Background: Advances in intensive care have markedly improved survival after severe brain injury, leading to a growing population of patients with prolonged disorders of consciousness (pDOC). Current management of pDOC remains largely supportive, and evidence-based neuromodulatory interventions are limited; moreover, existing guidelines provide insufficiently explicit recommendations regarding mechanisms of action and objective biomarkers of treatment response. Transcutaneous auricular vagus nerve stimulation (taVNS) has emerged as a potential noninvasive intervention; however, its modulatory effects on brain function in pDOC are not yet well characterized, and the paucity of integrative mechanistic evidence has constrained its translation into routine clinical practice. Objectives: Within a multimodal assessment framework, this study aims to systematically elucidate the neurobiological mechanisms by which taVNS modulates brain function and autonomic activity in patients with pDOC, and to evaluate its clinical potential to enhance levels of consciousness. Methods: In this randomized controlled trial, 60 patients with vegetative state/minimally conscious state will be enrolled and randomly allocated to a taVNS group, a transcutaneous nonauricular vagus nerve stimulation group (sham), or a control group (n = 20 per group) for a 4-week intervention. The primary outcome will be changes in the Coma Recovery Scale-Revised scores from baseline to weeks 1, 2, and 4 of treatment. Secondary outcomes will include functional brain activity assessed by electroencephalography and functional near-infrared spectroscopy, as well as autonomic modulation indexed by heart rate variability. Functional prognosis will be evaluated using the Glasgow Outcome Scale-Extended at the end of treatment and at a 6-month follow-up. Safety will be assessed by continuous monitoring and documentation of adverse events throughout the study period. Results and discussion: By integrating electroencephalography–functional near-infrared spectroscopy with heart rate variability, this study will characterize the effects of taVNS on functional brain networks and consciousness recovery in pDOC across complementary behavioral, electrophysiological, hemodynamic, and autonomic domains, while interrogating potential sources of clinical and neurobiological heterogeneity. The findings are expected to provide a mechanistic and evidence-based foundation for the mechanism-driven clinical implementation of taVNS and the optimization of stimulation protocols in pDOC. Clinical trial registration: International Traditional Medicine Clinical Trial Registry, ITMCTR20250021041, https://itmctr.ccebtcm.org.cn.

Circular RNA(circRNA)is a single-stranded RNA with a covalently closed loop structure,which has a more stable structure and lower immunogenicity than linear RNA.Many studies have shown that circRNA has characteristics such as conservation,stability,and tissue specificity,and can act as a microRNA(miRNA)sponge to interact with proteins and translation templates,and regulate biological functions such as gene expression and signal transduction.Based on the characteristics and various bio-logical functions of circRNA,some endogenous circRNA plays an important regulatory role in the occur-rence and development of tumors and has the potential to be used as biomarkers and therapeutic targets.In addition,mRNA drugs have limitations such as instability,easy degradation,low translation efficien-cy,and immunogenicity in practical applications.Engineered translatable exogenous circRNA can solve some of the limitations of linear mRNA application and become a new type of potential efficient drug.In this paper,we introduce the circRNA biogenesis mechanisms,specific biological functions,and the cur-rent status of diagnosis and treatment applications in tumors.This includes the diagnostic application of endogenous circRNA in tumors,the design and synthesis strategies of exogenous circRNA,and the cur-rent progress in the design and application of engineered circRNA vaccines using their stable and efficient protein expression functions in the treatment of tumors.Finally,we discuss the current clinical diagnostic application problems of circRNA,the challenges of exogenous circRNA therapeutic applications,and the prospects of the field.

To prepare monoclonal antibody to the N protein of porcine reproductive and respiratory syndrome virus(PRRSV),BALB/c mice were immunized with the purified N protein of the PRRSV-2 strain expressed by prokaryotic expression system.Mouse splenocytes were fused with myeloma cells using hybridoma technique,hybridoma cells were identified by indirect ELISA method and indirect immunofluorescence assay(IFA),and positive hybridoma cells were screened for subclones using the limited dilution method.The results showed that a monoclonal antibody cell line 4A7 was successfully obtained,and the results of Western blot and IFA indicated that the monoclonal antibody could accurately recognize the N proteins of type 1 and type 2 PRRSV.Mean-while,the N protein gene was truncated and expressed by prokaryotic expression system,and the amino acid sequence of the B-cell antigenic epitope recognized by 4A7 was screened as 51EKPHF55 using Western blot.Comparison of epitope amino acids in the N protein gene sequences of different strains revealed that the antigenic epitope 51EKPHF55 recognized by the monoclonal antibody 4A7 has no amino acid difference in the sequences of three subtypes among the PRRSV-1 strains and nine lineages among the PRRSV-2 strains,indicating a high degree of conservation.The results of the study provide a foundation the development of PRRSV diagnostic kits and novel vaccines.

Objective To explore the CT and MRI manifestation of salivary gland mucosa-associated lymphoid tissue(MALT)lymphoma.Methods The preoperative CT and MRI images of 89 patients with salivary gland MALT lymphoma who were confirmed by surgical pathology were analyzed retrospectively.Results Salivary gland MALT lymphoma were mainly located in the parotid gland(64 cases,71.9％).The ratio of male to female was 1 ≤ 5.Sixty-nine cases(77.5％)showed concomitant Sjogren's syndrome(SS).Typical imaging findings were round or lobulated well-marginated solid masses with moderate to severe enhancement.Atypical imaging findings were all from parotid gland MALT lymphoma,including cyst-solid mixed masses(34 cases,38.2％),poorly marginated boundaries(9 cases,10.1％),and punctuate calcification(5 cases,5.6％).The average apparent diffusion coefficient(ADC)value of lesions was(0.59±0.09)× 10-3 mm2/s and the time-signal intensity curve(TIC)type was mainly type Ⅱ(66.7％).Conclusion Salivary gland MALT lymphoma exhibits certain clinical and imaging characteristics.But when occur in parotid gland,some lesions may present atypically,which require diagnosis with the aid of functional MRI.

Circular RNA(circRNA)is a single-stranded RNA with a covalently closed loop structure,which has a more stable structure and lower immunogenicity than linear RNA.Many studies have shown that circRNA has characteristics such as conservation,stability,and tissue specificity,and can act as a microRNA(miRNA)sponge to interact with proteins and translation templates,and regulate biological functions such as gene expression and signal transduction.Based on the characteristics and various bio-logical functions of circRNA,some endogenous circRNA plays an important regulatory role in the occur-rence and development of tumors and has the potential to be used as biomarkers and therapeutic targets.In addition,mRNA drugs have limitations such as instability,easy degradation,low translation efficien-cy,and immunogenicity in practical applications.Engineered translatable exogenous circRNA can solve some of the limitations of linear mRNA application and become a new type of potential efficient drug.In this paper,we introduce the circRNA biogenesis mechanisms,specific biological functions,and the cur-rent status of diagnosis and treatment applications in tumors.This includes the diagnostic application of endogenous circRNA in tumors,the design and synthesis strategies of exogenous circRNA,and the cur-rent progress in the design and application of engineered circRNA vaccines using their stable and efficient protein expression functions in the treatment of tumors.Finally,we discuss the current clinical diagnostic application problems of circRNA,the challenges of exogenous circRNA therapeutic applications,and the prospects of the field.

Objective To investigate the effect and mechanism of methyl oxofulnonone A(META)on high glucose(HG)-induced H9c2 cell injury.Methods H9c2 cells were divided into control group(normal culture),model group(55 mmol·L-1 glucose)and experimental-L,-M,-H groups(55 mmol·L-1 glucose+12.5,25.0,50.0 μmol·L-1 META).Cell viability was detected by cell counting kit-8;intracellular reactive oxygen species(ROS)level was detected by DCFH-DA fluorescent probe;intracellular adenosine triphosphate(ATP)content was detected by luciferase;and autophagy-related protein expression was detected by Western blotting.Results The optical density values of 72-hour cells in the control group,model group and experimental-M,-H groups were 0.91±0.03,0.61±0.01,0.69±0.02 and 0.72±0.03;the ROS levels were(40.75±1.53)％,(43.73±1.30)％,(30.87±1.27)％and(28.28±1.43)％;the ATP contents were(8.16±0.71),(4.03±0.29),(5.29±0.31)and(5.83±0.31)nmol·mg-1;the relative expression levels of autophagy-related gene 5 protein were 1.05±0.06,1.46±0.09,0.98±0.11 and 0.89±0.09;the relative expression levels of ubiquitin-binding protein were 1.05±0.10,0.55±0.13,0.89±0.04 and 0.98±0.04;the ratios of microtubule-associated protein 1 light chain 3 11/Ⅰ protein were 1.09±0.09,1.82±0.05,1.67±0.29 and 1.09±0.15,respectively.Among the above indicators,there were statistically significant differences between the model group and the control and experimental-M,-H groups(P＜0.05,P＜0.01).Conclusion META significantly ameliorates H9c2 cardiomyocyte damage caused by high glucose,ameliorates oxidative stress,protects mitochondrial respiration and inhibits autophagy.

Triple-negative breast cancer (TNBC) represents a distinctive subtype, characterized by the absence of estrogen receptors, progesterone receptors, and human epidermal growth factor receptor 2 (HER2). Due to its high inter-tumor and intra-tumor heterogeneity, TNBC poses significant chanllenges for personalized diagnosis and treatment. The advant of clustered regular interspaced short palindromic repeats (CRISPR) technology has profoundly enhanced our understanding of the structure and function of the TNBC genome, providing a powerful tool for investigating the occurrence and development of diseases. This review focuses on the application of CRISPR/Cas technology in the personalized diagnosis and treatment of TNBC. We begin by discussing the unique attributes of TNBC and the limitations of current diagnostic and treatment approaches: conventional diagnostic methods provide limited insights into TNBC, while traditional chemotherapy drugs are often associated with low efficacy and severe side effects. The CRISPR/Cas system, which activates Cas enzymes through complementary guide RNAs (gRNAs) to selectively degrade specific nucleic acids, has emerged as a robust tool for TNBC research. This technology enables precise gene editing, allowing for a deeper understanding of TNBC heterogeneity by marking and tracking diverse cell clones. Additionally, CRISPR facilitates high-throughput screening to promptly identify genes involved in TNBC growth, metastasis, and drug resistance, thus revealing new therapeutic targets and strategies. In TNBC diagnostics, CRISPR/Cas was applied to develop molecular diagnostic systems based on Cas9, Cas12, and Cas13, each employing distinct detection principles. These systems can sensitively and specifically detect a variety of TNBC biomarkers, including cell-specific DNA/RNA and circulating tumor DNA (ctDNA). In the realm of precision therapy, CRISPR/Cas has been utilized to identify key genes implicated in TNBC progression and treatment resistance. CRISPR-based screening has uncovered potential therapeutic targets, while its gene-editing capabilities have facilitated the development of combination therapies with traditional chemotherapy drugs, enhancing their efficacy. Despite its promise, the clinical translation of CRISPR/Cas technology remains in its early stages. Several clinical trials are underway to assess its safety and efficacy in the treatment of various genetic diseases and cancers. Challenges such as off-target effects, editing efficiency, and delivery methods remain to be addressed. The integration of CRISPR/Cas with other technologies, such as 3D cell culture systems, human induced pluripotent stem cells (hiPSCs), and artificial intelligence (AI), is expected to further advance precision medicine for TNBC. These technological convergences can offer deeper insights into disease mechanisms and facilitate the development of personalized treatment strategies. In conclusion, the CRISPR/Cas system holds immense potential in the precise diagnosis and treatment of TNBC. As the technology progresses and becomes more costs-effective, its clinical relevance will grow, and the translation of CRISPR/Cas system data into clinical applications will pave the way for optimal diagnosis and treatment strategies for TNBC patients. However, technical hurdles and ethical considerations require ongoing research and regulation to ensure safety and efficacy.

Objective:To summarize the genotype and clinical phenotype characteristics of children with epilepsy associated with the SCN1B gene encoding the sodium channel β1 subunit. Methods:The genotypes and clinical phenotypes of patients with SCN1B variants among suspected genetic epilepsy cases treated at the Children′s Medical Center of Peking University First Hospital between May 2016 and July 2024 were analyzed. These variants were identified using next-generation sequencing and subsequently validated by Sanger sequencing or quantitative polymerase chain reaction methods. Results:A total of 17 patients were analyzed, including 8 males and 9 females. Ten cases of missense variations (including 2 with the same variations), 4 cases of deletion variations, and 1 case each of nonsense variations, splice site variations, and exons 4-5 deletions were identified. Among them, 6 cases had novel SCN1B variations. The variants in 11 cases were inherited from 1 parent. Eleven types of gene variants have not been reported yet. Onset of epilepsy ranged from 3 months to 5 years and 3 months old (median age: 14 months). Types of seizures included generalized tonic-clonic seizures (GTCS) in 14 cases, focal seizures in 9 cases, myoclonic seizures in 3 cases, atypical absence seizures in 2 cases and epilepsy spasms, tonic seizures and atonic seizures in 1 case each. Eleven cases had diverse seizure types. Fourteen cases (14/17) demonstrated fever sensitivity. Electroencephalography revealed focal discharges in 3 cases, coinciding with focal and generalized discharges in 3 additional cases, and multifocal discharges in 6 cases. Seizures were identified in 4 cases: 1 case of myoclonic seizures, 1 case of GTCS, 1 case of atypical absence seizures, and 1 case exhibiting both myoclonic and tonic seizures. Nine cases (9/17) were diagnosed with genetic epilepsy with febrile seizures plus, 1 case diagnosed with myoclonic epilepsy in infancy and 1 diagnosed with infant epileptic spasms syndrome. There were 2 cases of nonspecific developmental epileptic encephalopathy, while the remaining 4 cases could not be diagnosed with a specific epileptic syndrome. Effective antiseizure medications (ASMs) included valproate in 8 cases, levetiracetam in 5 cases, topiramate in 3 cases, clobazam in 2 cases, clonazepam and vigabatrin in 1 case each. Sodium channel blockers exacerbated seizures in 3 cases, specifically oxcarbazepine in 2 cases and lamotrigine in 1 case. At the last follow-up, seizures were controlled for at least 6 months in 14 patients (14/17), while seizures remained uncontrolled in 3 patients despite trialing 2 or more ASMs. Thirteen patients exhibited normal development, while 4 experienced developmental delays. Conclusions:The heterozygous variants in children with SCN1B gene-related epilepsy include missense, deletion, nonsense, splice site variants, and exon deletions. The correlation between different genetic variants and clinical phenotypes remains unclear. These variants are associated with epilepsy onset from infancy to early childhood, presenting with various seizure types, with GTCS being the most common. Phenotypic manifestations can vary significantly in severity, ranging from benign febrile seizures or febrile seizures plus to developmental epileptic encephalopathy. Valproic acid demonstrates the highest effectiveness rate, while the use of sodium channel blockers may worsen seizures in certain patients, necessitating cautious administration.

To prepare monoclonal antibody to the N protein of porcine reproductive and respiratory syndrome virus(PRRSV),BALB/c mice were immunized with the purified N protein of the PRRSV-2 strain expressed by prokaryotic expression system.Mouse splenocytes were fused with myeloma cells using hybridoma technique,hybridoma cells were identified by indirect ELISA method and indirect immunofluorescence assay(IFA),and positive hybridoma cells were screened for subclones using the limited dilution method.The results showed that a monoclonal antibody cell line 4A7 was successfully obtained,and the results of Western blot and IFA indicated that the monoclonal antibody could accurately recognize the N proteins of type 1 and type 2 PRRSV.Mean-while,the N protein gene was truncated and expressed by prokaryotic expression system,and the amino acid sequence of the B-cell antigenic epitope recognized by 4A7 was screened as 51EKPHF55 using Western blot.Comparison of epitope amino acids in the N protein gene sequences of different strains revealed that the antigenic epitope 51EKPHF55 recognized by the monoclonal antibody 4A7 has no amino acid difference in the sequences of three subtypes among the PRRSV-1 strains and nine lineages among the PRRSV-2 strains,indicating a high degree of conservation.The results of the study provide a foundation the development of PRRSV diagnostic kits and novel vaccines.

Objective To investigate the effect of miR-10a-5p downregulation on cisplatin(DDP)resistance in bladder cancer T24 cells and elucidate the underlying molecular mechanism.Methods Quantitative real-time PCR quantified the expression of miR-10a-5p in the cisplatin-resistant bladder cancer cell line T24/DDP and its parental cell line T24.T24/DDP cells were divided into control,inhibitor NC,miR-10a-5p inhibitor,miR-10a-5p inhibitor+si-NC,and miR-10a-5p inhibitor+si-Apaf1 groups.Different concentrations of DDP were administered for 24 h.Cell proliferative activity was detected using the MTT assay,and the drug resistance index was calculated.Apop-tosis was analyzed using flow cytometry.The protein expression levels of Apaf1,cleaved caspase-9,cleaved caspase-3,and cytochrome C(Cyt C)were analyzed using Western blotting.A dual-luciferase reporter gene assay confirmed the target binding relationship between miR-10a-5p and Apaf1.Results The expression of miR-10a-5p was significantly higher in drug-resistant T24/DDP cells than in parental T24 cells(P<0.05).Compared with the inhibitor NC group,the miR-10a-5p inhibitor group exhibited increased T24/DDP cells sensitivity to DDP,decreased drug resistance index,and elevated apoptosis levels.The protein expression levels of Apaf1,cleaved caspase-9,cleaved caspase-3,and cytoplasmic Cyt C proteins were upregulated in the miR-10a-5p inhibitor group compared with the inhibitor NC group(all P<0.05).The DDP sensitivity of T24/DDP cells was reduced in the miR-10a-5p inhibitor+si-Apaf1 group compared to the miR-10a-5p inhibitor+si-NC group,accompanied by an increase in the drug resistance index and a decrease in apoptosis(all P<0.05).The double-lu-ciferase reporter gene assay results confirmed that Apaf1 was a downstream target gene regulated by miR-10a-5p.Conclusion miR-10a-5p knockdown targeting the upregulation of Apaf1reversed DDP resistance in T24/DDP cells.