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.

BACKGROUND: Several studies have demonstrated the occurrence of secondary tumors as a rare but significant complication of chimeric antigen receptor T (CAR-T) cell therapy, underscoring the need for a detailed investigation. Given the limited variety of secondary tumor types reported to date, a comprehensive characterization of the various secondary tumors arising after CAR-T therapy is essential to understand the associated risks and to define the role of the immune microenvironment in malignant transformation. This study aims to characterize the immune microenvironment of a newly identified secondary tumor post-CAR-T therapy, to clarify its pathogenesis and potential therapeutic targets. METHODS: In this study, the bone marrow (BM) samples were collected by aspiration from the primary and secondary tumors before and after CD19 CAR-T treatment. The CD45 + BM cells were enriched with human CD45 microbeads. The CD45 + cells were then sent for 10× genomics single-cell RNA sequencing (scRNA-seq) to identify cell populations. The Cell Ranger pipeline and CellChat were used for detailed analysis. RESULTS: In this study, a rare type of secondary chronic myelomonocytic leukemia (CMML) were reported in a patient with diffuse large B-cell lymphoma (DLBCL) who had previously received CD19 CAR-T therapy. The scRNA-seq analysis revealed increased inflammatory cytokines, chemokines, and an immunosuppressive state of monocytes/macrophages, which may impair cytotoxic activity in both T and natural killer (NK) cells in secondary CMML before treatment. In contrast, their cytotoxicity was restored in secondary CMML after treatment. CONCLUSIONS This finding delineates a previously unrecognized type of secondary tumor, CMML, after CAR-T therapy and provide a framework for defining the immune microenvironment of secondary tumor occurrence after CAR-T therapy. In addition, the results provide a rationale for targeting macrophages to improve treatment strategies for CMML treatment.
Humans ; Lymphoma, Large B-Cell, Diffuse/therapy* ; Tumor Microenvironment/genetics* ; Antigens, CD19/metabolism* ; Leukemia, Myelomonocytic, Chronic/genetics* ; Immunotherapy, Adoptive/adverse effects* ; Male ; Single-Cell Analysis/methods* ; Female ; Sequence Analysis, RNA/methods* ; Receptors, Chimeric Antigen ; Middle Aged

Guided by the concept of quality by design(QbD), this study optimizes the calcination and quenching process of calcined Magnetitum and establishes the XRD characteristic spectra of calcined Magnetitum, providing a scientific basis for the formulation of quality standards. Based on the processing methods and quality requirements of Magnetitum in the Chinese Pharmacopoeia, the critical process parameters(CPPs) identified were calcination temperature, calcination time, particle size, laying thickness, and the number of vinegar quenching cycles. The critical quality attributes(CQAs) included Fe mass fraction, Fe~(2+) dissolution, and surface color. The weight coefficients were determined by combining Analytic Hierarchy Process(AHP) and the criteria importance though intercrieria correlation(CRITIC) method, and the calcination process was optimized using orthogonal experimentation. Surface color was selected as a CQA, and based on the principle of color value, the surface color of calcined Magnetitum was objectively quantified. The vinegar quenching process was then optimized to determine the best processing conditions. X-ray diffraction(XRD) was used to establish the characteristic spectra of calcined Magnetitum, and methods such as similarity evaluation, cluster analysis, and orthogonal partial least squares-discriminant analysis(OPLS-DA) were used to evaluate the quality of the spectra. The optimized calcined Magnetitum preparation process was found to be calcination at 750 ℃ for 1 h, with a laying thickness of 4 cm, a particle size of 0.4-0.8 cm, and one vinegar quenching cycle(Magnetitum-vinegar ratio 10∶3), which was stable and feasible. The XRD characteristic spectra analysis method, featuring 9 common peaks as fingerprint information, was established. The average correlation coefficient ranged from 0.839 5-0.988 1, and the average angle cosine ranged from 0.914 4 to 0.995 6, indicating good similarity. Cluster analysis results showed that Magnetitum and calcined Magnetitum could be grouped together, with similar compositions. OPLS-DA discriminant analysis identified three key characteristic peaks, with Fe_2O_3 being the distinguishing component between the two. The final optimized processing method is stable and feasible, and the XRD characteristic spectra of calcined Magnetitum was initially established, providing a reference for subsequent quality control and the formulation of quality standards for calcined Magnetitum.
X-Ray Diffraction/methods* ; Drugs, Chinese Herbal/chemistry* ; Quality Control ; Particle Size

Incomplete debridement of chronic osteomyelitis is the main factor leading to recurrence. For the treatment of chronic osteomyelitis, the complete elimination of the source of infection is the key to preventing recurrence. This process includes not only the complete removal of infected lesions, dead bone, accreted scar tissue and granulation tissue, but also the elimination of dead space and improved local blood circulation. In these steps, debridement is a core procedure, and judging the scope of debridement is the premise of whether it could be completely debridement. This article systematically reviewed the application of different imaging techniques in evaluating the scope of chronic osteomyelitis infection, and discusses its future development trend. Although traditional plain X-ray film could preliminarily indicate osteomyelitis, it is difficult to determine the infection scope. CT scan has the function of accurate anatomic localization, which is important for preoperative assessment of the scope of bone infection, but the recognition of soft tissue information is limited. MRI, with its high sensitivity, clearly distinguishes between infected bone and soft tissue, which plays an important role in the evaluation of soft tissue infection, but may overestimate the extent of bone infection. Nuclide techniques such as 18F-FDG PET/CT and SPECT/CT show great potential for accurately assessing the extent of infection before surgery. In the future, by optimizing the combination of different imaging technologies, combining clinical symptoms, intraoperative conditions and pathological results, and developing an image analysis platform based on artificial intelligence, it will be able to more accurately assess the scope of infection, provide more effective and personalized treatment plans for patients with chronic osteomyelitis, enhance treatment effects, and significantly improve quality of life of patients.
Humans ; Osteomyelitis/diagnosis* ; Chronic Disease ; Magnetic Resonance Imaging ; Tomography, X-Ray Computed

OBJECTIVES: To investigate the clinical features of children with STAT gene mutations, and to explore corresponding immunotherapy strategies. METHODS: A retrospective analysis was performed for the clinical data of 10 children with STAT gene mutations who were admitted to the Department of Pediatrics of the First Affiliated Hospital of Guangzhou Medical University, from October 2015 to October 2024. Exploratory immunotherapy was implemented in some refractory cases, and the changes in symptoms, imaging manifestations, and cytokine levels were assessed after treatment. RESULTS: For the 10 children, the main clinical manifestations were recurrent rash since birth (7/10), cough (8/10), wheezing (5/10), expectoration (4/10), and purulent nasal discharge (4/10). Genotyping results showed that there was one child with heterozygous loss-of-function (LOF) mutation in the STAT1 gene, four children with heterozygous LOF mutation in the STAT3 gene, and five children with heterozygous gain-of-function (GOF) mutation in the STAT3 gene. Two children with LOF mutation in the STAT3 gene showed decreased interleukin-6 levels and improved clinical symptoms and imaging findings after omalizumab treatment. Three children with GOF mutation in the STAT3 gene achieved effective disease control after treatment with methylprednisolone (0.5 mg/kg per day). Two children with GOF mutation in the STAT3 gene received treatment with JAK inhibitor and then showed some improvement in symptoms. CONCLUSIONS STAT gene mutation screening should be considered for children with recurrent rash and purulent respiratory tract infections. Targeted immunotherapy may improve prognosis in patients with no response to conventional treatment.
Humans ; Male ; Immunotherapy ; Female ; Child, Preschool ; Child ; Gain of Function Mutation ; Retrospective Studies ; Infant ; Loss of Function Mutation ; STAT Transcription Factors/genetics*

OBJECTIVE: Photodynamic therapy has become an important method in clinical tumor treatment. This study aimed to investigate the effects of hematoporphyrin on multiple myeloma (MM) and its potential applications. METHODS: The MM cell line RPMI 8226 was treated with hematoporphyrin derivative (HPD), and CCK-8 assay was used to determine cell viability, apoptosis was detected by flow cytometry, intracellular reactive oxygen species (ROS) levels were measured using a detection kit combined with flow cytometry, and Western blot assay was used to detect apoptosis-related proteins and key signaling pathway protein levels. RESULTS: The optimal incubation time for the maximum absorption of HPD in RPMI 8226 cells was 4 hours. HPD significantly inhibited the proliferation of RPMI 8226 cells in a dose- and illumination time-dependent manner ( r =0.981; r =0.961). Additionally, HPD induced apoptosis in RPMI 8226 cells, but had no significant inhibitory effect on peripheral blood mononuclear cells derived from healthy individuals. HPD combined with illumination treatment significantly increased the intracellular ROS level, upregulated the expression of apoptosis-related proteins such as cleaved PARP, cleaved caspase-3 and Bax, and down-regulated the expression of proteins that maintain cell survival, such as NF-κB and Akt. CONCLUSION The HPD can inhibit the proliferation and induce apoptosis of multiple myeloma cells.
Humans ; Multiple Myeloma/pathology* ; Hematoporphyrins/pharmacology* ; Apoptosis/drug effects* ; Cell Line, Tumor ; Reactive Oxygen Species/metabolism* ; Cell Proliferation/drug effects* ; Photochemotherapy ; Cell Survival/drug effects* ; Signal Transduction

OBJECTIVE: To evaluate the efficacy of shockwave therapy, acupuncture, hyperthermia, biofeedback therapy, electrical nerve stimulation, magnetotherapy and ultrasound therapy in the treatment of chronic prostatitis/chronic pelvic pain syndrome(CP/CPPS), and to provide evidence-based support for clinical decision-making. METHODS: Two researchers independently searched PubMed, Web of Science, Embase, Cochrane Library, CNKI, Wanfang, VIP and Chinese Biomedical Literature databases for randomized controlled trials(RCTs) on the effects of different interventions on CP/CPPS from the establishment of the databases to August 2024. We evaluated the quality of the included literature and extracted the relevant data according to the Cochrane Handbook for Systematic Reviews of Interventions, followed by network meta-analysis using Revman 5.3, R 4.33 and Stata17 software. RESULTS: A total of 25 RCTs involving 1 794 cases were included. The results of network meta-analysis showed that electrical nerve stimulation, shockwave therapy, biofeedback therapy, magnetotherapy, ultrasound therapy and acupuncture were significantly superior to conventional medication and placebo in the total NIH-CPSI scores(P< 0.05), and so were electrical nerve stimulation and shockwave therapy to acupuncture and hyperthermia(P< 0.05), magnetic therapy to hyperthermia, and ultrasound therapy to placebo(P< 0.05). Shockwave therapy, biofeedback therapy, electrical nerve stimulation, magnetotherapy and ultrasound therapy achieved remarkably better clinical efficacy than conventional medication and placebo in the treatment of CP/CPPS, and so did shockwave therapy than electrical nerve stimulation, hyperthermia, ultrasonic therapy, magnetotherapy and acupuncture. CONCLUSION For the treatment of CP/CPPS, electrical nerve stimulation is advantageous over the other interventions in improving total NIH-CPSI scores, and shockwave therapy is advantageous in relieving pain symptoms and clinical efficacy. This conclusion, however, needs to be further verified by more high-quality clinical studies.
Humans ; Acupuncture Therapy ; Biofeedback, Psychology ; Chronic Disease ; Electric Stimulation Therapy ; Extracorporeal Shockwave Therapy ; Magnetic Field Therapy ; Pelvic Pain/therapy* ; Prostatitis/therapy* ; Randomized Controlled Trials as Topic ; Ultrasonic Therapy

BACKGROUND: The association between uric acid-albumin ratio (UAR) with different diseases has been evaluated before. However, the association between UAR with spontaneous reperfusion (SR) in patients with ST-segment elevation myocardial infarction (STEMI) has not been explored. METHODS: STEMI patients admitted to our department and underwent primary coronary angiography between 1^st November 2018 and 31^st December 2020 were retrospectively enrolled. The patients were divided into the SR group and the non-SR group according to the index coronary angiography results. The association between UAR and SR was evaluated by uni-variable and multi-variable logistic analysis. Receiver operating characteristic curve analysis was used to determine the optimum cut-off level of UAR in predicting SR. RESULTS: Three hundred and fifty-seven patients were finally enrolled in our study, 55 patients were divided into the SR group and 302 patients were divided into the non-SR group. In uni-variable analysis, patients with SR were older (P = 0.032), with higher red blood cell distribution width (P < 0.001) and red blood cell distribution width-to-platelet ratio (P < 0.001), higher level of C-reactive protein (P = 0.046), higher level of uric acid (P < 0.001) compared with patients without SR. Patients with SR had a lower level of platelets (P = 0.008), lower level of on-admission B-type natriuretic peptide (P < 0.001). As for the level of UAR, STEMI patients with SR had significantly higher levels of UAR compared with STEMI patients without SR [11.1 (8.9-13.4) vs. 8.3 (6.6-10.0), P < 0.001]. Further multi-variable logistic analysis reveals that UAR was the independent risk factor of SR in different models after adjusting different variables. Receiver operating characteristic analysis showed that UAR had good predictive value in SR (AUC = 0.75, 95% CI: 0.702-0.794, P < 0.01). CONCLUSIONS Our study shows that UAR is an independent risk factor for predicting SR in STEMI patients.

OBJECTIVE: To investigate the mechanism of electroacupuncture (EA) in treating diabetic gastroparesis (DGP) by inhibiting the activation of Nod-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome and pyroptosis mediated via NLRP3/cysteinyl aspartate specific proteinase-1 (caspase-1)/gasdermin D (GSDMD) signaling pathway. METHODS: Forty Sprague-Dawley rats were randomly divided into 4 groups including the control, DGP model, EA, and MCC950 groups. The DGP model was established by a one-time high-dose intraperitoneal injection of 2% streptozotocin and a high-glucose and high-fat diet for 8 weeks. EA intervention was conducted at Zusanli (ST 36), Liangmen (ST 21) and Sanyinjiao (SP 6) with sparse-dense wave for 15 min, and was administered for 3 courses of 5 days. After intervention, the blood glucose, urine glucose, gastric emptying, and intestinal propulsive rate were observed. Besides, HE staining was used to observe histopathological changes in gastric antrum tissues, and TUNEL staining was utilized to detect DNA damage. Protein expression levels of NLRP3, apoptosis-associated speck-like protein containing CARD (ASC), pro-caspase-1, caspase-1 and GSDMD were measured by Western blot. Immunofluorescence staining was employed to assess the activity of GSDMD-N. Lactate dehydrogenase (LDH) levels were detected by using a biochemical kit. RESULTS: DGP rats showed persistent hyperglycemia and a significant decrease in gastrointestinal motility (P<0.05 or P<0.01), accompanied by pathological damage in their gastric antrum tissues. Cellular DNA was obviously damaged, and the expressions of NLRP3, ASC, pro-caspase-1, caspase-1 and GSDMD proteins were significantly elevated, along with enhanced fluorescence signals of GSDMD-N and increased LDH release (P<0.01). EA mitigated hyperglycemia, improved gastrointestinal motility in DGP rats and alleviated their pathological injury (P<0.05). Furthermore, EA reduced cellular DNA damage, lowered the protein levels of NLRP3, ASC, pro-caspase-1, caspase-1 and GSDMD, suppressed GSDMD-N activity, and decreased LDH release (P<0.05 or P<0.01), demonstrating effects comparable to MCC950. CONCLUSION EA promotes gastrointestinal motility and repairs the pathological damage in DGP rats, and its mechanism may be related to the inhibition of NLRP3 inflammasome and pyroptosis mediated by NLRP3/caspase-1/GSDMD pathway.
Animals ; Electroacupuncture ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Pyroptosis ; Rats, Sprague-Dawley ; Caspase 1/metabolism* ; Gastroparesis/physiopathology* ; Signal Transduction ; Male ; Diabetes Mellitus, Experimental/physiopathology* ; Phosphate-Binding Proteins/metabolism* ; Gastrointestinal Motility ; Rats ; Intracellular Signaling Peptides and Proteins/metabolism* ; Diabetes Complications/physiopathology* ; Gasdermins

OBJECTIVE: To evaluate the efficacy and safety of Shexiang Tongxin Dropping Pill (STDP) in treating stable angina patients with phlegm-heat and blood-stasis syndrome by exercise duration and metabolic equivalents. METHODS: This multicenter, randomized, double-blind, placebo-controlled clinical trial enrolled stable angina patients with phlegm-heat and blood-stasis syndrome from 22 hospitals. They were randomized 1:1 to STDP (35 mg/pill, 6 pills per day) or placebo for 56 days. The primary outcome was the exercise duration and metabolic equivalents (METs) assessed by the standard Bruce exercise treadmill test after 56 days of treatment. The secondary outcomes included the total angina symptom score, Chinese medicine (CM) symptom scores, Seattle Angina Questionnaire (SAQ) scores, changes in ST-T on electrocardiogram and adverse events (AEs). RESULTS: This trial enrolled 309 patients, including 155 and 154 in the STDP and placebo groups, respectively. STDP significantly prolonged exercise duration with an increase of 51.0 s, compared to a decrease of 12.0 s with placebo (change rate: -11.1% vs. 3.2%, P<0.01). The increase in METs was significantly greater in the STDP group than in the placebo group (change: -0.4 vs. 0.0, change rate: -5.0% vs. 0.0%, P<0.01). The improvement of total angina symptom scores (25.0% vs. 0.0%), CM symptom scores (38.7% vs. 11.8%), reduction of nitroglycerin consumption (100.0% vs. 11.3%), and all domains of SAQ, were significantly greater with STDP than placebo (all P<0.01). The changes in Q-T intervals at 28 and 56 days from baseline were similar between the two groups (both P>0.05). Twenty-five participants (16.3%) with STDP and 16 (10.5%) with placebo experienced AEs (P=0.131), with no serious AEs observed. CONCLUSION STDP could improve exercise tolerance in patients with stable angina and phlegm-heat and blood stasis syndrome, with a favorable safety profile. (Registration No. ChiCTR-IPR-15006020).
Humans ; Double-Blind Method ; Drugs, Chinese Herbal/adverse effects* ; Male ; Female ; Middle Aged ; Angina, Stable/physiopathology* ; Aged ; Syndrome ; Treatment Outcome ; Placebos ; Tablets