Cuproptosis-Related lncRNA Gene Signature Establishes a Prognostic Model of Gastric Adenocarcinoma and Evaluate the Effect of Antineoplastic Drugs.

BACKGROUND: One of the most frequent malignancies of the digestive system is stomach adenocarcinoma (STAD). Recent research has demonstrated how cuproptosis (copper-dependent cell death) differs from other cell death mechanisms that were previously understood. Cuproptosis regulation in tumor cells could be a brand-new treatment strategy. Our goal was to create a cuproptosis-related lncRNA signature. Additionally, in order to evaluate the possible immunotherapeutic advantages and drug sensitivity, we attempted to study the association between these lncRNAs and the tumor immune microenvironment of STAD tumors. METHODS: The TCGA database was accessed to download the RNA sequencing data, genetic mutations, and clinical profiles for TCGA STAD. To locate lncRNAs related to cuproptosis and build risk-prognosis models, three techniques were used: co-expression network analysis, Cox-regression techniques, and LASSO techniques. Additionally, an integrated methodology was used to validate the models' predictive capabilities. Then, using GO and KEGG analysis, we discovered the variations in biological functions between each group. The link between the risk score and various medications for STAD treatment was estimated using the tumor mutational load (TMB) and tumor immune dysfunction and rejection (TIDE) scores. RESULT: We gathered 22 genes linked to cuproptosis based on the prior literature. Six lncRNAs related to cuproptosis were used to create a prognostic marker (AC016394.2, AC023511.1, AC147067.2, AL590705.3, HAGLR, and LINC01094). After that, the patients were split into high-risk and low-risk groups. A statistically significant difference in overall survival between the two groups was visible in the survival curves. The risk score was demonstrated to be an independent factor affecting the prognosis by both univariate and multivariate Cox regression analysis. Different risk scores were substantially related to the various immunological states of STAD patients, as further evidenced by immune cell infiltration and ssGSEA analysis. The two groups had differing burdens of tumor mutations. In addition, immunotherapy was more effective for STAD patients in the high-risk group than in the low-risk group, and risk scores for STAD were substantially connected with medication sensitivity. CONCLUSIONS: We discovered a marker for six cuproptosis-associated lncRNAs linked to STAD as prognostic predictors, which may be useful biomarkers for risk stratification, evaluation of possible immunotherapy, and assessment of treatment sensitivity for STAD.

Multiplex gene precise editing and large DNA fragment deletion by the CRISPR-Cas9-TRAMA system in edible mushroom Cordyceps militaris.

The medicinal mushroom Cordyceps militaris contains abundant valuable bioactive ingredients that have attracted a great deal of attention in the pharmaceutical and cosmetic industries. However, the development of this valuable mushroom faces the obstacle of lacking powerful genomic engineering tools. Here, by excavating the endogenous tRNA-processed element, introducing the extrachromosomal plasmid and alongside with homologous template, we develop a marker-free CRISPR-Cas9-TRAMA genomic editing system to achieve the multiplex gene precise editing and large synthetic cluster deletion in C. militaris. We further operated editing in the synthetases of cordycepin and ergothioneine to demonstrate the application of Cas9-TRAMA system in protein modification, promoter strength evaluation and 10 kb metabolic synthetic cluster deletion. The Cas9-TRAMA system provides a scalable method for excavating the valuable metabolic resource of medicinal mushrooms and constructing a mystical cellular pathway to elucidate the complex cell behaviours of the edible mushroom.

Enhancement of ergothioneine production by discovering and regulating its metabolic pathway in Cordyceps militaris.

BACKGROUND: Cordyceps militaris is a traditional medicinal fungus contains a variety of functional ingredients and has been developed as an important mushroom food recently. Ergothioneine, one of the antioxidative compounds in C. militaris, is benefits on aging-related diseases and therefore became a novel functional food nutritive fortifier. Currently, the main diet source of ergothioneine is mushroom food. However, the mushroom farming faces the problems such as rather low ingredient yield and spontaneous degeneration associated fruiting body that restricts large scale production of ergothioneine. RESULTS: In this study, we excavated the ergothioneine synthetases in mushroom and modified the genes in C. militaris to construct a new ergothioneine synthesis pathway. By further introducing this pathway into C. militaris genome, we succeeded to increase the ingredients' production of engineering strain, the highest amount of ergothioneine and cordycepin were up to 2.5 g/kg dry weight and 2 g/L, respectively. Additionally, the expression of ergothioneine synthetase genes in the shape-mutated degenerative C. militaris could recover the ability of degenerative strain to produce high amount of ingredients, suggesting the metabolic regulation of ergothioneine might release the symptom of mushroom degeneration. CONCLUSION: This study reveals a new pathway to fulfill the market needs of functional mushroom food and food fortifier ergothioneine. It implied the mycelium of C. militaris could be engineered as a novel medicinal mushroom food which could produce higher amount of valuable ingredients.

Yanghe Decoction Effectively Alleviates Lung Injury and Immune Disorder in Asthmatic Mice Induced by Ovalbumin.

Objective: To probe into the ameliorative effect of Yanghe Decoction on pulmonary injury and immunologic derangement in asthmatic mice. Methods: C57BL/6 mice were randomized into control (Con), Model, and Yanghe Decoction (YHF) groups, with 12 in each. The asthma model of adult female mice was induced by ovalbumin in the Model group, and the YHF group was treated by Yanghe Decoction on the basis of asthma modeling. The Con group received the same amount of normal saline. Inspiratory resistance (Ri), expiratory resistance (Re), lung compliance (CL), and maximal voluntary ventilation (MVV) were measured after modeling. Lung tissue was collected for the measurement of interleukin (IL)-4, IL-5, IL-6, IL-10, IL-13, and tumor necrosis factor-α (TNF-α) by ELISA kits. Combined with HE staining and PAS staining, the pathological alterations of the lung in each group were observed, and CD4+, Th2, and Th1 contents were determined by flow cytometry (FCM). Results: The pulmonary function (PF) test revealed notably reduced Ri and Re as well as enhanced CL and MVV in asthmatic mice after the application of Yanghe Decoction. Yanghe Decoction dramatically ameliorated the pathological changes of lung tissue in asthmatic mice, as demonstrated by the staining results. ELISA results showed that Yanghe Decoction validly reduces lung tissue IL-4, IL-5, IL-6, IL-13, TNF-α and upregulates IL-10 in asthmatic mice. FCM indicated that Yanghe Decoction obviously reduced the number of Th1 and Th2 cells in asthmatic mice, although it caused the decrease of CD4+ cells, but the difference was not statistically significant. Conclusions: Yanghe Decoction can effectively ameliorate the inflammatory reaction, immune cell disorder, and PF injury in ovalbumin-induced asthmatic mice.

Lonicerin attenuates house dust mite-induced eosinophilic asthma through targeting Src/EGFR signaling.

Eosinophilic asthma is the predominant phenotype of asthma, and although these patients are sensitive to glucocorticoid therapy, they also experience many side effects. Lonicerin is a kind of bioflavonoid isolated from the Chinese herb Lonicera japonica Thunb, which has anti-inflammatory and immunomodulatory effects. The aim of this study was to elucidate the effects of lonicerin on eosinophilic asthma and its potential mechanisms. Here, we established a house dust mite (house dust mite)-induced eosinophilic asthma model in BALB/c mouse, and evaluated the effects of lonicerin on it. Our results showed that lonicerin significantly reduced airway hyperresponsiveness the number of inflammatory cells (especially eosinophils) and the elevation of interleukin (IL)-4, IL-5, IL-13 and eotaxin in bronchoalveolar lavage fluid (BALF) supernatants of mice. Additionally, lonicerin also eminently blunted inflammatory infiltration and mucus secretion, as well as mRNA levels of Mucin 5AC (MUC5AC) in lung tissue. Furthermore, results of network pharmacology and molecular docking revealed that Src kinase and epidermal growth factor receptor may be the potential targets responsible for the effects of lonicerin. Finally, in vivo experiments confirmed that lonicerin inhibited activation of the Src/EGFR pathway by decreasing their phosphorylation. Taken together, the present study demonstrated that lonicerin could suppress HDM-induced eosinophilic asthma in mice through inhibiting the activation of Src/EGFR pathway, which also provides a basis for further research as a new potentially therapeutic agent for eosinophilic asthma and its underlying mechanisms in the future.

Jia-Wei-Yu-Ping-Feng-San Attenuates Group 2 Innate Lymphoid Cell-Mediated Airway Inflammation in Allergic Asthma.

The incidence of asthma has increased in recent decades. Although corticosteroids and bronchodilators are used in clinical practice, the control of asthma remains a challenge. Allergic asthma is characterized airway inflammation mediated by type 2 immune response. Group 2 innate lymphoid cells (ILC2s) are an important source of type 2 cytokines IL-5 and IL-13, which contribute to the progress of asthma. Jia-Wei-Yu-Ping-Feng-San (JWYPFS), a traditional Chinese medicine, has been widely used to treat asthma in China. In this study we investigated the mechanisms of JWYPFS in the treatment of asthma, especially the effect on ILC2s important in airway inflammation. Female C57BL/6 mice were sensitized and challenged with OVA to establish a model of allergic asthma. Airway hyperresponsiveness was examined by direct airway resistance analysis. Inflammatory cell counts were determined in bronchoalveolar lavage fluid (BALF). Inflammatory cell infiltration and mucus hypersecretion in lung tissue sections was observed by HE and PAS staining, respectively. The numbers and proportions of ILC2s as well as the ILC2s-related transcription factors GATA3, IRF4, and type 2 cytokines were measured in lung tissue samples. Additionally, ILC2s were collected from mouse lung; ILC2s-related cytokines and GATA3 and IRF4 were evaluated after IL-33-induced activation of ILC2s in vitro. Elevated inflammatory cells, mucus secretion, airway hyperresponsiveness and type 2 cytokines in the OVA-treated asthma group indicated that an allergic asthma model had been established. JWYPFS treatment attenuated airway resistance and reduced inflammatory cells including eosinophils, and inhibited mucus production and type 2 cytokines in these asthmatic mice. Moreover, JWYPFS treatment dramatically decreased the numbers and proportions of ILC2s and the mRNA levels of GATA3 and IRF4. In an in vitro experiment JWYPFS significantly suppressed GATA3, IRF4 and type 2 cytokine expression, including IL-5 and IL-13 in IL-33-stimulated ILC2s. JWYPFS alleviates ILC2s-mediated airway inflammation, suggesting that JWYPFS might be an effective agent to treat allergic asthma.

Linggan Wuwei Jiangxin formula ameliorates airway hyperresponsiveness through suppression of IL-1ß and IL-17A expression in allergic asthmatic mice especially with diet-induced obesity.

BACKGROUND: Obese asthma represents a disease phenotype, which is associated with worse disease control and unresponsiveness to standard anti-inflammatory regimens, including inhaled corticosteroids. Obesity-related innate airway hyperresponsiveness (AHR) plays a role in this asthma phenotype via activation of the IL-1ß/innate lymphoid cell 3 (ILC3)/IL-17A pathway. Linggan Wuwei Jiangxin (LGWWJX) formula may be a promising therapeutic option for obese asthma according to traditional Chinese medicine theory, clinical experience and related research. METHODS: The murine model of allergic asthma with obesity was induced by ovalbumin (OVA) sensitization and challenge in combination with a high fat diet (HFD). LGWWJX formula intervention was oral administrated. AHR and bronchoalveolar lavage fluid (BALF) cellularity were measured. Lung and liver histopathology assessment was performed by haematoxylin and eosin (H&E) staining. IL-1ß and IL-17A in BALF and serum were evaluated by ELISA. Additionally, the influence of different concentrations of LGWWJX formula on IL-1ß stimulated IL-17A mRNA expression in ILC3 cells was evaluated in vitro. RESULTS: LGWWJX treatment significantly reduced AHR and allergic airway inflammatory responses in asthmatic mice, as measured by pulmonary histopathology and BALF cellularity, and these effects were more pronounced in obese asthmatic mice. While eosinophil infiltration in BALF was suppressed with LGWWJX treatment in non-obese asthmatic mice, neutrophils and basophils were significantly decreased in obese asthmatic mice. Notably, LGWWJX also demonstrated remarkable efficacy for weight loss and improvements in hepatic steatosis in mice fed with a HFD. Furthermore, the protein levels of IL-1ß in both serum and BALF, as well as those of BALF IL-17A, declined with LGWWJX intervention in both obese and non-obese asthmatic mice, and results from ex-vivo experiments found that LGWWJX significantly attenuated the expression of IL-17A in ILC3 cells with or without stimulation by IL-1ß. CONCLUSIONS: LGWWJX may exert a protective effect on asthmatic individuals, especially those with concurrent obesity, most likely through mechanisms including the inhibition of the IL-1ß/ILC3/IL-17A/AHR axis, anti-inflammatory effects, weight loss, and the regulation of lipid metabolism. This suggests a promising role of LGWWJX, alone or in combination with anti-inflammatory agents, for the treatment of obese asthma.

Transcriptome Analysis Reveals the Flexibility of Cordycepin Network in Cordyceps militaris Activated by L-Alanine Addition.

Cordycepin, isolated from the traditional medicinal fungus Cordyceps militaris, has gained much attention due to its various clinical functions. Previous reports of L-alanine addition could significantly improve cordycepin production, but the molecular mechanism remains unknown. In this study, transcriptome analysis of C. militaris with doubled cordycepin production induced by L-alanine addition provides an insight into the flexibility of the cordycepin network. The biopathways of energy generation and amino acid conversion were activated so that cordycepin substrate generation was consequently improved. Specific genes of rate-limiting enzymes in these pathways, as well as related transcription factors, were figured out. Two key Zn2Cys6-type transcription factors CmTf1 and CmTf2 were verified to play the roles of doubling the cordycepin production by overexpression of their coding genes in C. militaris wild type. These results provide a complete map of the cordycepin network in C. militaris with a distinct understanding of the flexibility of joints, giving a better foundation for increasing cordycepin yield and strain breeding in the future.

Optimized method to harvest both kidneys from one donor rat for transplantation.

BACKGROUND: Rat renal transplantation is an essential experimental model for studies of transplantation immunobiology. Harvesting both kidneys from one donor rat for transplantation is widely used to reduce the number of experimental animals. Using the conventional method, both kidneys of the donor rat are harvested simultaneously, which leads to the prolonged warm ischemic times during transplantation of the second donor kidney. Prolonged warm ischemia time is the main risk factor for delayed graft function. METHODS: Two different approaches are compared. Method 1, conventional method: both kidneys of the donor rat are harvested simultaneously and then transplanted into two recipients. During transplantation, the first and second donor kidneys were regarded as Group 1 and 2, respectively. Method 2, step-by-step method: after left nephrectomy, the donor rat survives, and we perform left renal transplantation (Group 3). Then, the right kidney of the surviving donor rat is incised and transplanted into the left side of the second recipient (Group 4). RESULTS: The success rates were 86.7, 93.3, 93.3 and 86.7% in groups 1, 2, 3 and 4, respectively. The warm ischemia times increased significantly in group 2 compared with the other 3 groups (p < 0.05) but differed non-significantly between groups 3 and 4 (p > 0.05). Serum creatinine levels, blood urea nitrogen and 24-h urine protein level obviously increased after kidney transplantation in group 2 compared with other groups (p < 0.05). CONCLUSIONS: We developed an optimized method for reducing warm ischemia time, thereby minimizing delayed graft function.