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.

ObjectiveMigraine is a leading neurological disorder and the fourth most common cause of years lived with disability worldwide, affecting nearly 116 million individuals. Although pharmacological treatments are available, their efficacy is often limited by side effects and variable response rates. Repetitive transcranial magnetic stimulation (rTMS) over the dorsolateral prefrontal cortex (DLPFC) offers a safe, non-invasive alternative for migraine management. However, the neurophysiological mechanisms, particularly how rTMS modulates local cortical excitability and distributed pain-related circuits, remain poorly understood. Elucidating these mechanisms is essential for optimizing treatment protocols and improving clinical outcomes. MethodsThis study employed concurrent transcranial magnetic stimulation and electroencephalography (TMS-EEG) to investigate neuroplastic and neurocircuitry mechanisms of DLPFC-rTMS in migraine. Study 1 compared 30 migraineurs and 28 healthy controls to identify abnormalities in TMS-evoked potentials (TEPs) and significant current density (SCD) within sensory-discriminative regions including the primary somatosensory cortex (S1) and posterior insula (pINS), cognitive-affective regions including the anterior insula (aINS) and midcingulate cortex (MCC), and a descending modulatory region, the periaqueductal gray (PAG). Study 2 used a single-blind, crossover, sham-controlled design in 34 healthy participants. Each participant received both active (10 Hz, 80% RMT, 1 500 pulses) and sham DLPFC-rTMS in counterbalanced order. TMS-EEG and cold pain tolerance were assessed before and after each session. ResultsIn Study 1, migraineurs showed a significantly less negative N120 amplitude compared to healthy controls (P=0.027, Cohen’s d=0.60), indicating local intracortical disinhibition. No group differences were observed for N40, P60, or P180 components. At the source level, migraineurs exhibited significantly higher SCD in the S1, pINS, aINS, and MCC (allQ<0.05), but not in the ventroposterior thalamus (vpTHAL), mediodorsal thalamus (mdTHAL), or PAG. In Study 2, active rTMS significantly reduced SCD from pre- to post-stimulation in the S1, aINS, and MCC (all Q<0.05). Sham stimulation also reduced SCD in the S1 (Q<0.05) but not in the aINS or MCC. Although no significant group-level analgesic effect was observed between active and sham conditions (P=0.107), correlation analyses revealed that greater SCD reductions in the S1 and MCC were significantly associated with higher post-rTMS pain tolerance (R=-0.487 and -0.495, both Q<0.01) and larger improvements in pain tolerance(R=-0.487 and -0.451, both Q<0.05). No such correlations were found following sham stimulation, suggesting that the behavioural relevance of neural changes is specific to active rTMS. ConclusionThis study provides novel evidence that migraineurs exhibit both local neuroplastic abnormalities (reduced N120 amplitude) and hyperactivity in key pain-processing regions (S1, pINS, aINS, MCC). A single session of DLPFC-rTMS reduced hyperactivity in the aINS, MCC, and S1. Notably, greater reductions in the S1 and MCC were associated with improved pain tolerance. These findings identify distinct cortical circuitries, particularly within the cognitive-affective pain network, that may serve as potential biomarkers for optimizing rTMS treatment in migraine and other chronic pain conditions. Future studies should validate these results in patient populations experiencing spontaneous migraine attacks and explore multi-session or accelerated rTMS protocols.

Objective: To retrospectively analyze the supply and demand of RhD-negative red blood cell (RBC) components, explore the dynamic changes in clinical demand and application trends of RhD-negative RBC products, and provide evidence for optimizing the preparation and supply management of various RhD-negative RBCs products. Methods: The total supply of RBCs and the demand and supply of RhD-negative RBC products from 2021 to 2025 were retrieved from the information management system. The trend chi-square test was performed using SPSS 22.0 statistical software to analyze the annual supply rate and repeat reservation rate of RhD-negative RBC products. Meanwhile, the clinical transfusion indications for RhD-negative RBC products in 2025 were further analyzed. Results: From 2021 to 2025, the supply rate of RhD-negative RBC products gradually increased from 0.42% (1 904.5 U/448 787 U) to 0.48% (1945U/402088.5U), showing an upward trend across years (P<0.05). The actual demand and supply of RhD-negative RBC products remained balanced, while the repeat reservation rate increased significantly from 29.73% (804 U/2 704 U) to 43.62% (1 505 U/3 450 U), showing an upward trend year by year (P<0.05). Fresh RhD-negative RBC products accounted for 87.09% (8 270 U/9 495.5 U) of the total supply, while RhD-negative frozen RBC products accounted for 12.91% (1 225.5 U/9 495.5 U). In 2025, therapeutic transfusion, elective surgery (including obstetric transfusion), and emergency transfusion accounted for 55.40% (1 077.5 U/1 945 U), 38.56% (750 U/1 945 U), and 6.04% (117.5 U/1 945 U) of the total supply of RhD-negative RBC products, respectively. RhD-negative frozen RBC products accounted for 67.23% (79 U/117.5 U) of emergency use and 29.47% (221 U/750 U) of elective surgery (including obstetrics) use; fresh RhD-negative RBC suspensions accounted for 93.92% (1 012 U/1 077.5 U) of therapeutic use and 70.56% (529 U/750 U) of elective surgery (including obstetrics) use. In recent years, RhD-negative washed RBC products and concentrated washed RBC products have been introduced as new products, and their clinical utilization has shown an increasing trend. Conclusion: Despite a decline in total blood collection volume, the security management system of RhD-negative RBC components in Guangzhou Blood Center has functioned largely meeting clinical demand. Advances in hospital technology had led to the routine application of RhD-negative concentrated washed RBCs. It is recommended that concentrated washed RBCs should be standardized with a formal definition and corresponding national quality standards should be established.

ObjectiveThis study aimed to investigate the anti-Mycoplasma pneumoniae (MP) activity of luteolin and elucidate its underlying mechanisms. MethodsLuteolin was identified as the primary active compound from the polyphenol extract ofF. diotrys using network pharmacology. Its efficacy was evaluated against two MP strains: the standard strain M129 and the multidrug-resistant strain M19. A modified culture medium with visual characteristics was employed to determine the minimum inhibitory concentration (MIC) of luteolin. The expression of key proteins involved in MP growth and pathogenicity was assessed by qRT-PCR following luteolin treatment. Additionally, the viability of A549 cells infected with MP was compared between luteolin-treated and untreated groups. In vivo anti-MP activity was evaluated using a mouse model, and the expression of inflammatory cytokines in lung tissues was analyzed. ResultsLuteolin effectively inhibited both MP strains, with MIC₉₀ values of 100 mg/L for M19 and M129. Treatment with luteolin significantly downregulated the expression of adhesion proteins P1 and P30 in both strains. However, the expression of P65, HMW3, TrmB, and CARDS TX was reduced only in the M19 strain following luteolin intervention. Luteolin also enhanced the growth and viability of A549 cells infected with MP. In the mouse model, luteolin treatment resulted in steady weight gain and was well tolerated. The bacteriostatic rate of luteolin in lung tissues was 50.7%, significantly higher than the 25.2% observed in the roxithromycin group. Furthermore, luteolin reduced the expression of inflammatory factors, including IL-6, TNF-α, and HMGB1, in MP-infected mice. ConclusionLuteolin effectively and safely inhibits the proliferation and pathogenicity of MP, particularly the drug-resistant M19 strain, by downregulating the expression of toxicity-associated proteins (P1, P30, P65, HMW3, TrmB, CARDS TX) and modulating host inflammatory responses. These findings suggest that luteolin may offer a novel therapeutic strategy for treating MP infections, especially those caused by drug-resistant strains.

ObjectiveThis study aimed to investigate the anti-Mycoplasma pneumoniae (MP) activity of luteolin and elucidate its underlying mechanisms. MethodsLuteolin was identified as the primary active compound from the polyphenol extract ofF. diotrys using network pharmacology. Its efficacy was evaluated against two MP strains: the standard strain M129 and the multidrug-resistant strain M19. A modified culture medium with visual characteristics was employed to determine the minimum inhibitory concentration (MIC) of luteolin. The expression of key proteins involved in MP growth and pathogenicity was assessed by qRT-PCR following luteolin treatment. Additionally, the viability of A549 cells infected with MP was compared between luteolin-treated and untreated groups. In vivo anti-MP activity was evaluated using a mouse model, and the expression of inflammatory cytokines in lung tissues was analyzed. ResultsLuteolin effectively inhibited both MP strains, with MIC₉₀ values of 100 mg/L for M19 and M129. Treatment with luteolin significantly downregulated the expression of adhesion proteins P1 and P30 in both strains. However, the expression of P65, HMW3, TrmB, and CARDS TX was reduced only in the M19 strain following luteolin intervention. Luteolin also enhanced the growth and viability of A549 cells infected with MP. In the mouse model, luteolin treatment resulted in steady weight gain and was well tolerated. The bacteriostatic rate of luteolin in lung tissues was 50.7%, significantly higher than the 25.2% observed in the roxithromycin group. Furthermore, luteolin reduced the expression of inflammatory factors, including IL-6, TNF-α, and HMGB1, in MP-infected mice. ConclusionLuteolin effectively and safely inhibits the proliferation and pathogenicity of MP, particularly the drug-resistant M19 strain, by downregulating the expression of toxicity-associated proteins (P1, P30, P65, HMW3, TrmB, CARDS TX) and modulating host inflammatory responses. These findings suggest that luteolin may offer a novel therapeutic strategy for treating MP infections, especially those caused by drug-resistant strains.

Background: The hypotension prediction index (HPI) is an algorithm designed to predict hypotension. Some studies have reported that HPI-guided hemodynamic management strategies decrease intraoperative hypotension and complications; however, the effect of HPI on reducing perioperative complications is controversial. This meta-analysis aimed to assess the efficacy of the HPI in reducing major complications and intraoperative hypotension. Methods: We conducted this meta-analysis according to the PRISMA statement and Cochrane Handbook guidelines. A comprehensive literature review was conducted to identify studies focusing on the efficacy of HPI-guided management in reducing intraoperative hypotension and postoperative complications. The PubMed, Embase, Scopus, and Web of Science databases were searched, and the resulting data were combined to calculate the pooled mean differences or risk ratios (RRs) with 95% CIs of both randomized controlled trials (RCTs) and retrospective studies, as appropriate. Heterogeneity and potential publication bias were also assessed. Results: Nineteen articles (12 RCTs and 7 retrospective studies) with 2570 recruited patients were included in this meta-analysis. The critical evaluation of the study quality revealed a low risk of bias in the included RCTs. Among the non-randomized trials, one was rated 7, two were rated 8, and the remaining four were rated 9 on the Newcastle-Ottawa Scale, indicating high quality and a low risk of bias. HPI-guided management significantly reduced intraoperative hypotension and associated major complications (RR = 0.79, 95% CI [0.69–0.90], I2 = 0, P < 0.001). Blood loss and length of hospital stay were comparable between the groups. Conclusions HPI-guided management significantly reduced intraoperative hypotension and major complications.

Objective To summarize the clinical features and treatment strategies of acute mercury poisoning caused by intramuscular injection of metallic mercury. Methods The clinical data of a patient with acute mercury poisoning following intramuscular injection of metallic mercury were collected, and a retrospective analysis of the diagnosis and treatment process was conducted. Results The patient was a 14-year-old boy who self-injected 20 g of metallic mercury into the left upper arm muscle. Clinical manifestations included fever, chest tightness, cough, and a painful mass at the injection site. Laboratory tests of the patient indicated that blood mercury level was 4.160 μmol/L, urinary mercury level was 6 545.50 μg/g creatinine, urinary retinol-binding protein was 6 556 μg/L, urinary β₂-microglobulin was 8 726.48 μg/g creatinine, and urinary α1-microglobulin was 41.60 mg/L at day 11 after injection. Thoracoabdominal imaging revealed mercury deposition in the lungs, heart, liver, kidneys, and other organs. The patient's clinical symptoms were markedly relieved after one course of treatment with sodium dimercaptopropanesulfonate for mercury chelation. Subsequent treatment included continued mercury chelation therapy, debridement of muscle tissue at the injection site, removal of retained foreign bodies, and other supportive measures. Reexamination showed a blood mercury level of 2.810 μmol/L and urinary mercury level of 5 280 μg/L at day 127 after injection. Conclusion The clinical manifestations of intramuscular injection of metallic mercury poisoning differ from those of occupational mercury poisoning. Debridement of the injection site combined with sodium dimercaptopropanesulfonate chelation therapy can alleviate systemic symptoms and reduce mercury levels in such patients.

The drug resistance in the treatment of solid tumors is closely related to abnormalities in the tumor vascular microenvironment (TVM). The TVM is dynamically composed of multiple stromal, vascular, and immune cell populations together with extracellular matrix. Its structural disorder and functional imbalance can lead to abnormal angiogenesis, perfusion disorders, limited drug delivery, and insufficient immune cell infiltration, thereby creating a microenvironment conducive to tumor progression and treatment resistance. Therefore, the coordinated regulation of TVM through vascular normalization and immune remodeling has become an important research direction for improving the treatment effect of solid tumors. Tumor necrosis factor superfamily member 14 (TNFSF14), also known as LIGHT (homologous to lymphotoxin, inducible, competes with herpes simplex virus (HSV) glycoprotein D for herpesvirus entry mediator (HVEM), expressed by T lymphocyte), is an important molecule connecting tumor vascular remodeling and immune regulation. LIGHT can bind to the lymphotoxin β receptor (LTβR) to activate the NF-κB (nuclear factor kappa B)-related signaling pathway, regulate the functions of vascular endothelial cells and stromal cells, and induce the expression of adhesion molecules and chemokines such as VCAM-1(vascular cell adhesion molecule 1), ICAM-1(intercellular adhesion molecule 1), CCL19 (C-C motif chemokine ligand 19), CCL21 (C-C motif chemokine ligand 21), and CXCL13 (C-X-C motif chemokine ligand 13), promoting the formation of high endothelial venules (HEVs). HEVs can improve the vascular access of immune cells into tumor tissues and further promote the formation of tertiary lymphoid structures (TLS), providing a structural basis for the recruitment of effector T cells and sustained anti-tumor immune responses. At the same time, LIGHT interacts with HVEM to provide costimulatory signals to T cells, enhancing their activation, proliferation, and cytotoxic function. Thus, LIGHT can link tumor vascular normalization, immune cell recruitment, and T cell activation through the dual actions mediated by LTβR and HVEM, forming a coordinated regulation model of “vascular remodeling-immune infiltration-immune activation”, providing new molecular targets for the treatment of solid tumors. Despite LIGHT’s good anti-tumor potential, its therapeutic application is still limited by factors such as insufficient stability in vivo, short half-life, and potential off-target effects due to systemic exposure. In recent years, researchers have developed various strategies for the precise delivery of LIGHT, including fusion proteins, bacterial vectors, viral vectors, nanoparticles, chimeric antigen receptor T cells (CAR-T), and mesenchymal stem cells (MSCs), to enhance the enrichment, expression, or sustained release of LIGHT in tumor sites through different approaches, thereby improving its therapeutic efficacy and safety. This article systematically reviews the structural characteristics, biological functions, and molecular mechanisms mediating TVM remodeling by LIGHT, with a focus on the signal transduction mediated by LTβR and HVEM and their roles in tumor immune regulation. Subsequently, it systematically outlines the molecular mechanisms by which LIGHT regulates the tumor vascular microenvironment and its intrinsic connection with immune cell recruitment and activation, from the perspectives of TVM abnormalities, vascular normalization, HEV formation, and TLS induction. Furthermore, it categorizes and summarizes precise delivery strategies such as LIGHT fusion proteins, bacterial vectors, viral vectors, nanoparticles, CAR-T cells, and MSCs according to delivery vectors and modes of action, and compares their functional characteristics, antitumor effects, and potential advantages and limitations. On this basis, the article further discusses the key issues that LIGHT therapeutic strategies still face in terms of targeting, controllability,in vivo stability, safety, and clinical translation, and anticipates the potential value of its combination with immune checkpoint inhibitors, anti-angiogenic therapy, and other tumor treatment methods. This article aims to deepen the understanding of the antitumor mechanism of LIGHT from the perspective of synergistic regulation of tumor vascular microenvironment remodeling and immune activation, providing theoretical basis and new research ideas for the design of solid tumor treatment strategies based on precise regulation of TVM and the clinical translation of LIGHT-related treatment systems.

AIM To investigate the effects of Yunpi Tongchang Formula on intestinal mucosal barrier damage in rats with opioid-induced constipation(OIC)of Spleen-Kidney Yang Deficiency Syndrome.METHODS In contrast to the 10 rats of the blank group,the 50 rats of the modeling group were induced into models of OIC of Spleen-Kidney Yang Deficiency Pattern by 7 days consecutive administration of both subcutaneous loperamide injection and alternating gavage of activated carbon ice water and vinegar.Following successful modeling,rats were randomly allocated into the model group,the mosapride citrate tablet group(1.35 mg/kg),and the high-dose,medium-dose,and low-dose Yunpi Tongchang Formula groups(15.12,7.56,3.78 g/kg),with 8 mice in each group.Upon the completion of the 14 days treatment,the rats had their TCM Syndrome scores assessed;their fecal water content,initial black stool excretion time,and small intestine propulsion rate measured;their colon tissue morphology observed by HE staining;their serum levels of IL-6,TNF-α,and IL-1β detected by ELISA;their expressions of occludin and zonula occludens-1(ZO-1)in colon tissues detected by immunohistochemistry;their mRNA expressions of MyD88,TLR4 and NF-κB p65 in the colon tissues detected by RT-qPCR;and their protein expressions of MyD88,TLR4 and NF-κB p65 in the colon tissues detected by Western blot.RESULTS Compared to the blank group,the model group had higher TCM Syndrome scores(P＜0.01);lower fecal water content and small intestine propulsion rate(P＜0.05,P＜0.01);longer initial black stool excretion time(P＜0.01);more mucosal edema in colon tissue,obvious inflammatory infiltration,and glandular disorder;increased serum levels of IL-6,TNF-α and IL-1 β(P＜0.05);decreased colon expressions of ZO-1 and occludin(P＜0.01);and increased mRNA and protein expressions of TLR4,MyD88 and NF-κB p65(P＜0.01).Compared to the model group,both the medium-dose Yunpi Tongchang Formula group and the mosapride citrate tablet group demonstrated effectively reduced TCM syndrome scores(P＜0.01);increased fecal water content and small intestine propulsion rate(P＜0.05,P＜0.01);and shorter initial black stool excretion time(P＜0.01);improved colon mucosal edema and inflammatory infiltration;decreased serum levels of IL-6,TNF-α and IL-1β(P＜0.01);upregulated protein expressions of ZO-1 and occludin(P＜0.01);and downregulated mRNA and protein expressions of TLR4,MyD88 and NF-κB p65(P＜0.05,P＜0.01).CONCLUSION Yunpi Tongchang Formula significantly ameliorates constipation symptoms in OIC rat models of Spleen-Kidney Yang Deficiency Syndrome because of its efficacy in attenuating intestinal inflammation and preserving the integrity of intestinal epithelial barrier structure,with its mechanistic action in downregulating TLR4/MyD88/NF-κB signaling pathway activation.

Postoperative infection of internal fixation of closed fractures the lower limbs in adults represents a devastating complication, characterized by diagnostic challenges, prolonged treatment duration and high disability rates. Current management of these infections faces multiple challenges, such as difficulties in early accurate diagnosis, and various controversies about the treatment plan, leading to poor overall diagnosis and treatment results. To address these issues, based on evidence-based medicine and principles with emphasis on scientific rigor, clinical applicability and innovation, the Trauma Branch of the Chinese Medical Association, Orthopedic Branch of the Chinese Medical Doctor Association, Orthopedics Branch of the Chinese Medical Association, and Trauma Orthopedics and Polytrauma Group of the Resuscitation and Emergency Committee of the Chinese Medical Doctor Association have collaboratively organized a panel of relevant experts to develop the Guideline for diagnosis and treatment of infection after internal fixation of closed lower limb fractures in adults ( version 2025). The guideline proposed 10 recommendations, aiming to provide a foundation for standardized diagnosis and treatment of postoperative infection in adults with closed lower limb fractures.