The discovery of a novel single-function intermolecular Diels-Alder enzyme for the biosynthesis of hetero-dimer lithocarpins.

The deep-sea fungus Phomopsis lithocarpus FS508 produces tenellone-macrolide conjugated hetero-dimer lithocarpins A-G with anti-tumor activities. The deficiency of new intermolecular Diels-Alder (DA) enzymes hindered the development of new bioactive hetero-dimers. A novel single-function intermolecular DA enzyme, g7882, was initially discovered in this study. The deletion of g7882 led to the disappearance of lithocarpin A and an increase in precursor level . the overexpression of g7882 significantly improved lithocarpin A yield. The in vitro function of g7882DA was also confirmed by biochemical reaction using tenellone B as a substrate. Additionally, the knockout of KS modules of PKS in cluster 41 and cluster 81 (lit cluster) eliminated the production of lithocarpins, which firstly explains the biosynthetic process of hetero-dimer lithocarpins mediated by DA enzyme in FS508. Furthermore, the removal of a novel acetyltransferase GPAT in cluster 41 and the oxidoreductase, prenyltransferase in cluster81 resulted in the reduction of lithocarpin A in P. lithocarpus. The overexpression of gpat in P. lithocarpus FS508 improved the yield of lithocarpin A significantly and produced a new tenellone derivative lithocarol G. This study offers a new DA enzyme tool for the biosynthesis of novel hetero-dimer and biochemical clues for the biosynthetic logic elucidation of lithocarpins.

MiR-155 enhances phagocytosis of alveolar macrophages through the mTORC2/RhoA pathway.

Alveolar macrophage phagocytosis is significantly reduced in Chronic obstructive pulmonary disease, and cigarette smoke extract is one of the chief reasons for this decrease. Nevertheless, the specific underlying mechanism remains elusive. In this study, the role and possible mechanism of miR-155-5p/mTORC2/RhoA in the phagocytosis of mouse alveolar macrophages (MH-S) were explored. Our results revealed that cigarette smoke extract intervention reduced MH-S cell phagocytosis and miR-155-5p expression. Meanwhile, the dual-luciferase reporter assay validated that Rictor is a target of miR-155-5p. On the one hand, transfecting miR-155-5p mimic, mimic NC, miR-155-5p inhibitor, or inhibitor NC in MH-S cells overexpressing miR-155-5p increased the Alveolar macrophage phagocytotic rate, up-regulated the expression level of RhoA and p-RhoA, and down-regulated that of mTOR and Rictor mRNA and protein. On the other hand, inhibiting the expression of miR-155-5p lowered the phagocytotic rate, up-regulated the expression of mTOR, Rictor mRNA, and protein, and down-regulated the expression of RhoA and p-RhoA, which taken together, authenticated that miR-155-5p participates in macrophage phagocytosis via the mTORC2/RhoA pathway. Finally, confocal microscopy demonstrated that cells overexpressing miR-155-5p underwent cytoskeletal rearrangement during phagocytosis, and the phagocytic function of cells was enhanced, signaling that miR-155-5p participated in macrophage skeletal rearrangement and enhanced alveolar macrophage phagocytosis by targeting the expression of Rictor in the mTORC2/RhoA pathway.

TSHR-based chimeric antigen receptor T cell specifically deplete auto-reactive B lymphocytes for treatment of autoimmune thyroid disease.

Graves' disease (GD) is a prominent antibody-mediated autoimmune disorder characterized by stimulating antibodies (TRAb) that target the thyroid-stimulating hormone receptor (TSHR). Targeting and eliminating TRAb-producing B lymphocytes hold substantial therapeutic potential for GD. In this study, we engineered a novel chimeric antigen receptor T cell (CAR-T) therapy termed TSHR-CAR-T. This CAR-T construct incorporates the extracellular domain of the TSH receptor fused with the CD8 transmembrane and intracellular signal domain (4-1BB). TSHR-CAR-T cells demonstrated the ability to recognize and effectively eliminate TRAb-producing B lymphocytes both in vitro and in vivo. Leveraging this autoantigen-based chimeric receptor, our findings suggest that TSHR-CAR-T cells offer a promising and innovative immunotherapeutic approach for the treatment of antibody-mediated autoimmune diseases, including GD.

Fibrinogen function indexes are potential biomarkers for evaluating the occurrence and severity of diabetic foot.

BACKGROUND AND OBJECTIVES: Research suggests that fibrinogen (Fib) concentrations are used to assess the occurrence and severity of diabetic foot (DF) and to monitor the progression of diabetic foot in patients. However, its correlation with Fib function has not been reported. Here, angle α and k value, reflecting the Fib function, were used to analyse its correlation with DF, and their potential as biological indicators for evaluating the occurrence and severity of DF was explored. SUBJECTS AND METHODS: This clinical study enrolled 163 type 2 diabetes mellitus (T2DM) patients, who were divided into the diabetes with DF (84 cases) group, diabetes with no DF (79 cases) group. Meanwhile, 90 healthy unrelated subjects were enrolled as controls. RESULTS: Angle α and fibrinogen levels increased greatly in subjects with DF compared with those without. The k value levels greatly decreased in subjects with DF compared with those without (P < 0.01). Spearman correlation analysis showed that angle α and fibrinogen were positively correlated with DF grading (r = 0.635, P < 0.01; r = 0.616, P < 0.01), k value was negatively correlated with DF (r= - 0.589, P < 0.01). ROC curve analysis showed that the optimal cut-off point for angle α to distinguish patients with DF from those without was 62.85 deg, with a sensitivity of 78.6% and specificity of 78.7%. The optimal cut-off point for k value was 1.75 min, with a sensitivity of 82.1% and specificity of 65.8%. The optimal cut-off point for fibrinogen was 3.85 g/l, with a sensitivity of 63.1% and specificity of 98.2%. The optimal cut-off point for angle α to evaluate the risk of diabetic foot progression was 70.20 deg, with a sensitivity of 73.2% and specificity of 90.7%. The optimal cut-off point for k value was 1.25 min, with a sensitivity of 67.9% and specificity of 90.8%. The optimal cut-off point for fibrinogen was 4.12 g/l, with a sensitivity of 85.7% and specificity of 93.5%. CONCLUSION: Angle α, k-value and fibrinogen have clinical significance on the risk of occurrence and development of diabetic foot, which can contribute to early diagnosis and early clinical intervention in DF.

Resveratrol attenuates cigarette smoke extract induced cellular senescence in human airway epithelial cells by regulating the miR-34a/SIRT1/NF-κB pathway.

Chronic obstructive pulmonary disease (COPD) is characterized by accelerated lung aging. Smoking is the critical risk factor for COPD. Cellular senescence of airway epithelial cells is the cytological basis of accelerated lung aging in COPD, and the regulation of microRNAs (miRNAs) is the central epigenetic mechanism of cellular senescence. Resveratrol (Res) is a polyphenol with anti-aging properties. This study investigated whether Res attenuates cigarette smoke extract (CSE)-induced cellular senescence in human airway epithelial cells (BEAS-2B) through the miR-34a/SIRT1/nuclear factor-kappaB (NF-κB) pathway. BEAS-2B cells were treated with Res, CSE and transfected with miR-34a-5p mimics. Cellular senescence was evaluated by senescence -related ß-galactosidase (SA-ß-gal) staining and expression of senescence-related genes (p16, p21, and p53). The expressions of miR-34a-5p, SIRT1, and NF-κB p65 were examined using quantitative real time polymerase chain reaction and western blotting. The senescence-associated secretory phenotype (SASP) cytokines (IL-1ß, IL-6, IL-8, TNF-α) were assessed by enzyme-linked immunosorbent assay. The binding between miR-34a-5p and SIRT1 was confirmed by dual-luciferase reporter assay. The results showed that CSE dose-dependently decreased cell viability and elevated cellular senescence, characterized by increased SA-ß-gal staining and senescence-related gene expressions (p16, p21, and p53). Further, CSE dose-dependently increased the expression of miR-34a-5p and SASP cytokines (IL-1ß, IL-6, IL-8, TNF-α) in BEAS-2B cells. Pretreatment with Res inhibited CSE-induced cellular senescence and secretion of SASP cytokines (IL-1ß, IL-6, IL-8, TNF-α) in a dose-dependent manner. Moreover, Res reversed the CSE-induced down-regulation of SIRT1 and up-regulation of miR-34a-5p and NF-κB p65. SIRT1 is a target of miR-34a-5p. Overexpression of miR-34a-5p via transfection with miR-34a-5p mimic in BEAS-2B cells attenuated the inhibitory effect of Res on cellular senescence, accompanied by reversing the expression of SIRT1 and NF-κB p65. In conclusion, Res attenuated CSE-induced cellular senescence in BEAS-2B cells by regulating the miR-34a/SIRT1/NF-κB pathway, which may provide a new approach for COPD treatment.

Corticosteroids showed more efficacy in treating hospitalized patients with COVID-19 than standard care but the effect is minimal: A systematic review and meta-analysis.

Background: During the ongoing coronavirus disease 2019 (COVID-19) pandemic, the use of corticosteroids for COVID-19 has ignited worldwide debate. Previous systematic reviews, including randomized controlled trials (RCTs) and retrospective observational studies, found that corticosteroids have beneficial effects in treating COVID-19. Aim: This systematic review and meta-analysis only included RCTs to assess the effectiveness and safety of corticosteroids in hospitalized patients with COVID-19. Methods: Comprehensive research strategies (PubMed, Embase, MEDLINE, and Coherence Library) were used to search for RCTs from December 2019 to January 2021. Results: Five RCTs were included with 7,235 patients, of which 2,508 patients were receiving corticosteroid treatments (dexamethasone or methylprednisolone), and 4,727 received standard care. The primary outcome was mortality within 28 days. The use of corticosteroids decreased the 28-day mortality of patients with COVID-19, but the findings were not statistically significant (RR, 0.91; 95% CI, 0.78-1.06, p = 0.24). The secondary outcome was the duration of hospitalization; no differences were found between the corticosteroid and standard care groups. However, corticosteroids were associated with a higher hospital discharge rate than standard treatment, but the result was not statistically significant (RR, 1.36; 95% CI, 0.95-1.96, p = 0.09). Conclusions: The results suggest that corticosteroids are comparable to standard care in terms of safety in treating COVID-19. Corticosteroids showed greater efficacy than standard care; however, the effect was minimal.

Circular RNA HIPK3 contributes to hyperglycemia and insulin homeostasis by sponging miR-192-5p and upregulating transcription factor forkhead box O1.

It has been shown that circular RNAs, a class of non-coding RNA molecules, play an important role in the regulation of glucose and lipid homeostasis. In the present study, we sought to investigate the function of circular RNA HIPK3 (circHIPK3) in diabetes-associated metabolic disorders, including hyperglycemia and insulin resistance. Results show that oleate stimulated circHIPK3 increase, and that circHIPK3 enhanced the stimulatory effect of oleate on adipose deposition, triglyceride (TG) content, and cellular glucose content in HepG2 cells. MiR-192-5p was the potential target of circHIPK3, since circHIPK3 significantly decreased miR-192-5p mRNA level, whereas anti-circHIPK3 significantly increased miR-192-5p mRNA level. Further study shows that transcription factor forkhead box O1 (FOXO1) was a downstream regulator of miR-192-5p, since miR-192-5p significantly decreased FOXO1 expression, whereas circHIPK3 significantly increased FOXO1 expression. Notably, the inhibitory effect of miR-192-5p was significantly reversed by circHIPK3. In vivo study shows that anti-miR-192-5p significantly increased blood glucose content, which was significantly inhibited by FOXO1 shRNA. MiR-192-5p significantly decreased adipose deposition and TG content in HepG2 cells, which was significantly reversed by the co-treatment with circHIPK3. Forskolin/dexamethasone (FSK/DEX) significantly increased cellular glucose, mRNA level of phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase), and this stimulatory effect of FSK/DEX was significantly inhibited by miR-192-5p. In the presence of circHIPK3, however, the inhibitory effect of miR-192-5p was totally lost. In summary, the present study demonstrated that circHIPK3 contributes to hyperglycemia and insulin resistance by sponging miR-192-5p and up-regulating FOXO1.