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.

In modern society， the comorbidity of insomnia with amnesia has shown a significantly increasing incidence and become increasingly severe， emerging as a public health issue that endangers people's physical and mental health and quality of life. Anmei Dan （AMD）， originally recorded in the Shishi Milu by CHEN Shiduo in the Qing Dynasty， possesses profound theoretical foundations and distinct clinical advantages in ameliorating insomnia with amnesia. Basic research has unequivocally confirmed its definite therapeutic effects on insomnia with amnesia and revealed partial mechanisms. AMD is therefore a veritable classic prescription embodying the principle of treating different diseases with the same method for insomnia and amnesia. However， few researchers have reviewed the related research progress. This article systematically summarized the theoretical connotations of traditional Chinese medicine in treating insomnia with amnesia， delineated the multi-component material basis of AMD in ameliorating insomnia with amnesia （including various chemical components such as ginsenosides， jujubosides， and salvianolic acids）， and evaluate its multi-dimensional clinical efficacy （e.g.， prolonging total sleep time， improving sleep efficiency， and enhancing memory）. Moreover， it systematically summarizes the multi-pathway mechanisms of action. The results indicate that AMD has a clear material basis， definite clinical efficacy， and a favorable safety profile in ameliorating insomnia and amnesia. AMD may exert the therapeutic effect by regulating sleep-wake rhythms， enhancing synaptic plasticity， inhibiting neuroimmune inflammation， and modulating autophagy， oxidative stress， and energy metabolism. In conclusion， AMD exerts synergistic effects through multi-component， multi-dimensional， and multi-pathway approaches， serving as an important prescription for enriching and developing the theory of treating different diseases with the same method for insomnia with amnesia.

Alzheimer’s disease (AD) is a central neurodegenerative disease characterized by progressive cognitive decline and memory impairment in clinical. Currently, there are no effective treatments for AD. In recent years, a variety of therapeutic approaches from different perspectives have been explored to treat AD. Although the drug therapies targeted at the clearance of amyloid β-protein (Aβ) had made a breakthrough in clinical trials, there were associated with adverse events. Neuroinflammation plays a crucial role in the onset and progression of AD. Continuous neuroinflammatory was considered to be the third major pathological feature of AD, which could promote the formation of extracellular amyloid plaques and intracellular neurofibrillary tangles. At the same time, these toxic substances could accelerate the development of neuroinflammation, form a vicious cycle, and exacerbate disease progression. Reducing neuroinflammation could break the feedback loop pattern between neuroinflammation, Aβ plaque deposition and Tau tangles, which might be an effective therapeutic strategy for treating AD. Traditional Chinese herbs such as Polygonum multiflorum and Curcuma were utilized in the treatment of AD due to their ability to mitigate neuroinflammation. Non-steroidal anti-inflammatory drugs such as ibuprofen and indomethacin had been shown to reduce the level of inflammasomes in the body, and taking these drugs was associated with a low incidence of AD. Biosynthetic nanomaterials loaded with oxytocin were demonstrated to have the capability to anti-inflammatory and penetrate the blood-brain barrier effectively, and they played an anti-inflammatory role via sustained-releasing oxytocin in the brain. Transplantation of mesenchymal stem cells could reduce neuroinflammation and inhibit the activation of microglia. The secretion of mesenchymal stem cells could not only improve neuroinflammation, but also exert a multi-target comprehensive therapeutic effect, making it potentially more suitable for the treatment of AD. Enhancing the level of TREM2 in microglial cells using gene editing technologies, or application of TREM2 antibodies such as Ab-T1, hT2AB could improve microglial cell function and reduce the level of neuroinflammation, which might be a potential treatment for AD. Probiotic therapy, fecal flora transplantation, antibiotic therapy, and dietary intervention could reshape the composition of the gut microbiota and alleviate neuroinflammation through the gut-brain axis. However, the drugs of sodium oligomannose remain controversial. Both exercise intervention and electromagnetic intervention had the potential to attenuate neuroinflammation, thereby delaying AD process. This article focuses on the role of drug therapy, gene therapy, stem cell therapy, gut microbiota therapy, exercise intervention, and brain stimulation in improving neuroinflammation in recent years, aiming to provide a novel insight for the treatment of AD by intervening neuroinflammation in the future.

In recent years,significant breakthroughs have been achieved in the immunotherapy for Alzheimer's disease.In line with global advancements,two anti-amyloid-β monoclonal antibodies have been approved and successfully launched in China for clinical use.Lecanemab and Donanemab were officially used in June 2024 and April 2025 in China,respectively.In order to standardize the rational and safe application of anti-amyloid-β monoclonal antibodies for Alzheimer's disease in China,this article integrates recom-mendations from the clinical trials and real-world experience from the author's team and domestic peers to further update the recom-mendations for the clinical use of anti-amyloid-β monoclonal antibody based on the 2024 version.It includes indications for therapy,pre-treatment evaluation and preparation,administration protocols and safety measures during treatment,and post-treatment monitor-ing strategies.

Nitrogen oxides(NOx=NO+NO2)are important precursors of ozone(O3),and NOx and its oxides together constitute reactive nitrogen oxides(NOy)in the atmosphere.A comprehensive understanding of the total NOy level in the atmosphere is of great significance for a deeper understanding of the atmospheric nitrogen cycle and oxidation,as well as for formulating strategies for air pollution prevention and control.In this work,a thermal decomposition-broadband cavity enhanced absorption spectroscopy(TD-BBCEAS)technique for online measurement of total NOy in the atmosphere was developed.With this method,the NOy was efficiently converted into NO2,and the total NOy concentration in the atmosphere was indirectly obtained by measuring NO2.Focusing on the key factors affecting the measurement of total NOy,the influence of NO titration efficiency and other NOy component TD efficiency on measurement accuracy was emphasized.By changing the oxygen(O2)flow rate through the mercury lamp to alter the O3 concentration for titrating NO,the conversion efficiency of NO was evaluated.At O2 flow rate of 6 mL/min,the conversion efficiency of NO was greater than 99%.TD efficiency testing and analysis on NO2,peroxyacetyl nitrate(PAN),nitric acid(HNO3),and nitrous acid(HONO),which account for a large proportion of atmospheric NOy components,was carried out using 680℃as the optimal TD temperature for efficient conversion of NOy.With NO and HONO sample gases as typical verification gases,the conversion efficiency of NOy and the accuracy of NOy measurement by TD-BBCEAS system were verified by switching the on and off modes of mercury lamp and TD device.At integration time of 60 s,the detection limit of the system for NOy was 2.83×1010 molecules/cm3(60 s,2σ).A comparative measurement of actual atmospheric NOy was conducted between the TD-BBCEAS system and the NOy analyzer.The observation results showed a correlation coefficient(R2)of 0.98 and a slope of 0.93,further verifying the feasibility and accuracy of applying the TD-BBCEAS system to measurement of total NOy.

Objective To construct an aptamer-functionalized carboxylated graphene oxide(CGO)fluo-rescent sensor to achieve highly sensitive and specific detection of ketamine(KET)and its metabolite norketamine(NK)using an aptamer capable of simultaneously recognizing KET and NK.Methods A specific aptamer for simultaneous recognition of KET and NK was screened using graphene oxide-sys-tematic evolution of ligand by exponential enrichment(GO-SELEX)and molecular docking tech-niques.The aptamer,labeled with Cy5 fluorescence,was chemically conjugated to CGO to construct an aptamer-functionalized CGO fluorescent sensor.By optimizing detection conditions,including the mass concentration of CGO,aptamer concentration,reaction temperature,and incubation time,quantita-tive analysis of the target analytes was achieved using the ratio of fluorescence intensity changes be-fore and after target addition.The stability of the sensor in biological matrices was evaluated by moni-toring fluorescence intensity changes over incubation time in blank blood and urine,in comparison with the traditional physical adsorption-based CGO fluorescent sensor.Spiked recovery experiments in blank blood and urine were conducted to compare performance with that of HPLC-MS/MS.Results A specific aptamer A5 was selected and chemically conjugated with CGO to construct the aptamer-functionalized CGO fluorescent sensor.Under optimized conditions,the proposed fluorescent sensor ex-hibited a linear detection range of 1.0-5.0 ng/mL for KET,with a limit of detection(LOD)of 0.86 ng/mL;while for NK,the linear detection range was 1.0-5.0 ng/mL,with an LOD of 0.70 ng/mL.Com-pared with the CGO fluorescent sensor constructed via physical adsorption,this sensor demonstrated greater stability in blood and urine.The spiked recovery rates of KET and NK in blank blood and urine ranged from 81.50%to 110.03%,exhibiting detection performance comparable to that of HPLC-MS/MS.Conclusion The aptamer screening method offers a novel approach for selecting aptamers tar-geting drugs and their metabolites.The constructed aptamer-functionalized CGO fluorescent sensor pro-vides an efficient and reliable strategy for the high-performance detection of KET and NK.

Objective To observe the effect of a heavy load exercise on the ultrastructure,function and fission of skeletal muscle mitochondria in rats,and to analyze the changes of the phosphorylation expression of mitochondrial fission protein and upstream kinase at different times postexercise,and to explore the effect of acute heavy load exercise on mitochondrial fission in skeletal muscle of rats and its possible mechanism.Methods Forty-eight adult male Sprague-Dawley rats were randomly divided in-to a quiet control group(C,n=8)and an exercise group(E,n=40).Rats in the E group exercised on a treadmill down a 16° decline at 16 m/min for 90 min and were further divided into 0 h,12 h,24 h,48 h,and 72 h postexercise subgroups.Soleus muscle was isolated and mitochondria were ex-tracted at the corresponding time points after exercise.The ultrastructure of mitochondria in the soleus muscle was observed using transmission electron microscopy,and mitochondrial quantity and morphomet-ric analysis were conducted.Moreover,the colocalization and quantity of dynamin-related protein 1(Drp1)and cytochrome C oxidease subunit Ⅳ(COXⅣ)in the soleus muscle were detected using im-munofluorescence double-labeling techniques.Meanwhile,protein levels of soleus musclep-Drp1Ser616,p-Drp1Ser637,p-extracellular regulatory protein kinaseThr202/Tyr204(p-ERKThr202/Tyr204),p-protein kinaseAThr197(p-PKAThr197),and mitochondrial NADH of ubiquinone oxidoreductase subunit B8(NDUFB8)and ubiqui-nol-cytochrome C reductase core protein 2(UQCRC2)were determined by using Western blotting.An-other twenty-four rats were randomly divided into a DMSO group(CD),a U0126 group(CU),an Ex-ercise+DMSO group(ED),and an Exercise+U0126 group(EU).Six mice in each group were giv-en a single intra-bitoneal injection of DMSO or ERK inhibitor U0126 20 min before acute downhill running.Then,their phosphorylated expressions of ERKThr202/Tyr204 and Drp1Ser616 in soleus muscle were de-tected by Western blotting.Results(1)From 0 h to 48 h after exercise,the soleus muscle mitochon-dria showed swelling,rounding,and uneven distribution of mitochondria,among which the degree of mitochondrial damage was the most serious at 12 h and 24 h after exercise.Moreover,the protein ex-pression of NDUFB8 and UQCRC2 in the mitochondria fractions from soleus muscle was significantly lower at 12 h post-exercise(P<0.05).(2)The co-localization of Drp1 and COXⅣ in the skeletal muscle increased significantly at 12 h to 24 h after a heavy load exercise compared with group C and group E0(P<0.01).Moreover,the mitochondrial area,circumference,aspect ratio and Ferret diameter in the skeletal muscle were significantly lower at 12 h to 24 h postexercise(P<0.05).Meanwhile,the number of mitochondria was significantly higher at 24 h after exercise(P<0.01).(3)The phosphoryla-tion of ERKThr202/Tyr204,PKAThr197 and Drp1Ser616 was significantly higher at 24 h after exercise(P<0.01),while that of Drp1Ser637 was significantly lower at 48 h and 72 h post-exercise(P<0.01).However,the phosphorylated expressions of ERKThr202/Tyr204 and Drp1Ser616 were significantly down-regulated by U0126 treatment before exercise.Conclusion A session of heavy load exercise caused mitochondrial structure and function damage and induced mitochondrial fission in the skeletal muscle,and then to maintain the homeostasis of skeletal muscle cells by cleaving damaged mitochondria.The mechanism of promot-ing skeletal muscle repair may be related to the positive and negative regulation of Drp1 activity by the phosphorylation of Drp1Ser616 and Drp1Ser637,respectively.Among them,the activation of ERKThr202/Tyr204 mediates the phosphorylation activation of Drp1Ser616,but PKAThr197 is not an upstream kinase that medi-ates the inactivation of Drp1Ser637 phosphorylation.

Objective To investigate the clinical characteristics of severe pertussis in children.Methods Clinical data of 55 children with severe pertussis and admitted to the pediatric intensive care unit(PICU)of Maternal and Child Heath Hospital of Hubei Province from January 2022 to May 2024 were retrospectively analyzed.Patients were grouped according to age(≤3-month group and＞3-month group),vaccination status,and mixed infection status,differences in clinical characteristics among different groups of pediatric patients were compared.Results 55 children with severe pertussis were aged 1 month and 5 days-9 years,all had severe pneumonia,54 children were improved and discharged from hospital,1 died.26 children(47.3％)were aged＜3 months,45 children(81.8％)were unvaccinated,and 47 children(85.5％)had mixed infection.The rates of post-vomiting cough and blood ex-change transfusion in children in ≤3-month group were lower those in＞3-month group(30.8％vs 58.6％;11.5％vs 34.5％,respectively);The rate of elevation of cerebrospinal fluid protein in children in ≤3-month group was higher than that in＞3-month group(61.5％vs 31.0％);The pre-admission disease course of children in ≤3-month group was shorter than that in＞3-month group([10.15±5.64]days vs[14.24±8.90]days),differences were all statistically significant(all P≤0.05).The absolute counts of lymphocyte in the vaccinated group was lower than that in the unvaccinated group([9.92±5.92]× 109/L vs[17.93±11.41]× 109/L,P＜0.05).The usage rate of gamma-globulin in children in the mixed infection group was higher than that in the simple infection group(87.2％vs 50.0％).The length of hospital stay([15.11±6.53]days vs[9.50±4.69]days),length of PICU stay([10.53±5.26]days vs[5.88±4.16]days),and macrolides antibiotic use days([8.36±4.21]days vs[5.00±2.73]days)in children in the mixed infection group were all longer than those in the simple infection group,differences were all statistically significant(all P＜0.05).No independent influencing factors were found to prolong the length of PICU stay in children with severe pertussis.Conclusion Severe pertussis mostly occurs in unvaccinated children aged ≤3 months,with a high incidence of mixed infection,all presenting as severe pneumonia.Vaccination status,mixed infection,and complications are key factors affecting prognosis.

Objective To evaluate the clinical characteristics of human immunodeficiency virus (HIV) infected patients with peripheral lung masses (PLMs), and to assess the diagnostic utility of contrast-enhanced ultrasound (CEUS) in differentiating benign and malignant PLMs. Methods A retrospective analysis was performed on the clinical data of 69 patients with PLM treated in Shanghai Public Health Clinical Center from January 2020 to December 2023. All patients underwent percutaneous biopsy, and were categorized into benign group (n＝36) and malignant group (n＝33). 25 patients were HIV-positive and 44 patients were HIV-negative. The clinical features and CEUS parameters in patients were compared across these groups. Results Patients with malignant masses were significantly older than those with benign masses (P＜0.05). In the malignant group, HIV-negative patients exhibited significantly larger tumor diameters compared to HIV-positive patients (P＜0.05); in the HIV-positive patients, no significant difference in tumor size was observed between benign and malignant masses. 19 patients underwent CEUS. 10 malignant masses, irrespective of HIV status (10 positive and 9 negative), commonly presented with indistinct margins, delayed enhancement, heterogeneous perfusion, and delayed peak enhancement on CEUS. 9 benign masses showed earlier peak enhancement compared to 10 malignant masses (P＜0.05); no significant differences were observed in the initiation and washout time of enhancement between benign and malignant masses. In HIV-positive patients, 5 benign masses frequently demonstrated discrepancies between CEUS findings and pathological results. Conclusions The clinical and CEUS characteristics were different between benign and malignant PLMs. However, CEUS shows limited accuracy in distinguishing benign and malignant PLMs, underscoring the need for pathological confirmation.

Aim To explore the mechanism of the syn-ergistic effect of the ferroptosis inducer erastin com-bined with shikonin in promoting ferroptosis in human hypertrophic scar fibroblasts(HSFBs).Methods Hypertrophic scar tissues provided by the General Hos-pital of Ningxia Medical University were collected,and HSFBs were extracted.HSFBs were identified by HE staining and immunofluorescence.The inhibitory rates of Era and SHK on HSFBs at different concentrations were detected by CCK-8 assay,and the IC50 value was calculated.CompuSyn software was used to calculate the co-use index(CI).Control group,Erastin(Era)group,shikonin(SHK)group and Era+SHK group were set up,and the number and morphological chan-ges of cells were observed after 24 hours of interven-tion.The ability of cell migration and invasion was de-tected by scratch test and Transwell test.The changes of malondialdehyde(MDA),total iron ion and reactive oxygen species(ROS)were detected by corresponding biochemical kits.The expressions of collagen I,α-SMA and GOT1,SLC7A11,GPX4 and FTH1 were detected by Western blot.Results The IC50 value of Era and SHK of primary HSFBs was 2.22 μmol·L-1 and 3.94μmol·L-1 respectively,which was used as the single drug concentration for subsequent experiments.The CompuSyn software was employed to calculate the CI value when the two drugs were used in combination,and the concentrations corresponding to CI=0.39597(Era:1.2 μmol·L-1+SHK:1.5 μmol·L-1)were selected as subsequent combination concentrations(Because when CI was equal to 0.395 97,the concen-tration of each drug was lower than the concentration of single drug,and the inhibition rate of combined drug was greater than 50％).Compared with the monother-apy group,the number of HSFBs in the SHK+Era group was significantly reduced,cell membrane showed breakage and vesiculation,cell wrinkling became smal-ler,and cytoplasm was concentrated.The migration and invasion ability of HSFBs in the SHK+Era group were obviously weakened(P＜0.05),and the expres-sion of fibrosis-related proteins collagen Ⅰ and α-SMA was reduced(P＜0.05);the contents of MDA,total i-ron ions,and ROS in HSFBs of the SHK+Era group increased(P＜0.05),and the protein expression lev-els of SLC7A11,GOT1,GPX4,and FTH1 further de-creased(P＜0.05).Conclusions Erastin in combi-nation with shikonin can synergistically inhibit the pro-liferation,migration and fibrosis levels of HSFBs.The mechanism may be that erastin enhances the inhibition of shikotin on GOT1,increases the levels of cellular i-ron ions,ROS,and lipid peroxides,thereby promoting ferroptosis in HSFBs.