Chinese hamster ovary (CHO) cells are the most established and versatile mammalian expression system for the large-scale production of recombinant therapeutic proteins, owing to their genetic stability, adaptability to serum-free suspension culture, and ability to perform human-like post-translational modifications. More than 70% of biologics approved by the U.S. Food and Drug Administration rely on CHO-based production platforms, underscoring their central role in modern biopharmaceutical manufacturing. Despite these advantages, CHO systems continue to face three persistent bottlenecks that limit their potential for high-yield, reproducible, and cost-efficient production: excessive metabolic burden during high-density culture, heterogeneity of glycosylation patterns, and progressive loss of long-term expression stability. This review provides an integrated analysis of recent advances addressing these challenges and proposes a forward-looking framework for constructing intelligent and sustainable CHO cell factories. In terms of metabolic regulation, excessive lactate and ammonia accumulation disrupts energy balance and reduces recombinant protein synthesis efficiency. Optimization of culture parameters such as temperature, pH, dissolved oxygen, osmolarity, and glucose feeding can effectively alleviate metabolic stress, while supplementation with modulators including sodium butyrate, baicalein, and S-adenosylmethionine promotes specific productivity (qP) by modulating apoptosis and chromatin structure. Furthermore, genetic engineering strategies—such as overexpression of MPC1/2, HSP27, and SIRT6 or knockout of Bax, Apaf1, and IGF-1R—have demonstrated significant improvements in cell viability and product yield. The combination of multi-omics metabolic modeling with artificial intelligence (AI)-based prediction offers new opportunities for building self-regulating CHO systems capable of dynamic adaptation to environmental stress. Regarding glycosylation uniformity, which determines therapeutic efficacy and immunogenicity, gene editing-based glycoengineering (e.g., FUT8 knockdown or ST6Gal1 overexpression) has enabled the humanization of CHO glycan profiles, minimizing non-human sugar residues and enhancing drug stability. Process-level strategies such as galactose or manganese co-feeding and fine control of temperature or osmolarity further allow rational regulation of glycosyltransferase activity. Additionally, in vitro chemoenzymatic remodeling provides a complementary route to construct human-type glycans with defined structures, though industrial applications remain constrained by cost and scalability. The integration of model-driven process design and AI feedback control is expected to enable real-time prediction and correction of glycosylation deviations, ensuring batch-to-batch consistency in continuous biomanufacturing. Long-term expression stability, another critical challenge, is often impaired by promoter silencing, chromatin condensation, and random genomic integration. Molecular optimization—such as the use of improved promoters (CMV, EF-1α, or CHO endogenous promoters), Kozak and signal peptide refinement, and incorporation of chromatin-opening elements (UCOE, MAR, STAR)—helps maintain durable transcriptional activity, while site-specific integration systems including Cre/loxP, Flp/FRT, φC31, and CRISPR/Cas9 can enable single-copy, position-independent gene insertion at genomic safe-harbor loci, ensuring stable, predictable expression. Collectively, this review highlights a paradigm shift in CHO system optimization driven by the convergence of genome editing, synthetic biology, and artificial intelligence. The transition from empirical optimization to rational, data-driven design will facilitate the development of programmable CHO platforms capable of autonomous regulation of metabolic flux, glycosylation fidelity, and transcriptional activity. Such intelligent cell factories are expected to accelerate the transformation from laboratory-scale research to industrial-scale, high-consistency, and economically sustainable biopharmaceutical manufacturing, thereby supporting the next generation of efficient and customizable biologics manufacturing.

Chinese hamster ovary (CHO) cells are the most established and versatile mammalian expression system for the large-scale production of recombinant therapeutic proteins, owing to their genetic stability, adaptability to serum-free suspension culture, and ability to perform human-like post-translational modifications. More than 70% of biologics approved by the U.S. Food and Drug Administration rely on CHO-based production platforms, underscoring their central role in modern biopharmaceutical manufacturing. Despite these advantages, CHO systems continue to face three persistent bottlenecks that limit their potential for high-yield, reproducible, and cost-efficient production: excessive metabolic burden during high-density culture, heterogeneity of glycosylation patterns, and progressive loss of long-term expression stability. This review provides an integrated analysis of recent advances addressing these challenges and proposes a forward-looking framework for constructing intelligent and sustainable CHO cell factories. In terms of metabolic regulation, excessive lactate and ammonia accumulation disrupts energy balance and reduces recombinant protein synthesis efficiency. Optimization of culture parameters such as temperature, pH, dissolved oxygen, osmolarity, and glucose feeding can effectively alleviate metabolic stress, while supplementation with modulators including sodium butyrate, baicalein, and S-adenosylmethionine promotes specific productivity (qP) by modulating apoptosis and chromatin structure. Furthermore, genetic engineering strategies—such as overexpression of MPC1/2, HSP27, and SIRT6 or knockout of Bax, Apaf1, and IGF-1R—have demonstrated significant improvements in cell viability and product yield. The combination of multi-omics metabolic modeling with artificial intelligence (AI)-based prediction offers new opportunities for building self-regulating CHO systems capable of dynamic adaptation to environmental stress. Regarding glycosylation uniformity, which determines therapeutic efficacy and immunogenicity, gene editing-based glycoengineering (e.g., FUT8 knockdown or ST6Gal1 overexpression) has enabled the humanization of CHO glycan profiles, minimizing non-human sugar residues and enhancing drug stability. Process-level strategies such as galactose or manganese co-feeding and fine control of temperature or osmolarity further allow rational regulation of glycosyltransferase activity. Additionally, in vitro chemoenzymatic remodeling provides a complementary route to construct human-type glycans with defined structures, though industrial applications remain constrained by cost and scalability. The integration of model-driven process design and AI feedback control is expected to enable real-time prediction and correction of glycosylation deviations, ensuring batch-to-batch consistency in continuous biomanufacturing. Long-term expression stability, another critical challenge, is often impaired by promoter silencing, chromatin condensation, and random genomic integration. Molecular optimization—such as the use of improved promoters (CMV, EF-1α, or CHO endogenous promoters), Kozak and signal peptide refinement, and incorporation of chromatin-opening elements (UCOE, MAR, STAR)—helps maintain durable transcriptional activity, while site-specific integration systems including Cre/loxP, Flp/FRT, φC31, and CRISPR/Cas9 can enable single-copy, position-independent gene insertion at genomic safe-harbor loci, ensuring stable, predictable expression. Collectively, this review highlights a paradigm shift in CHO system optimization driven by the convergence of genome editing, synthetic biology, and artificial intelligence. The transition from empirical optimization to rational, data-driven design will facilitate the development of programmable CHO platforms capable of autonomous regulation of metabolic flux, glycosylation fidelity, and transcriptional activity. Such intelligent cell factories are expected to accelerate the transformation from laboratory-scale research to industrial-scale, high-consistency, and economically sustainable biopharmaceutical manufacturing, thereby supporting the next generation of efficient and customizable biologics manufacturing.

ObjectiveThe frequency difference limen (FDL) serves as a fundamental metric utilized for effectively quantifying the precise perceptual capabilities of the central auditory system. However, traditional measurement methods rely heavily on the active behavioral responses of subjects and are consequently highly susceptible to the negative influence of confounding subjective factors. Furthermore, existing research paradigms frequently employ uniform stimulus configurations that overlook critical individual perceptual differences. Based on brain-computer interface (BCI) technology, this comprehensive study aims to establish an objective and quantitative evaluation method for auditory frequency discrimination by systematically analyzing and decoding the specific neural responses elicited at the exact threshold state. MethodsWe designed a personalized rapid serial auditory presentation (RSAP) paradigm customized based on each individual’s precise FDL. A cohort of eleven healthy participants was recruited to evaluate the paradigm using pure-tone sequences at a baseline frequency of 4 000 Hz. This experimental paradigm simulates a realistic auditory perception environment through the continuous presentation of acoustic stimuli, thereby allowing for an in-depth investigation into the specific neural representations evoked by weak frequency deviations at the threshold state. Given that auditory stimulus-evoked response features exhibit complex and differentiated spatiotemporal distribution patterns across multiple frequency domains, this study further deeply integrates the cross-scale feature interaction module with the dynamic spatiotemporal attention allocation strategy, innovatively proposing the Multi-Scale Spatial-Temporal Dual Attention Network (MS-STAMNet). Specifically, the network constructs parallel processing branches with multiple receptive fields and introduces a dynamic adaptive weighting strategy to precisely localize core neural activity signals, further deeply integrating multi-scale information through cross-branch feature information interaction to achieve robust single-trial decoding of weak auditory evoked responses. ResultsThe comprehensive electrophysiological data analysis demonstrated that subtle auditory frequency deviation stimuli presented at the threshold level successfully elicited pronounced N2 and P3 event-related potential features, reflecting pre-attentive mismatch detection and subsequent cognitive evaluation, which were prominently distributed over the frontal, central, and temporal regions of the scalp. In the complex time-frequency domain, the extracted neural response characteristics exhibited distinct, statistically significant event-related synchronization within both the low-frequency δ and θ frequency bands, which was simultaneously accompanied by a widespread, prominent event-related desynchronization within the higher α band. A comparative analysis of model performance demonstrated that MS-STAMNet achieved an average unweighted average recall (UAR) of (69.67±6.12)% and area under the curve (AUC) of 0.761 8±0.07, significantly outperforming the established baseline models such as EEGNet and PLNet. Furthermore, a distinct dissociation phenomenon was verified between neural decoding and behavioral performance through regression analysis (R²=0.016, P=0.709), indicating that this model can effectively capture the implicit features of subtle frequency deviations, even when they fail to trigger explicit conscious responses. Additionally, attention weight visualization analysis further reveals the highly accurate focus of the network on key features concentrated over the bilateral temporal and fronto-parietal regions. ConclusionThis study systematically and comprehensively uncovers the multi-dimensional spatiotemporal evolutionary patterns of complex neural responses processing subtle acoustic variations under long-sequence threshold auditory stimulation. Concurrently, it verifies the efficacy and robustness of the proposed MS-STAMNet architecture in accurately deciphering weak, single-trial electroencephalogram signals amidst complex background noise. Ultimately, these neurophysiological and algorithmic findings lay a solid theoretical and methodological foundation for the objective and quantitative evaluation of individual auditory cognitive capabilities in clinical applications, transcending the fundamental limitations of traditional behavioral paradigms and providing robust technical support for future auditory research and related clinical assessments.

Immobilized enzyme-based enzyme electrode biosensors, characterized by high sensitivity and efficiency, strong specificity, and compact size, demonstrate broad application prospects in life science research, disease diagnosis and monitoring, etc. Immobilization of enzyme is a critical step in determining the performance (stability, sensitivity, and reproducibility) of the biosensors. Random immobilization (physical adsorption, covalent cross-linking, etc.) can easily bring about problems, such as decreased enzyme activity and relatively unstable immobilization. Whereas, directional immobilization utilizing amino acid residue mutation, affinity peptide fusion, or nucleotide-specific binding to restrict the orientation of the enzymes provides new possibilities to solve the problems caused by random immobilization. In this paper, the principles, advantages and disadvantages and the application progress of enzyme electrode biosensors of different directional immobilization strategies for enzyme molecular sensing elements by specific amino acids (lysine, histidine, cysteine, unnatural amino acid) with functional groups introduced based on site-specific mutation, affinity peptides (gold binding peptides, carbon binding peptides, carbohydrate binding domains) fused through genetic engineering, and specific binding between nucleotides and target enzymes (proteins) were reviewed, and the application fields, advantages and limitations of various immobilized enzyme interface characterization techniques were discussed, hoping to provide theoretical and technical guidance for the creation of high-performance enzyme sensing elements and the manufacture of enzyme electrode sensors.

Objective:This multicenter retrospective study aimed to analyze the clinicopathological features of duodenal adenocarcinoma (DA) and identify prognostic factors for postoperative survival.Methods:Demographic characteristics, clinicopathological features, treatment outcomes and survival of DA patients undergoing surgical treatment at 18 Chinese medical centers from January 2012 to December 2023 were retrospectively analyzed.Results:Among the 2 056 DA patients included, 46.8% (963) had extra-ampullary DA (EA-DA), and 53.2% (1 093) had peri-ampullary DA (PA-DA). The 1-, 3-, and 5-year overall survival (OS) rates for patients who underwent radical surgery were 93.2%, 71.0%, and 57.2%, respectively. The median overall survival was 76 months, and the median progression-free survival (PFS) was 65 months. No differences in survival were observed between the laparotomy group and minimally invasive surgery (MIS) group either before or after propensity score matching (OS: 76 vs. 75 months before PSM, P=0.986; OS: 75 vs. 75 months after PSM, P=0.602). Furthermore, there were no significant differences between-group in operation time and postoperative complications ( P＞0.05). The MIS group experienced less intraoperative blood loss and shorter hospital stays. The multivariate Cox regression analysis revealed that advanced age ( HR=1.43,95% CI:1.18-1.73), elevated carbohydrate antigen 19-9 levels ( HR=1.24,95% CI:1.02-1.51), perineural invasion ( HR=1.44,95% CI:1.14-1.81), vascular invasion ( HR=1.35,95% CI:1.07-1.71), advanced T stage (T3-4 vs. T1-2: HR=1.86,95% CI:1.49-2.31), regional lymph node metastasis ( HR=1.93,95% CI:1.58-2.36), preoperative biliary drainage ( HR=1.26,95% CI:1.04-1.53), intraoperative blood loss ( HR=1.34,95% CI:1.11-1.62), clinically significant postoperative pancreatic fistulas ( HR=1.53,95% CI:1.12-2.09), and postoperative hemorrhage ( HR=1.62,95% CI:1.14-2.29) were independent risk factors for poor prognosis after surgery (all P＜0.05). Conclusions:Radical surgery is associated with favorable overall survival among DA patients, and no difference in survival is observed between EA-DA and PA-DA patients. MIS is a reliable alternative for DA treatment.

Body weight abnormalities, including overweight, obesity, and underweight, have become a dual public health challenge in Chinese adults: overweight and obesity lead to a variety of chronic complications, while underweight increases the risks of malnutrition, sarcopenia, and organ dysfunction. To systematically address these issues, multidisciplinary experts in endocrinology, sports science, nutrition, and psychiatry from various regions have held multiple weight management seminars. Based on the latest epidemiological data and clinical evidence, they expanded the guideline to include assessment and intervention strategies for underweight, in addition to the core content of obesity management. This guideline outlines the etiological mechanisms, evaluation methods, and multidimensional management strategies for overweight and obesity, covering key areas such as diagnosis and assessment, medical nutrition therapy, exercise prescription, pharmacological intervention, and psychological support. It is intended to provide a scientific and standardized approach to weight management across the adult population, aiming to curb the rising prevalence of obesity, mitigate complications associated with abnormal body weight, and improve nutritional status and overall quality of life.

Objective:To explore the feasibility of obtaining three-dimensional (3D) models of intraosseous and periosteal arteries of hallux phalanx using micro-arteriography with micro-CT scan.Methods:From January 2022 to April 2025, the Department of Orthopaedic, the 988th Hospital of the Joint Logistics Support Force of the Chinese PLA conducted a study on 7 fresh-frozen specimens of distal lower limb (right lower limb) from an 85-year-old male, and both lower limbs from an 82-year-old male, a 78-year-old female and a 66-year-old male in the Department of Human Anatomy & Histology and Embryology, Peking University School of Basic Medical Sciences. Red lead oxide powder (Pb 3O 4) was ground and filtered through a 300 mesh, and then mixed with turpentine at ratios of 1 g ∶ 1.5 ml, 1 g ∶ 1.0 ml, and 1 g ∶ 0.5 ml to prepare lead-based contrast agent suspensions. After thawing the specimens at room temperature, the suspensions were injected via the popliteal artery in ascending order of concentration. After injections, the specimens were fixed in 10% methanal for 2 weeks. The proximal and distal phalanges of the hallux, with the surrounding periosteum preserved intact, were then harvested. The harvested specimens were scanned using micro-CT at an ultimate resolution of 12 μm. Subsequently, Mimics Medical software was used to reconstruct 3D models of the intraosseous and periosteal arteries within the phalanges. Results:Periosteal arteries in the proximal phalanx were primarily distributed near the joint region. A consistently large trunk artery entered from plantar side, supplying most of the diaphysis and head. There was a rich periosteal arterial network on both sides of the distal phalanx, which communicates with each other through the arterial arch in the bone groove. However, trunk intraosseous artery could be absent. Intraosseous arteries in the proximal ends of both the proximal and distal phalanges originated from periosteal arteries. These formed an interconnected arteriosomes and coursed parallel to the articular surfaces.Conclusion:The micro-arteriography acquired by micro-CT scan effectively visualizes intraosseous and periosteal arteries and enables the reconstruction and analysis of 3D models of the arteriosomes. The characteristics of arteriolar distribution provide a theoretical basis for osteotomy or internal fixation procedures involving a hallux phalanx.

Objective To investigate the comparative neuroprotective effects of human umbilical cord mesenchymal stem cells(hUC-MSCs-Exos)administered via different routes on hypoxic ischemic brain damage(HIBD)in neonatal mice.Methods Healthy one-week-old SPF-grade BALB/c mice were randomly divided into 4 groups:sham operation group(n=6),model group(n=6),exosome group 1(n=8),exosome group 2(n=8).HIBD was induced using the Rice-Vannucci method.Exosome group 1 and Exosome group 2 were intraperitoneal injection/intranasal drip of phosphate buffer(PBS)100 μl containing 10 μl exosomes within 24 h after successful modeling,respectively.Sham operation and model groups were intraperitoneal injection of PBS 100 μl.On the 7th day after the intervention,neuromotor function was assessed using the horizontal grid test and pole climbing test.On the 2nd day after the evaluation,all mice were killed and their brains were removed by decapitation.HE staining was used to observe the pathological injury of brain tissue,toluidine blue staining was used to observe the survival of neurons in cerebral cortex,and TUNEL staining was used to observe the apoptosis of cerebral cortex cells.Results Compared with sham operation group,model group,exosome group 1 and exosome group 2 exhibited increased hind limb drops in horizontal grid test and climbing scores(P<0.05).No significant difference was found in model group,exosome group 1 and exosome group 2 in these measures(P<0.05).Significant pathology was observed in model group,exosome group 1 and exosome group 2 compared to sham operation group(P<0.05),with significantly reduced damage in exosome group 1 and exosome group 2 compared to model group(P<0.05).Compared with sham operation group,Nissl body count was lower in model group and exosome group 1 and exosome group 2,with a higher count in exosome group 2 compared to exosome group 1(P<0.05).Compared with sham operation group,apoptotic cells were higher in model group and exosome group 1 and exosome group 2,with a significant reduction in exosome group 1 and exosome group 2 compared to model group,and the lowest in exosome group 2(P<0.05).Conclusions hUC-MSCs-Exos can improve the neuronal motor function,promote neuron repair and inhibit apoptosis in HIBD mice.Intranasal administration of hUC-MSCs-Exos is more effective than intraperitoneal administration for reducing neuronal apoptosis in HIBP neonatal mice,offering a convenient and rapid method suitable for clinical application.

Objective To investigate the diagnostic value of 4 novel inflammatory markers related to routine blood tests,namely neutrophil-to-lymphocyte ratio(NLR),red blood cell distribution width(RDW),hemoglobin-to-RDW ratio(HRR)and systemic immune-inflammation index(SII),in elderly patients with chronic cardiovascular disease(CVD)complicated with frailty.Methods Retrospectively analyze 110 patients with chronic stable CVD who were hospitalized in the cadre ward of cardiovascular medicine at the Air Force Characteristic Medical Center from January 2022 to June 2023.According to the assessment results of the Fried scale,they were divided into three groups:non-frailty group(Fried score=0,n=30),the pre-frailty group(Fried score 1 or 2,n=40)and frailty group(Fried score≥3,n=40).The differences in general information,the impairment rate of daily living activities,miniature nutritional assessment-short form(MNA-SF)scores,mini-mental state examination(MMSE)scores,and the indicators such as NLR,RDW,HRR,and SII among the three groups were compared.Spearman rank correlation was used to analyze the correlation between NLR,RDW,HRR,SII and frailty scores as well as each frailty indicator.Multivariate logistic regression analysis was performed to identify the independent risk factors for frailty in elderly patients with chronic CVD,and the receiver operating characteristic(ROC)curve was used to assess the clinical diagnostic value of NLR and HRR in elderly patients with chronic CVD complicated with frailty.Results Compared with non-frailty group and pre-frailty group,patients in frailty group were older,with higher impaired rates of daily living activities,NLR,RDW,and SII,and lower MNA-SF scores,MMSE scores,and HRR,and differences were statistically significant(P<0.05).Spearman rank correlation analysis showed that the frailty score was positively correlated with NLR(rs=0.354,P<0.001),and RDW(rs=0.448,P<0.001),negatively correlated with HRR(rs=-0.232,P=0.024),and had no significant correlation with SII(rs=0.144,P=0.167).Further analysis of the correlation between the above novel inflammatory markers and the 5 components of frailty showed that NLR was positively correlated with fatigue(rs=0.228,P=0.017),slowed walking speed(rs=0.299,P<0.001),and low physical function(rs=0.319,P<0.001);RDW was positively correlated with decreased grip strength(rs=0.321,P<0.001),slowed walking speed(rs=0.422,P<0.001),and low physical function(rs=0.246,P=0.001);and HRR was negatively correlated with slowed walking speed(rs=-0.230,P=0.025),and low physical function(rs=-0.299,P=0.003).Multivariate logistic regression analysis showed that MNA-SF score(OR=0.577,95%CI 0.342-0.973)was an independent protective factor for pre-frailty in elderly patients with chronic CVD(P<0.05);NLR(OR=7.866,95%CI 1.101-56.185)was an independent risk factor for frailty,while HRR(OR=0.344,95%CI 0.120-0.983)and MNA-SF score(OR=0.292,95%CI 0.146-0.580)were independent protective factors for frailty in elderly CVD patients(P<0.05).The area under the ROC curve of NLR and HRR for diagnosing frailty in elderly patients with chronic CVD were 0.778 and 0.749,respectively.Conclusion NLR and HRR have high clinical diagnostic value for frailty in elderly patients with chronic CVD,and are expected to become effective inflammatory markers for screening elderly patients with chronic CVD complicated with frailty.

Objective To investigate the role of the neutrophil-to-lymphocyte ratio(NLR)in predicting the in-hospital mortality risk of patients with acute aortic dissection(AAD)in multicenter hospitals.Methods A multicenter retrospective cohort study was conducted.Clinical data were collected from 2642 AAD patients who were hospitalized in five teaching hospitals:Tongji Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology,Henan Provincial People's Hospital,Fuwai Central China Cardiovascular Hospital,the Third Affiliated Hospital of Xinxiang Medical University,and the Second Affiliated Hospital of Chongqing Medical University between August 2010 and December 2021.According to the quartiles of serum NLRlevels,the patients were divided into four groups:first quartile(Q1,n=660),second quartile(Q2,n=661),third quartile(Q3,n=661),and fourth quartile(Q4,n=660).The clinical characteristics and biochemical indicators of each group were compared.Partial correlation analysis was used to assess the relationship between NLR and cardiovascular parameters.Restricted cubic splines,Kaplan-Meier survival analysis,and Cox regression models were employed to evaluate the association between NLR levels and in-hospital mortality risk in AAD patients.Results The median age of all patients was 54[interquartile range(IQR):46-63]years,including 2096 males and 546 females.Compared with Q1-Q3 groups,patients inQ4group had a lower incidence of smoking history and diabetes history,and were more likely to have DeBakey type Ⅰ AAD(P＜0.05).Additionally,the levels of aspartate aminotransferase,high-density lipoprotein cholesterol,creatinine,and D-dimer in Q4 group were higher,while the levels of triglycerides and C-reactive protein(CRP)were lower(P＜0.01).The results of partial correlation analysis showed that the plasma NLR level was positively correlated with D-dimer(r=0.43,P＜0.01)and creatinine(r=0.16,P＜0.01).The restricted cubic spline function in the Cox model revealed a significant non-linear relationship between the plasma NLR level and clinical outcomes in AAD patients(P＜0.01).Kaplan-Meier survival analysis indicated that patients in Q4 group had the highest in-hospital mortality rate compared with Q1-Q3 groups(P＜0.0001).Furthermore,multivariate Cox regression analysis demonstrated that compared with Q1 group,the hazard ratio(HR)of NLR in Q4 group was 1.77(95％CI 1.33-2.37,P＜0.001),which was an independent risk factor for the primary endpoint events.Conclusion A higher plasma NLR level is significantly associated with the occurrence of cardiovascular events in AAD patients,and this association remains significant even after adjusting for potential confounding factors such as the multicenter visiting hospitals.