Definitive chemoradiotherapy (dCRT) has become a cornerstone in the treatment of locally advanced esophageal cancer; however, local control remains suboptimal, and persistent lesions or locoregional recurrences after treatment are not uncommon. For patients without distant metastases but with local failure, whether surgical intervention can still offer curative potential remains a major clinical dilemma. Salvage esophagectomy (SE) offers potential long-term survival for selected patients, but this procedure is performed in the context of severe fibrosis, impaired local blood supply, and obscured anatomical planes following chemoradiotherapy, resulting in significantly higher perioperative risk compared to primary esophagectomy. Consequently, controversies exist regarding patient selection, preoperative restaging, choice of surgical approach, extent of lymphadenectomy, gastrointestinal reconstruction, and perioperative management. In recent years, with the refinement of restaging modalities such as PET/CT, the accumulation of experience in high-volume centers, and emerging evidence from clinical studies, the clinical role of SE has gradually shifted from a "high-risk salvage measure" to a "selective curative strategy aimed at achieving long-term survival in carefully selected patients". Nevertheless, standardized guidelines for patient selection, technical approaches, and perioperative management are still lacking. Based on current evidence and clinical experience, experts organized by the Integrated Esophageal Cancer Committee of Chinese Anti-Cancer Association systematically reviewed key issues regarding SE, including its definition, indications, preoperative evaluation, choice of surgical approach, lymphadenectomy, gastrointestinal reconstruction, and perioperative management, and formulated a Chinese expert consensus. This consensus aims to provide guidance for standardized assessment, appropriate referral, individualized surgical decision-making, and optimized perioperative management of patients with locoregional failure after dCRT. Ultimately, this will increase the likelihood of R0 resection, reduce the risk of severe complications, and promote the safer, more judicious, and standardized implementation of SE in high-risk scenarios.

BACKGROUND: Electroacupuncture (EA) may affect the severity of hot flashes (HFs) associated with natural menopause and provide additional benefits for postmenopausal women. However, the evidence for its effectiveness in the management of early postmenopausal HFs remains inadequately understood. OBJECTIVE: We designed this trial to assess the efficacy and safety of EA for relieving early postmenopausal HFs. DESIGN, SETTING, PARTICIPANTS AND INTERVENTIONS: This randomized sham-controlled trial involved 72 women with HFs. The participants were divided equally into the intervention and control groups. The intervention group was treated with EA, while the control group was treated with sham acupuncture. The main acupoints used were Hegu (LI4), Guanyuan (RN4), Sanyinjiao (SP6), Taixi (KI3), Fuliu (KI7) and Shenshu (BL23). All participants received 18 treatment sessions, distributed across a 6-week period. The treatment was administered on three occasions per week, adhering to a fixed weekday schedule (Monday, Wednesday, Friday or Tuesday, Thursday, Saturday) with a minimum interval of one day between sessions. Each patient received a 12-week follow-up. MAIN OUTCOME MEASURES: The HF score was the primary outcome. Participants documented the frequency and severity of HFs in a 7-day symptom diary, which provided data for calculating the HF score. Secondary outcomes were the Menopause Rating Scale (MRS), Menopause-Specific Quality of Life Questionnaire (MENQOL), Pittsburgh Sleep Quality Index (PSQI) and Traditional Chinese Medicine Syndrome Score Scale (TCMSSS), as well as estradiol (E₂), luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels. RESULTS: Both groups demonstrated significant reductions in HF scores after the treatment and during the follow-up (P < 0.001). Immediately after completion of the 6-week treatment cycle and at 12 weeks post-intervention, the HF scores were similar in both groups. At week 6, the intervention group showed significantly greater improvements in MRS, MENQOL (vasomotor, psychosocial, and physical), PSQI and TCMSSS scores (P < 0.05). The improvements in the MENQOL (vasomotor, and psychosocial) and PSQI total scores persisted through the follow-up (P < 0.05). However, the results showed no significant inter- or intragroup differences in sexual scores on the MENQOL (P > 0.05). EA did not significantly decrease E₂, LH or FSH levels compared to placebo. The incidence of adverse events was similar in both groups. CONCLUSION: EA does not significantly improve HFs in early postmenopausal patients. However, it enhances the quality of sleep and decreases menopausal symptoms across vasomotor, psychosocial and physical domains. TRIAL REGISTRATION Chinese Clinical Trial Registry (http://www.chictr.org.cn); Trial ID: ChiCTR2300072002. Please cite this article as: Wang HX, Yu XT, Hu J, Chen JJ, Mei YT, Chen YF. Electroacupuncture for hot flashes in early menopause: A randomized sham-controlled trial. J Integr Med. 2025; 23(5):519-527.
Humans ; Female ; Electroacupuncture ; Hot Flashes/therapy* ; Middle Aged ; Acupuncture Points ; Quality of Life ; Menopause ; Treatment Outcome ; Adult

OBJECTIVE: CRISPR-Cas protects bacteria from exogenous DNA invasion and is associated with bacterial biofilm formation and pathogenicity. METHODS: We analyzed the type I-F CRISPR-Cas system of Legionella pneumophila WX48, including Cas1, Cas2-Cas3, Csy1, Csy2, Csy3, and Cas6f, along with downstream CRISPR arrays. We explored the effects of the CRISPR-Cas system on the in vitro growth, biofilm-forming ability, and pathogenicity of L. pneumophila through constructing gene deletion mutants. RESULTS: The type I-F CRISPR-Cas system did not affect the in vitro growth of wild-type or mutant strains. The biofilm formation and intracellular proliferation of the mutant strains were weaker than those of the wild type owing to the regulation of type IV pili and Dot/Icm type IV secretion systems. In particular, Cas6f deletion strongly inhibited these processes. CONCLUSION The type I-F CRISPR-Cas system may reduce biofilm formation and intracellular proliferation in L. pneumophila.
Legionella pneumophila/pathogenicity* ; CRISPR-Cas Systems ; Biofilms/growth & development* ; Phenotype ; Bacterial Proteins/metabolism* ; Gene Deletion

The emergence of clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated proteins (Cas) system represents a revolutionary paradigm shift in molecular diagnostics, offering transformative potential for RNA analysis within the rigorous demands of forensic science. Conventional forensic RNA detection methodologies, such as reverse transcription-quantitative polymerase chain reaction (RT-qPCR) or microarray analysis, are significantly hampered by inherent limitations including complex, multi-step protocols requiring sophisticated laboratory infrastructure, pronounced susceptibility to inhibitors prevalent in complex forensic matrices (e.g., humic acids, heme, indigo dyes), and often inadequate sensitivity for trace or degraded samples typical of crime scenes, thereby failing to meet the critical operational imperatives of forensic practice: rapidity, high specificity, sensitivity, portability, and robustness against interference. This review posits that CRISPR-Cas-based RNA detection technology provides a groundbreaking solution by leveraging the programmable, sequence-specific recognition conferred by the synergistic interaction between a designed guide RNA (gRNA) and Cas effector proteins (e.g., Cas12a, Cas13a, Cas14). Upon target RNA binding, specific Cas enzymes undergo conformational activation, exhibiting collateral cleavage activity―a unique catalytic amplification mechanism where the enzyme non-specifically cleaves surrounding reporter molecules, enabling ultra-high sensitivity. To further enhance detection limits, CRISPR-Cas systems are strategically integrated with isothermal pre-amplification techniques like recombinase polymerase amplification (RPA) or loop-mediated isothermal amplification (LAMP), which efficiently amplify target RNA at constant temperatures, eliminating the need for thermal cyclers. This powerful cascade―isothermal pre-amplification followed by CRISPR-mediated sequence-specific recognition and collateral signal amplification―achieves exceptional sensitivity, often down to the single-molecule (attomolar) level, while drastically reducing analysis time to potentially 30-60 min. Crucially, the compatibility of CRISPR-Cas detection with simple, equipment-free readout systems, such as lateral flow strips (LFS) for visual colorimetric results or portable fluorescence/electrochemical sensors, facilitates true point-of-need (PON) forensic analysis directly at crime scenes, morgues, or field labs. This enables rapid applications like specific body fluid identification (e.g., distinguishing menstrual blood via miRNA, identifying saliva via mRNA), post-mortem interval (PMI) estimation through RNA degradation/expression patterns, donor age inference via age-related RNA markers, tissue identification, and microbial forensics, thereby accelerating investigative leads, minimizing sample degradation risks, and optimizing resource allocation. However, significant challenges impede widespread adoption, including persistent environmental interference inhibiting enzymes, fluctuations in Cas/amplification enzyme activity affecting reproducibility, a critical lack of standardized protocols and validated quality assurance/quality control (QA/QC) frameworks essential for forensic reliability and court admissibility, and current limitations in multiplex detection capability. Consequently, future research must prioritize overcoming multiplexing bottlenecks for comprehensive analysis, enhancing system robustness through Cas protein engineering and optimized reagents, developing fully integrated, sample-to-answer microfluidic or lateral flow devices for user-friendly field deployment, and collaboratively establishing universally accepted validation guidelines, performance standards, and stringent QA/QC procedures. Furthermore, the urgent development of clear ethical guidelines governing the use of this highly sensitive technology, particularly concerning RNA data privacy and potential misuse, is imperative. This review systematically outlines the principles, forensic applications, current limitations, and future trajectories of CRISPR-RNA detection, with the authors’ conviction that focused efforts addressing these challenges will translate this technology into a cornerstone of next-generation forensic practice, driving unprecedented efficiency and innovation in field investigations and laboratory analysis to enhance justice delivery.

This paper aims to study the effect of Ziziphi Spinosae Semen extracts on chronic unpredictable mild stress(CUMS)-induced depression-like and insomnia behavior models of mice. The CUMS-induced depression-like and insomnia behavior model of mice was established by CUMS treatment for three weeks. The mice were randomly divided into control group, model group, positive drug diazepam group(2 mg·kg~(-1)), as well as low-dose group(1.95 g·kg~(-1)), medium-dose group(3.9 g·kg~(-1)), and high-dose group(7.8 g·kg~(-1)) of Ziziphi Spinosae Semen extracts, with 18 mice in each group. On the 15th day of modeling, the drug was administered intragastrically once a day for one week. Then, the pentobarbital sodium cooperative righting experiment, open field experiment, and elevated plus maze experiment were carried out, respectively. The contents of neurotransmitters 5-hydroxytryptamine(5-HT) and 5-hydroxyindoleacetic acid(5-HIAA) in serum and thalamus of mice, as well as the levels of corticotropin releasing hormone(CRH), adrenocorticotropic hormone(ACTH), and corticosterone(CORT) in serum, were determined by enzyme-linked immunosorbent assay(ELISA). The neuron damage in the hippocampus of mice was observed by hematoxylin-eosin(HE) staining and Nissl staining. Western blot was used to detect the expressions of tryptophan hydroxylase 2(TPH2), serotonin transporter(SERT), monoamine oxidase A(MAOA), five prime repressors under dual repression binding protein 1(Freud1), synaptic plasticity-related proteins [cellular gene FOS(C-FOS), postsynaptic density protein 95(PSD95), synapsin 1(SYN1), and activity-regulated cytoskeleton-associated gene(ARC)], blood-brain barrier(BBB) permeability-related proteins [zonula occludens 1(ZO-1), occludin, and claudin 1], inflammatory factors [NOD-, LRR-and pyrin domain-containing protein 3(NLRP3), apoptosis-associated speck-like protein(ASC), gasdermin D(GSDMD), caspase-3, and caspase-8], and antioxidant factors [nuclear factor erythroid 2-related factor 2(NRF2) and heme oxygenase 1(HO1)] in thalamic tissue of mice. The results indicated that compared with that in the model group, the sleep latency was significantly shortened, and the sleep duration was significantly prolonged in each dose group of Ziziphi Spinosae Semen extracts. The number of visits to the central area of the open field and the distance and time of visits were significantly increased in each dose group of Ziziphi Spinosae Semen extracts. In addition, the proportion of distance and time of entering the open arm area of the elevated plus maze was significantly increased in each dose group of Ziziphi Spinosae Semen extracts. The contents of 5-HT and 5-HIAA in serum and thalamus of mice increased to varying degrees in each dose group of Ziziphi Spinosae Semen extracts; the contents of CRH, ACTH, and CORT in serum of mice were significantly decreased. The protein expression of TPH2 was significantly increased. The protein expression of MAOA, SERT, and Freud1 was significantly decreased. Ziziphi Spinosae Semen extracts could also significantly reduce the protein expression of C-FOS but significantly increase the protein expression of PSD95, ARC, and SYN1. They could reduce the pathological damage of the hippocampus in mice and significantly increase the protein expression of ZO-1, occluding, and claudin 1. The protein expression of NLRP3, GSDMD, ASC, caspase-3, and caspase-8 in the thalamic tissue of mice was significantly decreased, and the protein expression of HO1 and NRF2 was significantly increased. In conclusion, Ziziphi Spinosae Semen extracts could effectively improve sleep disorders and depression-like behaviors in CUMS-induced model mice, which may be related to regulating the 5-HT anabolism process and hypothalamic-pituitary-adrenal(HPA) axis-related hormone levels, reducing pathological damage in the hippocampus, improving synaptic plasticity, repairing BBB integrity, and alleviating inflammatory response and oxidative stress damage.
Animals ; Ziziphus/chemistry* ; Mice ; Male ; Depression/psychology* ; Drugs, Chinese Herbal/administration & dosage* ; Sleep Initiation and Maintenance Disorders/psychology* ; Stress, Psychological/complications* ; Behavior, Animal/drug effects* ; Humans ; Disease Models, Animal

High-performance liquid chromatography(HPLC) was used to determine the content of three major components in Citri Reticulatae Semen(CRS), including limonin, nomilin, and obacunone. The chromaticity of the CRS sample during salt processing and stir-frying was measured using a color difference meter. Next, the relationship between the color and content of the salt-processed CRS sample was investigated through correlation analysis. By integrating the oil bath technique for processing simulation with HPLC, the changes in the relative content of nomilin and its transformation products were analyzed, with its structural transformation pattern during processing identified. Additionally, RAW264.7 cells were induced with lipopolysaccharides(LPSs) to establish an inflammatory model, and the anti-inflammatory activity of nomilin and its transformation product, namely obacunone was evaluated. The results indicated that as processing progressed, E~*ab and L~* values showed a downward trend; a~* values exhibited a slow increase over a certain period, followed by no significant changes, and b~* values remained stable with no significant changes over a certain period and then started to decrease. The limonin content remained barely unchanged; the nomilin content decreased, and the obacunone increased significantly. The changing trends in content and color parameters during salt-processing and stir-frying were basically consistent. The content of nomilin and obacunone was significantly correlated with the colorimetric values(L~*, a~*, b~*, and E~*ab), while limonin content showed no significant correlation with these values. By analyzing HPLC patterns of nomylin at different heating temperatures and time, it was found that under conditions of 200-250 ℃ for heating of 5-60 min, the content of nomilin significantly decreased, while the obacunone content increased pronouncedly. The in vitro anti-inflammatory activity results indicated that compared to the model group, the group with a high concentration of nomilin and the groups with varying concentrations of obacunone showed significantly reduced release of nitric oxide(NO)(P<0.01). When both were at the same concentration, obacunone showed better performance in inhibiting NO release. In this study, the obvious correlation between the color and content of major components during the processing of CRS samples was identified, and the dynamic patterns of quality change in CRS samples during processing were revealed. Additionally, the study revealed and confirmed the transformation of nomilin into obacunone during processing, with the in vitro anti-inflammatory activity of obacunone significantly greater than that of nomilin. These findings provided a scientific basis for CRS processing optimization, tablet quality control, and its clinical application.
Mice ; Animals ; Drugs, Chinese Herbal/pharmacology* ; RAW 264.7 Cells ; Limonins/chemistry* ; Chromatography, High Pressure Liquid ; Citrus/chemistry* ; Color ; Benzoxepins/chemistry* ; Anti-Inflammatory Agents/chemistry*

This paper aimed to explore the effects of Duzhong Decoction(DZD)-containing serum on the proliferation and osteoblast differentiation of MC3T3-E1 cells through L-type voltage-gated calcium channels(L-VGCCs). L-VGCCs inhibitors, nifedipine and verapamil, were used to block L-VGCCs in osteoblasts. MC3T3-E1 cells were divided into a control group, a low-dose DZD-containing serum(L-DZD) group, a medium-dose DZD-containing serum(M-DZD) group, a high-dose DZD-containing serum(H-DZD) group, a nifedipine group, a H-DZD + nifedipine group, verapamil group, and a H-DZD + verapamil group. The CCK-8 method was used for cell proliferation analysis, alkaline phosphatase(ALP) assay kits for intracellular ALP activity measurement, Western blot for protein expression level in cells, real-time fluorescence quantitative PCR technology for intracellular mRNA expression level determination, fluorescence spectrophotometer for free Ca~(2+) concentration determination in osteoblasts, and alizarin red staining(ARS) for mineralized nodule formation in osteoblasts. The experimental results show that compared to the control group, DZD groups can promote MC3T3-E1 cell proliferation, ALP activity, and mineralized nodule formation, increase intracellular Ca~(2+) concentrations, and upregulate the protein expression of bone morphogenetic protein 2(BMP2), collagen Ⅰ(COL1), α2 subunit protein of L-VGCCs(L-VGCCα2), and the mRNA expression of Runt-related transcription factor 2(RUNX2), and BMP2. After blocking L-VGCCs with nifedipine and verapamil, the intervention effects of DZD-containing serum were inhibited to varying degrees. Both nifedipine and verapamil could inhibit ALP activity, reduce mineralized nodule areas, and downregulate the expression of bone formation-related proteins. Moreover, the effects of DZD-containing serum on increasing MC3T3-E1 cell proliferation, osteoblast differentiation, and Ca~(2+) concentrations, upregulating the mRNA expression of osteoprotegerin(OPG) and protein expression of phosphorylated protein kinase B(p-Akt) and phosphorylated forkhead box protein O1(p-FOXO1), and upregulating phosphatase and tensin homolog(PTEN) expression were reversed by nifedipine. The results indicate that DZD-containing serum can increase the Ca~(2+) concentration in MC3T3-E1 cells to promote bone formation, which may be mediated by L-VGCCs and the PTEN/Akt/FoxO1 signaling pathway, providing a new perspective on the mechanism of DZD in treating osteoporosis.
Animals ; Osteoblasts/metabolism* ; Cell Proliferation/drug effects* ; Cell Differentiation/drug effects* ; Mice ; Drugs, Chinese Herbal/pharmacology* ; Calcium Channels, L-Type/genetics* ; Alkaline Phosphatase/genetics* ; Serum/chemistry* ; Cell Line ; Osteogenesis/drug effects* ; Bone Morphogenetic Protein 2/genetics*

To investigate the effects of phosphorus fertilizer on the morphological traits, active ingredients and rhizosphere soil microbial community of Polygala tenuifolia. The phosphorus fertilizer was calculated in terms of P_2O_5. Five treatments were set up: 0(CK), 17(P1), 34(P2), 51(P3), and 68(P4) kg per Mu(1 Mu≈667 m~2). A randomized block design was adopted. Samples of P. tenuifolia and its rhizosphere soil were collected under different superphosphate fertilizer treatments. Illumina high-throughput sequencing was used to analyze the rhizosphere soil microbial community, 9 morphological traits were measured and the content of 11 active ingredients were determined. The results showed that the whole plant weight, shoot fresh weight, root weight, and root peel thickness were the highest under P1 treatment, increasing by 34.41%, 38.80%, 39.21%, and 3.17% respectively compared to CK. Under P2 treatment, the plant height, stem diameter, root thickness, and core thickness were significantly higher than CK. Phosphorus fertilizer had a significant impact on the content of tenuifolin, sibiricose A5, sibiricose A6, arillanin A, 3,6'-disinapoyl sucrose, and polygalaxanthone Ⅲ. Correlation analysis results showed that the relative abundance of Arthrobacter, Bacillus, norank＿f＿Vicinamibacteraceae, norank＿o＿Vicinamibacterales, MND1 and other bacteria, as well as the relative abundance of Neocosmospora, Paraphoma and other fungi were positively correlated with root diameter, wood core diameter, the whole plant weight, root weight, shoot fresh weight of P. tenuifolia. Bacillus, Neocosmospora, Subulicystidium were significantly positively correlated with oligosaccharides such as 3,6'-disinapoyl sucrose, sibiricose A5、sibiricose A6、glomeratose A、arillanin A and tenuifoliside C. Arthrobacter, Humicola, Aspergillus, Paraphoma were positively correlated with tenuifolin and norank＿f＿Vicinamibacteraceae, norank＿o＿Vicinamibacterales, Fusarium were positively correlated with polygalaxanthone Ⅲ. Evidently, appropriate phosphorus application is conducive to the growth and quality improvement of P. tenuifolia, and can increase the abundance of beneficial microorganisms in the soil.
Rhizosphere ; Phosphorus/pharmacology* ; Soil Microbiology ; Polygala/anatomy & histology* ; Fertilizers/analysis* ; Bacteria/metabolism* ; Soil/chemistry* ; Microbiota/drug effects* ; Plant Roots/metabolism*

The chemical components of Carthami Flos were investigated by using macroporous resin, silica gel column chromatography, reversed-phase octadecylsilane(ODS) column chromatography, Sephadex LH-20, and semi-preparative high-performance liquid chromatography(HPLC). The planar structures of the compounds were established based on their physicochemical properties and ultraviolet-visible(UV-Vis), infrared(IR), high-resolution electrospray ionization mass spectrometry(HR-ESI-MS), and nuclear magnetic resonance(NMR) spectroscopic technology. The absolute configurations were determined by comparing the calculated and experimental electronic circular dichroism(ECD). Six flavonoid C-glycosides were isolated from the 30% ethanol elution fraction of macroporous resin obtained from the 95% ethanol extract of Carthami Flos, and identified as saffloquinoside F(1), 5-hydroxysaffloneoside(2), iso-5-hydroxysaffloneoside(3), isosafflomin C(4), safflomin C(5), and vicenin 2(6). Among these, the compounds 1 to 3 were new chalcone C-glycosides. The compounds 1, 2, 4, and 5 could significantly increase the viability of H9c2 cardiomyocytes damaged by oxygen-glucose deprivation/reoxygenation(OGD/R) at a concentration of 50 μmol·L~(-1), showing their good cardioprotective activity.
Glycosides/pharmacology* ; Flowers/chemistry* ; Drugs, Chinese Herbal/pharmacology* ; Carthamus tinctorius/chemistry* ; Chalcones/pharmacology* ; Animals