SARS-CoV-2 variants and possible lines of transmission. This information is vital for infection prevention and control. We used a Cas12a-based RT-PCR combined with CRISPR on-site rapid detection system (RT-CORDS) platform to detect the key mutations in SARS-CoV-2 variants, such as 69/70 deletion, N501Y, and D614G. We used type-specific CRISPR RNAs (crRNAs) to identify wild-type (crRNA-W) and mutant (crRNA-M) sequences of SARS-CoV-2. We successfully differentiated mutant variants from wild-type SARS-CoV-2 with a sensitivity of 10-17 M (approximately 6 copies/µL). The assay took just 10 min with the Cas12a/crRNA reaction after a simple RT-PCR using a fluorescence reporting system. In addition, a sensitivity of 10-16 M could be achieved when lateral flow strips were used as readouts. The accuracy of RT-CORDS for SARS-CoV-2 variant detection was 100% consistent with the sequencing data. In conclusion, using the RT-CORDS platform, we accurately, sensitively, specifically, and rapidly detected SARS-CoV-2 variants. This method may be used in clinical diagnosis.

[Changes of mean platelet volume, fibrinogen content and blood rheology in peripheral blood of youth patients with cerebral infarction].

This study was aimed to explore the correlation of mean platelet volume (MPV), fibrinogen (FIB) and blood rheology with the youth patients with cerebral infarction, so as to provide the basis for the clinical early diagnosis and treatment. The 109 patients with cerebral infarction aged between 18 - 45 were divided into three group: the large (> 10 cm(3)), middle (4 - 10 cm(3)) and small (< 4 cm(3)) area infarction; 30 healthy persons were served as control group. All the four groups were subjected to 16 examinations, such as MPV, FIB, and rheology (Lηb, Mηb, Hηb, ηp, Lηr, Mηr, Hηr, KVE, EAI, ERI, EDI, EEI, HCT, ESR). The results showed that all the MPV, FIB and rheology indexes of the different infarction groups were higher than those of healthy control group (P < 0.05). The MPV, FIB and rheology indexes in the large area infarction group were all higher than those in the small area infarction group (P < 0.05). The indexes of MPV, FIB and rheology in the various cerebral infarction area groups obviously decreased, but those did not reach to the level in the healthy control group (P < 0.05). The MPV, FIB content and rheology level correlated with infarction areas (r = 0.36, 0.29 and 0.48, respectively). It is concluded that the serious intensity of youth patients with cerebral infarction positively correlated with the levels of MPV, FIB and rheology indexes. Regular examination of above mentioned index may be useful to prevent youth patients from cerebral infraction.

Effects of Tat peptide on intracellular delivery of arsenic trioxide albumin microspheres.

The current study was designed to evaluate the ability of cell-penetrating peptides to deliver arsenic trioxide albumin microspheres (AsAMs) into bladder cancer cells. The transactivating transcriptional activator (Tat) peptide was labeled with the enhanced green fluorescent protein (EGFP) using eukaryotic vector construction and fusion gene expression techniques. Arsenic trioxide albumin mirospheres were prepared using the chemical crosslink and solidification method. The conjugate, Tat-EGFP-As2O3-AMs (TEAsAMs), was synthesized using the amine-reactive heterobifunctional linker agent N-succinimidyl-3-(2-pyridyldithio) propionate and verified by electrophoresis under reducing conditions and fluorescence microscopy. The intracellular delivery of TEAsAMs was evaluated by laser confocal microscopy and transmission electron microscopy. The arsenic content in the bladder cancer EJ cells was assayed to evaluate the efficiency of delivery. Gene sequencing showed that the pET-Tat-EGFP expression vector was constructed successfully. The expression of the Tat-EGFP fusion protein was verified by matrix-assisted laser desorption/ionization-time of flight analysis, and the protein was transduced into cell cytoplasm as observed under a fluorescence microscope. Electrophoresis under reducing conditions demonstrated the covalent linkage between Tat-EGFP and AsAMs. Under a laser confocal microscope and a transmission electron microscope, TEAsAMs surrounded by green fluorescence were shown to enter the cells faster than EGFP-As2O3-AMs, with an increase in the intracellular arsenic content being observed in cells treated with TEAsAMs compared with those treated with EGFP-As2O3-AMs. These results suggest that Tat peptide promotes the cellular uptake of large albumin microspheres with encapsulated arsenicals.