Constructing an efficient and easy-to-operate multigene expression system is currently a crucial part of plant genetic engineering. In this study, a fragment carrying three independent gene expression cassettes and the expression unit of the gene-silencing suppressor protein(RNA silencing suppressor 19 kDa protein, P19) simultaneously was designed and constructed. This fragment was cloned into the commonly used plant expression vector pCAMBIA300, and the plasmid pC1300-TP2-P19 was obtained. Each gene expression cassette consists of different promoters, fusion tags, and terminators. The target gene can be flexibly inserted into the corresponding site through enzymatic digestion and ligation or recombination and fused with different protein tags, which provides great convenience for subsequent detection. The enhanced green fluorescent protein(eGFP) reporter gene was individually constructed into each expression cassette to verify the feasibility of this vector system. The results of tobacco transient expression and laser-confocal microscopy showed that each expression cassette presented independent and normal expression. Meanwhile, the three key enzyme genes in the betanin synthesis pathway, BvCYP76AD, BvDODA1, and DbDOPA5GT, were constructed into the three expression cassettes. The results of tobacco transient expression phenotype, protein immunoblotting(Western blot), and chemical detection of product demonstrated that the three exogenous genes were highly expressed, and the target compound betanin was successfully produced. The above results indicated that the constructed multigene expression system for plants in this study was efficient and reliable and can achieve the co-transformation of multiple plant genes. It can provide a reliable vector platform for the analysis of plant natural product synthesis pathways, functional verification, and plant metabolic engineering.
Nicotiana/metabolism* ; Genetic Vectors/metabolism* ; Gene Expression Regulation, Plant ; Plant Proteins/metabolism* ; Plants, Genetically Modified/metabolism* ; Genetic Engineering/methods* ; Green Fluorescent Proteins/metabolism* ; Gene Expression

Premature ovarian insufficiency(POI) is a manifestation of ovarian aging, with a global incidence of 3.5%. If not addressed in time, POI can rapidly develop into premature ovarian failure(POF). The incidence of POI is mainly related to genetic factors, iatrogenic factors, autoimmunity, aging, infection, psychological factors, and other influences. POI not only causes menstrual disorders, amenorrhea, infertility, and dyspareunia but also tends to present with symptoms such as mood swings, insomnia, hot flashes, fatigue, as well as osteoporosis, coronary heart disease, diabetes, and other conditions, resulting in long-term psychological and physical health concerns for affected women. From traditional Chinese medicine(TCM)'s perspective, POI is primarily attributed to kidney Yin deficiency, with the main pathogenesis rooted in kidney deficiency, which affects the heart, liver, and spleen. It manifests in different syndrome types, including kidney deficiency with liver Qi stagnation, kidney deficiency with blood stasis, and spleen-kidney Yang deficiency. TCM employs a holistic view, utilizing multi-component TCM, multi-site acupuncture, and multi-target and multi-pathway interventions to treat POI. It offers unique advantages such as strong personalization, high safety, and good efficacy. In this paper, the animal and clinical research literature on the prevention and treatment of POI in the past 10 years was systematically summarized and reviewed. It is found that TCM mainly treats POI and alleviates POI-caused issues such as menstrual disorders, infertility, and emotional instability by regulating the neuroendocrine system(hypothalamic-pituitary-ovarian axis, HPOA) and related signaling pathways, improving ovarian function and antioxidant capacity, enhancing immune function, maintaining mitochondrial energy metabolism, inhibiting ferroptosis, and controlling endoplasmic reticulum stress.
Humans ; Primary Ovarian Insufficiency/physiopathology* ; Female ; Drugs, Chinese Herbal/therapeutic use* ; Animals ; Medicine, Chinese Traditional

In this study, lipopolysaccharide(LPS), ovalbumin(OVA), and compound 48/80(C48/80) were administered to establish non-infectious pneumonia models under simulated clinical conditions, and the correlation between their pathological characteristics and traditional Chinese medicine(TCM) syndromes was compared, providing the basis for the selection of appropriate animal models for TCM efficacy evaluation. An acute pneumonia model was established by nasal instillation of LPS combined with intraperitoneal injection for intensive stimulation. Three doses of OVA mixed with aluminum hydroxide adjuvant were injected intraperitoneally on days one, three, and five and OVA was administered via endotracheal drip for excitation on days 14-18 to establish an OVA-induced allergic pneumonia model. A single intravenous injection of three doses of C48/80 was adopted to establish a C48/80-induced pneumonia model. By detecting the changes in peripheral blood leukocyte classification, lung tissue and plasma cytokines, immunoglobulins(Ig), histamine levels, and arachidonic acid metabolites, the multi-dimensional analysis was carried out based on pathological evaluation. The results showed that the three models could cause pulmonary edema, increased wet weight in the lung, and obvious exudative inflammation in lung tissue pathology, especially for LPS. A number of pyrogenic cytokines, inclading interleukin(IL)-6, interferon(IFN)-γ, IL-1β, and IL-4 were significantly elevated in the LPS pneumonia model. Significantly increased levels of prostacyclin analogs such as prostaglandin E2(PGE2) and PGD2, which cause increased vascular permeability, and neutrophils in peripheral blood were significantly elevated. The model could partly reflect the clinical characteristics of phlegm heat accumulating in the lung or dampness toxin obstructing the lung. The OVA model showed that the sensitization mediators IgE and leukotriene E4(LTE4) were increased, and the anti-inflammatory prostacyclin 6-keto-PGF2α was decreased. Immune cells(lymphocytes and monocytes) were decreased, and inflammatory cells(neutrophils and basophils) were increased, reflecting the characteristics of "deficiency", "phlegm", or "dampness". Lymphocytes, monocytes, and basophils were significantly increased in the C48/80 model. The phenotype of the model was that the content of histamine, a large number of prostacyclins(6-keto-PGE1, PGF2α, 15-keto-PGF2α, 6-keto-PGF1α, 13,14-D-15-keto-PGE2, PGD2, PGE2, and PGH2), LTE4, and 5-hydroxyeicosatetraenoic acid(5S-HETE) was significantly increased, and these indicators were associated with vascular expansion and increased vascular permeability. The pyrogenic inflammatory cytokines were not increased. The C48/80 model reflected the characteristics of cold and damp accumulation. In the study, three non-infectious pneumonia models were constructed. The LPS model exhibited neutrophil infiltration and elevated inflammatory factors, which was suitable for the efficacy study of TCM for clearing heat, detoxifying, removing dampness, and eliminating phlegm. The OVA model, which took allergic inflammation as an index, was suitable for the efficacy study of Yiqi Gubiao formulas. The C48/80 model exhibited increased vasoactive substances(histamine, PGs, and LTE4), which was suitable for the efficacy study and evaluation of TCM for warming the lung, dispersing cold, drying dampness, and resolving phlegm. The study provides a theoretical basis for model selection for the efficacy evaluation of TCM in the treatment of pneumonia.
Animals ; Disease Models, Animal ; Mice ; Pneumonia/genetics* ; Medicine, Chinese Traditional ; Male ; Humans ; Cytokines/immunology* ; Female ; Lipopolysaccharides/adverse effects* ; Lung/drug effects* ; Drugs, Chinese Herbal ; Ovalbumin ; Mice, Inbred BALB C

In-depth study of the components of polymyxins is the key to controlling the quality of this class of antibiotics. Similarities and variations of components present significant analytical challenges. A two-dimensional (2D) liquid chromatography-mass spectrometr (LC-MS) method was established for screening and comprehensive profiling of compositions of the antibiotic colistimethate sodium (CMS). A high concentration of phosphate buffer mobile phase was used in the first-dimensional LC system to get the components well separated. For efficient and high-accuracy screening of CMS, a targeted method based on a self-constructed high resolution (HR) mass spectrum database of CMS components was established. The database was built based on the commercial MassHunter Personal Compound Database and Library (PCDL) software and its accuracy of the compound matching result was verified with six known components before being applied to genuine sample screening. On this basis, the unknown peaks in the CMS chromatograms were deduced and assigned. The molecular formula, group composition, and origins of a total of 99 compounds, of which the combined area percentage accounted for more than 95% of CMS components, were deduced by this 2D-LC-MS method combined with the MassHunter PCDL. This profiling method was highly efficient and could distinguish hundreds of components within 3 h, providing reliable results for quality control of this kind of complex drugs.

Lung cancer exhibits the highest incidence and mortality rates among cancers globally, with a five-year overall survival rate alarmingly below 20%. Targeting autophagy, though a controversial therapeutic strategy, is extensively employed in clinical practice. Current research is actively pursuing various therapeutic strategies using small molecules to exploit the dual function of autophagy. Nevertheless, the pivotal question of enhancing or inhibiting autophagy in cancer therapy merits further attention. This review aims to provide a comprehensive overview of the mechanisms of autophagy in lung cancer. It also explores recent advances in targeting cytotoxic autophagy and inhibiting protective autophagy with small molecules to induce cell death in lung cancer cells. Notably, most autophagy-targeting drugs, primarily natural small molecules, have demonstrated that activating cytotoxic autophagy effectively induces cell death in lung cancer, as opposed to inhibiting protective autophagy. These insights contribute to identifying druggable targets and drug candidates for potential autophagy-related lung cancer therapies, offering promising approaches to combat this disease.

Laparoscopic subtotal cholecystectomy (LSC) has been a safe and viable alternative to conversion to laparotomy in cases of severe cholecystitis. The objective of this study is to determine the utility of intraoperative choledochoscopy in LSC for the exploration of the gallbladder, cyst duct, and subsequent stone clearance of the cystic duct in cases of severe cholecystitis. A total of 72 patients diagnosed with severe cholecystitis received choledochoscopy-assisted laparoscopic subtotal cholecystectomy (CALSC). A choledochoscopy was performed to explore the gallbladder cavity and/or cystic duct, and to extract stones using a range of techniques. The clinical records, including the operative records and outcomes, were subjected to analysis. No LSC was converted to open surgery, and no bile duct or vascular injuries were sustained. All stones within the cystic duct were removed by a combination of techniques, including high-frequency needle knife electrotomy, basket, and electrohydraulic lithotripsy. A follow-up examination revealed the absence of residual bile duct stones, with the exception of one common bile duct stone, which was extracted via endoscopic retrograde cholangiopancreatography. In certain special cases, CALSC may prove to be an efficacious treatment for the management of severe cholecystitis. This technique allows for optimal comprehension of the situation within the gallbladder cavity and cystic duct, facilitating the removal of stones from the cystic duct and reducing the residue of the non-functional gallbladder remnant.

Laparoscopic subtotal cholecystectomy (LSC) has been a safe and viable alternative to conversion to laparotomy in cases of severe cholecystitis. The objective of this study is to determine the utility of intraoperative choledochoscopy in LSC for the exploration of the gallbladder, cyst duct, and subsequent stone clearance of the cystic duct in cases of severe cholecystitis. A total of 72 patients diagnosed with severe cholecystitis received choledochoscopy-assisted laparoscopic subtotal cholecystectomy (CALSC). A choledochoscopy was performed to explore the gallbladder cavity and/or cystic duct, and to extract stones using a range of techniques. The clinical records, including the operative records and outcomes, were subjected to analysis. No LSC was converted to open surgery, and no bile duct or vascular injuries were sustained. All stones within the cystic duct were removed by a combination of techniques, including high-frequency needle knife electrotomy, basket, and electrohydraulic lithotripsy. A follow-up examination revealed the absence of residual bile duct stones, with the exception of one common bile duct stone, which was extracted via endoscopic retrograde cholangiopancreatography. In certain special cases, CALSC may prove to be an efficacious treatment for the management of severe cholecystitis. This technique allows for optimal comprehension of the situation within the gallbladder cavity and cystic duct, facilitating the removal of stones from the cystic duct and reducing the residue of the non-functional gallbladder remnant.

Objective: To explore the association between dietary patterns and overweight and obesity among primary and middle school students, so as to provide the guidance of diet balance and obesity prevention for students. Methods: Students from 11 primary and middle schools in Tongzhou District, Beijing Municipality, were selected using the stratified cluster sampling method. Demographic information was collected through a general questionnaire, and dietary intake types and frequencies over the past week were assessed using a food frequency questionnaire. Dietary patterns were determined using factor analysis, and the dietary pattern factor scores were divided into Q1, Q2, Q3, and Q4 groups based on quartiles. Height and weight were collected through physical examinations, and Z-scores of body mass index adjusted for gender and age were calculated to assess overweight and obesity. The association between dietary patterns and overweight and obesity was analyzed using a multivariable logistic regression model. Results: A total of 1 485 students were surveyed, including 745 males (50.17%) and 740 females (49.83%). The mean age was (12.87±2.86) years. Three dietary patterns were identified: animal and plant protein dietary pattern, high-fat and high-sugar dietary pattern, and vegetable-fruit-dairy dietary pattern. The detection rates of overweight and obesity were 42.57%, 48.42%, and 34.75%, respectively, with statistically significant differences (P<0.05). Multivariable logistic regression analysis showed that compared with the vegetable-fruit-dairy dietary pattern, the animal and plant protein dietary pattern (OR=1.406, 95%CI: 1.084-1.823) and the high-fat and high-sugar dietary pattern (OR=2.137, 95%CI: 1.643-2.779) were associated with a higher risk of overweight and obesity among primary and middle school students. Compared with the Q1 group of dietary pattern factor scores, the Q3 group (OR=1.631, 95%CI: 1.206-2.208) and Q4 group (OR=1.965, 95%CI: 1.446-2.671) of the high-fat and high-sugar dietary pattern had an increased risk of overweight and obesity, while the Q4 group of the vegetable-fruit-dairy dietary pattern (OR=0.551, 95%CI: 0.406-0.747) had a reduced risk of overweight and obesity. There was no statistical association between factor scores of animal and plant protein dietary pattern and overweight and obesity (all P>0.05). Conclusions The animal and plant protein dietary pattern and the high-fat and high-sugar dietary pattern are associated with a higher risk of overweight and obesity among primary and middle school students. Within the same dietary pattern, the more inclined students are to the high-fat and high-sugar dietary pattern, the higher their risk of overweight and obesity, while the more inclined they are to the vegetable-fruit-dairy dietary pattern, the lower their risk of overweight and obesity.

To develop a novel polyamino acid-based nanohydrogel drug delivery system for dexamethasone to enhance its delivery efficiency to the inner ear.

Laparoscopic subtotal cholecystectomy (LSC) has been a safe and viable alternative to conversion to laparotomy in cases of severe cholecystitis. The objective of this study is to determine the utility of intraoperative choledochoscopy in LSC for the exploration of the gallbladder, cyst duct, and subsequent stone clearance of the cystic duct in cases of severe cholecystitis. A total of 72 patients diagnosed with severe cholecystitis received choledochoscopy-assisted laparoscopic subtotal cholecystectomy (CALSC). A choledochoscopy was performed to explore the gallbladder cavity and/or cystic duct, and to extract stones using a range of techniques. The clinical records, including the operative records and outcomes, were subjected to analysis. No LSC was converted to open surgery, and no bile duct or vascular injuries were sustained. All stones within the cystic duct were removed by a combination of techniques, including high-frequency needle knife electrotomy, basket, and electrohydraulic lithotripsy. A follow-up examination revealed the absence of residual bile duct stones, with the exception of one common bile duct stone, which was extracted via endoscopic retrograde cholangiopancreatography. In certain special cases, CALSC may prove to be an efficacious treatment for the management of severe cholecystitis. This technique allows for optimal comprehension of the situation within the gallbladder cavity and cystic duct, facilitating the removal of stones from the cystic duct and reducing the residue of the non-functional gallbladder remnant.