The functional realization of proteins and other biological macromolecules depends on conformational dynamics and allosteric regulation, and elucidating their molecular mechanisms is an important foundation for understanding life processes. Molecular dynamics simulations are a powerful tool for investigating conformational evolution at the atomic level. However, traditional methods are limited by simulation timescales and high free-energy barriers, making it difficult to effectively capture rare conformations and their transition pathways. As a result, the development of enhanced sampling techniques has become key to overcoming this bottleneck. As a classical enhanced sampling technique, metadynamics suffers from several shortcomings, including strong dependence on collective variables and significant errors caused by bias potential accumulation. This article reviews three major improvement strategies. The first combines stochastic resetting with metadynamics, using trajectory-resetting mechanisms to improve sampling efficiency while avoiding the difficulty of optimizing collective variables. The second, SinkMeta, employs a “sinking” bias effect to enable efficient exploration of specific regions and paths. The third, OPES-based hybrid methods, improve the stability of free-energy estimation by optimizing the target distribution or the way the bias is constructed. These methods provide new ideas for characterizing free-energy landscapes and studying conformational transitions in complex biological systems, while also promoting the continued development of enhanced sampling methodologies.

An assay was established for detection of toxigenic Clostridioides difficile by combining microfluidic chip analysis with immunochromatography,and its performance was evaluated and compared with those of the Xpert C.difficile/Epi and VIDAS CD AB tests.Primer pairs were designed according to the tcdB and tpi genes in C.difficile.The specificity,limit of detection,reproducibility,and stability were evaluated.A total of 215 stool samples from patients with diarrhea were collected and tested in parallel with the Xpert C.difficile/Epi,VIDAS CDAB,and our assay.C.difficile was isolated from samples,and the tcdB gene was identified when discrepant results were obtained from the three above assays.Our assay showed no cross-reaction with other diarrhea-associated pathogens.Its reproducibility was 100％in testing of two standard plasmids containing tcdB and tpi genes at two concentrations(105 and 102 copies/μL).Two standard plasmids were detected after the PCR and immunochromatography reagents had been stored for 3,6,9,and 12 months,and all the results were posi-tive.The limit of detection was 10 copies/μL for toxigenic C.difficile.Testing of 33 samples positive for C.difficile with our assay(33/215,15.3％)yielded findings statistically coherent with those of the Xpert C.difficile/Epi test(kappa value=0.965).The sensitivity,specificity,positive predictive value,and negative predictive value of our assay,with respect to Xpert C.difficile/Epi as the standard,were 94.3％,100.0％,100.0％,and 98.9％;these values were significantly higher than those of VIDAS CDAB(60.0％,98.9％,91.3％,and 92.7％)(Kappa=0.714,OR=157.50,95％CI:62.03-847.28,P=0.013).In conclusion,our newly developed assay is specific,stable,and reproducible,and may be used for rapid and accu-rate detection of toxigenic C.difficile.The assay could be used for C.difficile infection screening in outpatient and emergen-cy,community medical service center,and epidemiological settings.

Objective To analyze the risk factors for increased drainage volume after open transforaminal lumbar interbody fusion(TLIF),and to establish a predictive model and then validate it.Methods The clinical data of 680 patients who underwent open TLIF at the General Hospital of Northern Theater Command from January 2016 to December 2019 were collected and the patients were randomly divided into the training group(n=476)and the validation group(n=204).Taking the predictive factors screened out by LASSO regression analysis as independent variables,a multivariate Logistic regression predictive model was constructed.The model was internally validated through the receiver operating characteristic(ROC)curve,Hosmer-Lemeshow goodness-of-fit test,and calibration curve,and its clinical utility was assessed via decision curve analysis(DCA).Results LASSO regression analysis screened out four predictive variables:age,number of surgical segments,operative duration,and intraoperative blood loss.The multivariate Logistic regression predictive model demonstrated that age≥60 years,number of surgical segments≥4,operative duration≥2 hours,and intraoperative blood loss≥200 mL were independent influencing factors for the increased postoperative drainage volume in patients undergoing TLIF(P<0.05).ROC curve analysis revealed an area under the curve(AUC)of 0.816(95%CI:0.798 to 0.867)in the training group and 0.783(95%CI:0.685 to 0.823)in the validation group,indicating that the predictive model had good discriminatory ability.Additionally,the Hosmer-Lemeshow goodness-of-fit test and calibration curve indicated that the predictive model had a good degree of fit,and the predicted probability was basically consistent with the actual probability,demonstrating a good calibration.The DCA results confirmed that this predictive model could be applied in clinical practice.Conclusion The risk factors for increased drainage volume after open TLIF include age,number of surgical segments,operative duration,and intraoperative blood loss.The predictive model established based on these factors demonstrates good performance,and it can be applied in clinical guidance for the selection of drainage tube removal time after TLIF.

Aim To investigate the role of triggering receptor expressed on myeloidcells 2(TREM2)in syn-aptic plasticity induced by cocaine addiction.Methods C57BL/6J mice and Trem2 knockout mice were uti-lized in this study to evaluate the alterations in postsyn-aptic density protein 95(PSD-95)and synapsin 1(SYN1)within the cortex and hippocampus of co-caine-addicted mice by using immunological tech-niques.Results HE staining and Nissl staining showed increased neuronal damage in the hippocampus and cortex of mice after cocaine addiction.The results of immunohistochemistry and fluorescence of PSD-95 and SYN1 were consistent with the expression trend of Western blot.In the wild type mouse model,the ex-pression level of PSD-95 in the hippocampus and cortex was lower than that in the saline group,and the ex-pression of SYN1 was higher than that in the saline group.In the knockout mouse model,the expression levels of PSD-95 and SYN1 in the hippocampus and cortex were significantly higher than those in the saline group after cocaine addiction.The expression levels of PSD-95 and SYN1 in the hippocampus and cortex of cocaine knockout mice were higher than those of co-caine wild type mice.Conclusion Cocaine addiction can change the synaptic plasticity,and TREM2 plays a regulatory role in the synaptic plasticity of hippocampus and cortex in mice with cocaine injury.TREM2 is ex-pected to be a new target for studying the mechanism of cocaine addiction.

Objective To explore the predictive efficacy of several multimodal MRI-based machine learning models for the promoter methylation states of O6-methylguanine-DNA methyltransferase(MGMT)of glioblastoma muliforme(GBM)patients in terms of the GBM heterogeneity and the complexity of the tumor microenvironment.Methods Firstly,the multimodal MRI images of 317 GBM patients from The University of Pennsylvania Glioblastoma(UPENN-GBM)dataset were pre-processed,with four sequences involved in including T1-weighted imaging(T1WI)sequence,T1-weighted contrast-enhanced imaging(T1CE)sequence,T2-weighted imaging(T2WI)sequence and fluid-attenuated inversion recovery(FLAIR)sequence,and the radiomics features were extracted for two regions of interest(ROIs)such as the tumor core region and the tumor edema region.Secondly,the data of the 317 GBM patients were randomly divided into a training set(254 cases)and a test set(63 cases),which underwent normalization with Z-scores and feature selection and dimensionality reduction with Lasso regression.Finally,three models were established respectively with particle swarm optimization-support vector machine(PSO-SVM),C-support vector classification(C-SVC)and adaptive boosting(adaptive boosting(Adaboost)algorithms,and the predictive efficacy of the three models for glioblastoma multiforme MGMT promoter methylation states were evaluated in terms of accuracy and AUC.Results The Adaboost model based on T2WI sequence and radiomics features of the tumor core region had the highest predictive efficacy with accuracy and AUC values of 67％and 0.74,respectively,higher than those of other combinations of sequences,models and regions of interest.Conclusion The multimodal MRI-based machine learning models can be used for the prediction of glioblastoma multiforme MGMT promoter methylation states,which provides powerful support for personalized treatment and prognostic assessment of GBM.[Chinese Medical Equipment Journal,2025,46(6):7-13]

Objective To analyze the risk factors for increased drainage volume after open transforaminal lumbar interbody fusion(TLIF),and to establish a predictive model and then validate it.Methods The clinical data of 680 patients who underwent open TLIF at the General Hospital of Northern Theater Command from January 2016 to December 2019 were collected and the patients were randomly divided into the training group(n=476)and the validation group(n=204).Taking the predictive factors screened out by LASSO regression analysis as independent variables,a multivariate Logistic regression predictive model was constructed.The model was internally validated through the receiver operating characteristic(ROC)curve,Hosmer-Lemeshow goodness-of-fit test,and calibration curve,and its clinical utility was assessed via decision curve analysis(DCA).Results LASSO regression analysis screened out four predictive variables:age,number of surgical segments,operative duration,and intraoperative blood loss.The multivariate Logistic regression predictive model demonstrated that age≥60 years,number of surgical segments≥4,operative duration≥2 hours,and intraoperative blood loss≥200 mL were independent influencing factors for the increased postoperative drainage volume in patients undergoing TLIF(P<0.05).ROC curve analysis revealed an area under the curve(AUC)of 0.816(95%CI:0.798 to 0.867)in the training group and 0.783(95%CI:0.685 to 0.823)in the validation group,indicating that the predictive model had good discriminatory ability.Additionally,the Hosmer-Lemeshow goodness-of-fit test and calibration curve indicated that the predictive model had a good degree of fit,and the predicted probability was basically consistent with the actual probability,demonstrating a good calibration.The DCA results confirmed that this predictive model could be applied in clinical practice.Conclusion The risk factors for increased drainage volume after open TLIF include age,number of surgical segments,operative duration,and intraoperative blood loss.The predictive model established based on these factors demonstrates good performance,and it can be applied in clinical guidance for the selection of drainage tube removal time after TLIF.

Polygonati Rhizoma demonstrates significant potential for addressing both chronic and hidden hunger. The supply of high-quality seedlings is a primary factor influencing the development of the Polygonati Rhizoma industry. Warm water soaking is often used in agriculture to promote the rapid germination of seeds, while its application and molecular mechanism in Polygonati Rhizoma have not been reported. To rapidly obtain high-quality seedlings, this study treated Polygonatum kingianum var. grandifolium seeds with sand storage at low temperatures, warm water soaking, and cultivation temperature gradients. The results showed that the culture at 25 ℃ or sand storage at 4 ℃ for 2 months rapidly broke the seed dormancy of P. kingianum var. grandifolium, while the culture at 20 ℃ or sand storage at 4 ℃ for 1 month failed to break the seed dormancy. Soaking seeds in 60 ℃ warm water further increased the germination rate, germination potential, and germination index. Specifically, the seeds soaked at 60 ℃ and cultured at 25 ℃ without sand storage treatment(Aa25) achieved a germination rate of 78. 67%±1. 53% on day 42 and 83. 40%±4. 63% on day 77. The seeds pretreated with sand storage at 4 ℃ for 2 months, soaked in 60 ℃ water, and then cultured at 25 ℃ achieved a germination rate comparable to that of Aa25 on day 77. Transcriptomic analysis indicated that warm water soaking might promote germination by triggering reactive oxygen species( ROS), inducing the expression of heat shock factors( HSFs) and heat shock proteins( HSPs), which accelerated DNA replication, transcript maturation, translation, and processing, thereby facilitating the accumulation and turnover of genetic materials. According to the results of indoor controlled experiments and field practices, maintaining a germination and seedling cultivation environment at approximately 25 ℃ was crucial for the one-year seedling cultivation of P. kingianum var. grandifolium.
Germination ; Seedlings/genetics* ; Water/metabolism* ; Seeds/metabolism* ; Polygonatum/genetics* ; Temperature ; Plant Proteins/genetics* ; Plant Dormancy

The chemical constituents of the petroleum ether extract derived from the 90% ethanol extract of Elephantopus scaber were investigated. By silica gel column chromatography, C_(18), MCI column chromatography and semi-preparative high performance liquid chromatography, ten compounds were isolated. Their structures were identified as 3β-hydroxy-6β,7β-epoxytaraxeran-14-ene(1), 3β-hydroxyolean-12-en-28-oic acid(2), D-friedoolean-14-ene-3β,7α-diol(3), 3β-hydroxy-11α-methoxyolean-12-ene(4), 3β-hydroxyolean-11,13(18)-diene(5), 11α-hydroxy-β-amyrin(6), betulinic acid(7), 3β-hydroxy-30-norlupan-20-one(8), 6-acetonylchelerythrine(9), and 4',5'-dehydrodiodictyonema A(10) by analysis of the 1D NMR, 2D NMR, MS, and IR spectral data. Among them, compound 1 was a new triterpene and other compounds except compounds 2 and 7 were isolated from this plant for the first time.
Triterpenes/isolation & purification* ; Drugs, Chinese Herbal/isolation & purification* ; Molecular Structure ; Asteraceae/chemistry* ; Chromatography, High Pressure Liquid ; Magnetic Resonance Spectroscopy

This study aims to explore the scientific connotation of sinking Rehmanniae Radix has the best quality and compare the quality between floating and sinking fresh Rehmanniae Radix samples. Ultra-performance liquid chromatography tandem quadrupole electrostatic field Orbitrap high-resolution mass spectrometry(UHPLC-Q-Orbitrap HRMS) was employed to detect the chemical components in floating and sinking fresh Rehmanniae Radix samples. The fingerprint of fresh Rehmanniae Radix was established by high performance liquid chromatography(HPLC), and four index components were determined simultaneously. The cluster analysis, principal component analysis(PCA), and orthogonal partial least squares-discriminant analysis(OPLS-DA) were conducted to compare the quality of floating and sinking fresh Rehmanniae Radix samples. An evaporative light-scattering detector was used to compare the content of five sugars. The extract yield and drying rate were determined, and the quality connotation of sinking Rehmanniae Radix has the best quality was explained by multiple indicators. A total of 41 components were preliminarily identified from fresh Rehmanniae Radix by UHPLC-Q-Orbitrap HRMS, including 7 iridoid glycosides, 9 phenylethanol glycosides, 6 amino acids, 4 sugars, 3 phenolic acids, 5 nucleosides, 3 organic acids, 1 ionone, 1 furan, 1 coumarin, and 1 phenylpropanoid. The results showed that the main chemical components were consistent between floating and sinking fresh Rehmanniae Radix. Nine common peaks were identified in the fingerprints of 15 batches of floating and sinking fresh Rehmanniae Radix samples, and the similarity of fingerprints was greater than 0.9. The cluster analysis, PCA, and OPLS-DA classified floating and sinking fresh Rehmanniae Radix sasmples into two categories, indicating differences in the quality between them. The total content of catalpol, rehmannioside D, ajugol, and verbascoside in sinking fresh Rehmanniae Radix samples was higher than that in floating samples of the same batch and specification, and the main differential component was catalpol. The total content of fructose, glucose, sucrose, raffinose, and stachyose in sinking fresh Rehmanniae Radix samples was higher than that in floating samples of the same batch and specification, and the main differential component was stachyose. The extract yield and drying rate of the sinking samples were higher than those of floating samples. This study preliminarily showed that floating and sinking fresh Rehmanniae Radix samples had the same components but great differences in the content of medicinal substance basis. The total content of four glycosides and five sugars, extract yield, and drying rate of sinking fresh Rehmanniae Radix samples is higher than that of floating samples of the same batch and specification. These findings, to a certain extent, explains the scientificity of sinking Rehmanniae Radix has the best quality recorded in ancient books and provide a reference for the quality control and clinical application of fresh Rehmanniae Radix.
Chromatography, High Pressure Liquid/methods* ; Drugs, Chinese Herbal/chemistry* ; Rehmannia/chemistry* ; Chemometrics ; Mass Spectrometry/methods* ; Quality Control ; Principal Component Analysis ; Plant Extracts

This study aims to investigate the effect of Chaijin Jieyu Anshen Formula on the neuroinflammation of rats with insomnia complicated with depression through the regulation of triggering receptor expressed on myeloid cells 2(TREM2)/complement protein C1q signaling pathway. Rats were randomly divided into a normal group, a model group, a positive drug group, as well as a high, medium, and low-dose groups of Chaijin Jieyu Anshen Formula, with 10 rats in each group. Except for the normal group, the other groups were injected with p-chlorophenylalanine and exposed to chronic unpredictable mild stress to establish the rat model of insomnia complicated with depression. The sucrose preference experiment, open field experiment, and water maze test were performed to evaluate the depression in rats. Enzyme-linked immunosorbent assay was employed to detect serum 5-hydroxytryptamine(5-HT), dopamine(DA), and norepinephrine(NE) levels. Hematoxylin and eosin staining and Nissl staining were used to observe the damage in nucleus accumbens neurons. Western blot and immunofluorescence were performed to detect TREM2, C1q, postsynaptic density 95(PSD-95), and synaptophysin 1(SYN1) expressions in rat nucleus accumbens, respectively. Golgi-Cox staining was utilized to observe the synaptic spine density of nucleus accumbens neurons. The results show that, compared with the model group, Chaijin Jieyu Anshen Formula can significantly increase the sucrose preference as well as the distance and number of voluntary activities, shorten the immobility time in forced swimming test and the successful incubation period of positioning navigation, and prolong the stay time of space exploration in the target quadrant test. The serum 5-HT, DA, and NE contents in the model group are significantly lower than those in the normal group, with the above contents significantly increased after the intervention of Chaijin Jieyu Anshen Formula. In addition, Chaijin Jieyu Anshen Formula can alleviate pathological damages such as swelling and loose arrangement of tissue cells in the nucleus accumbens, while increasing the Nissl body numbers. Chaijin Jieyu Anshen Formula can improve synaptic damage in the nucleus accumbens and increase the synaptic spine density. Compared to the normal group, the expression of C1q protein was significantly higher in the model group, while the expression of TREM2 protein was significantly lower. Compared to the model group, the intervention with Chaijin Jieyu Anshen Formula significantly downregulated the expression of C1q protein and significantly upregulated the expression of TREM2. Compared with the model group, the PSD-95 and SYN1 fluorescence intensity is significantly increased in the groups receiving different doses of Chaijin Jieyu Anshen Formula. In summary, Chaijin Jieyu Anshen Formula can reduce the C1q protein expression, relieve the TREM2 inhibition, and promote the synapse-related proteins PSD-95 and SNY1 expression. Chaijin Jieyu Anshen Formula improves synaptic injury of the nucleus accumbens neurons, thereby treating insomnia complicated with depression.
Animals ; Male ; Rats ; Nucleus Accumbens/metabolism* ; Drugs, Chinese Herbal/administration & dosage* ; Depression/complications* ; Membrane Glycoproteins/genetics* ; Rats, Sprague-Dawley ; Sleep Initiation and Maintenance Disorders/complications* ; Neurons/metabolism* ; Receptors, Immunologic/genetics* ; Signal Transduction/drug effects* ; Synapses/metabolism*