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.

OBJECTIVES: To evaluate the clinical utility and translational potential of a remote jaundice monitoring system for home-based screening of neonatal hyperbilirubinemia. METHODS: A prospective self-controlled study was conducted, enrolling 538 newborns with gestational age ≥35 weeks, birth weight ≥2 000 g, and postnatal age ≤14 days at the Women's Hospital of Nanjing Medical University from March to October 2023. Four screening protocols with different predictive indicators were developed based on the Chinese Neonatal Transcutaneous Hourly Bilirubin Nomogram. The effectiveness of the system was evaluated, and the feasibility of using the remote jaundice monitoring system in actual home settings was analyzed. RESULTS: A total of 538 paired transcutaneous bilirubin (TcB) and total serum bilirubin (TSB) measurements showed a strong correlation (r=0.85, P<0.001), with 95.0% (511/538) of samples within the 95% limits of agreement. Using TcB ≥ the 95th percentile as the predictive indicator, the system achieved 100% sensitivity, 46.2% specificity, and an area under the receiver operating characteristic curve of 0.731 (95%CI: 0.682-0.780). This approach could reduce unnecessary hospital visits by 41.4% (221/538). CONCLUSIONS The system integrates the QBH-801 transcutaneous bilirubinometer, intelligent early warning, and remote guidance services, establishing a closed-loop "hospital-to-home" management model. It demonstrates high safety and feasibility, with significant clinical translational value.
Humans ; Infant, Newborn ; Female ; Male ; Bilirubin/blood* ; Feasibility Studies ; Prospective Studies ; Hyperbilirubinemia, Neonatal/diagnosis* ; Neonatal Screening/methods* ; Jaundice, Neonatal/diagnosis*

OBJECTIVE: To explore the clinical characteristics and prognosis of children with SIL-TAL1-positive T-cell acute lymphoblastic leukemia ( SIL-TAL1⁺ T-ALL). METHODS: The clinical data of 110 children with newly diagnosed T-ALL admitted to the pediatric department of our hospital from January 2010 to December 2018 were reviewed to compare the clinical characteristics, treatment response and prognosis between SIL-TAL1⁺ group and SIL-TAL1^-group. RESULTS: Among the 110 children with T-ALL, 25 cases (22.7%) were in the SIL-TAL1⁺ group and 85 cases (77.3%) in the SIL-TAL1^- group. The white blood cell (WBC) count in the SIL-TAL1⁺ group was significantly higher than that in the SIL-TAL1^- group (P < 0.05), while the other clinical characteristics and treatment response were not significantly different between the two groups. The 5-year overall survival (OS) rates of SIL-TAL1⁺ group and SIL-TAL1^- group were 80.0% and 75.5%, and 5-year disease-free survival (DFS) rates were 76.0% and 72.9%, respectively. There were no significant differences in OS rate and DFS rate between the two groups ( P >0.05). In children aged < 10 years, the 5-year OS rate of SIL-TAL1⁺ group and SIL-TAL1^- group was 100% and 75.1%, respectively, and the difference between the two groups was statistically significant (P < 0.05). CONCLUSION Although the WBC level is significantly higher in children with SIL-TAL1⁺ T-ALL than that in those with SIL-TAL1^- T-ALL, the treatment efficacy is similar between the two groups. In children aged < 10 years, the longterm survival rate is superior in the SIL-TAL1⁺ group.
Humans ; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/diagnosis* ; Prognosis ; Child ; Male ; Female ; Survival Rate ; T-Cell Acute Lymphocytic Leukemia Protein 1 ; Child, Preschool ; Oncogene Proteins, Fusion ; Leukocyte Count

OBJECTIVE: To evaluate the dynamic changes of glucocorticoid (GC) dose and the feasibility of GC discontinuation in rheumatoid arthritis (RA) patients under the background of Chinese medicine (CM). METHODS: This multicenter retrospective cohort study included 1,196 RA patients enrolled in the China Rheumatoid Arthritis Registry of Patients with Chinese Medicine (CERTAIN) from September 1, 2019 to December 4, 2023, who initiated GC therapy. Participants were divided into the Western medicine (WM) and integrative medicine (IM, combination of CM and WM) groups based on medication regimen. Follow-up was performed at least every 3 months to assess dynamic changes in GC dose. Changes in GC dose were analyzed by generalized estimator equation, the probability of GC discontinuation was assessed using Kaplan-Meier curve, and predictors of GC discontinuation were analyzed by Cox regression. Patients with <12 months of follow-up were excluded for the sensitivity analysis. RESULTS: Among 1,196 patients (85.4% female; median age 56.4 years), 880 (73.6%) received IM. Over a median 12-month follow-up, 34.3% (410 cases) discontinued GC, with significantly higher rates in the IM group (40.8% vs. 16.1% in WM; P<0.05). GC dose declined progressively, with IM patients demonstrating faster reductions (median 3.75 mg vs. 5.00 mg in WM at 12 months; P<0.05). Multivariate Cox analysis identified age <60 years [P<0.001, hazard ratios (HR)=2.142, 95% confidence interval (CI): 1.523-3.012], IM therapy (P=0.001, HR=2.175, 95% CI: 1.369-3.456), baseline GC dose ⩽7.5 mg (P=0.003, HR=1.637, 95% CI: 1.177-2.275), and absence of non-steroidal anti-inflammatory drugs use (P=0.001, HR=2.546, 95% CI: 1.432-4.527) as significant predictors of GC discontinuation. Sensitivity analysis (545 cases) confirmed these findings. CONCLUSIONS RA patients receiving CM face difficulties in following guideline-recommended GC discontinuation protocols. IM can promote GC discontinuation and is a promising strategy to reduce GC dependency in RA management. (Trial registration: ClinicalTrials.gov, No. NCT05219214).
Adult ; Aged ; Female ; Humans ; Male ; Middle Aged ; Arthritis, Rheumatoid/drug therapy* ; Glucocorticoids/therapeutic use* ; Medicine, Chinese Traditional ; Retrospective Studies

This study explores the effect of total secondary ginsenosides(TSG) on apoptosis and energy metabolism in H9c2 cells under hypoxia and its potential mechanisms. H9c2 cell viability was observed and the apoptosis rate was calculated to determine suitable intervention concentrations of TSG, antimycin A complex(AMA), and coenzyme Q10(CoQ10), along with the duration of hypoxia. H9c2 cells at the logarithmic phase were divided into a normal group, a model group, a TSG group, an AMA group, a TSG+AMA group, and a CoQ10 group. All groups, except the normal group, were treated with their respective intervention drugs and cultured under hypoxic conditions. Adenosine triphosphate(ATP) content and creatine kinase(CK) activity were measured using an ATP chemiluminescence assay kit and a CK colorimetric assay kit. Flow cytometry was used to assess apoptosis rates, and Western blot evaluated the expression levels of apoptosis-related proteins, including B-cell lymphoma 2(Bcl-2), Bcl-2-associated X protein(Bax), cysteinyl aspartate-specific protease(caspase)-3, caspase-8, and caspase-9, as well as mitochondrial biogenesis-related proteins peroxisome proliferator-activated receptor-γ coactivator 1α(PGC-1α), estrogen-related receptor-α(ERRα), nuclear respiratory factor(NRF)-1, NRF-2, peroxisome proliferator activated receptor-α(PPARα), and Na~+-K~+-ATPase. RT-PCR was employed to analyze the mRNA expression of mitochondrial biogenesis factors, including PGC-1α, ERRα, NRF-1, NRF-2, PPARα, mitochondrial transcription factor A(TFAM), mitochondrial cytochrome C oxidase 1(COX1), and mitochondrial NADH dehydrogenase subunit 1(ND1), ND2. The selected intervention concentrations were 7.5 μg·mL~(-1) for TSG, 10 μmol·L~(-1) for AMA, and 1×10~(-4) mol·L~(-1) for CoQ10, with a hypoxia duration of 6 h. Compared with the normal group, the model group showed decreased ATP content and CK activity, increased apoptosis rates, decreased Bcl-2 expression, and increased Bax, caspase-3, caspase-8, and caspase-9 expression in H9c2 cells. Additionally, the protein and mRNA expression levels of mitochondrial biogenesis-related factors(PGC-1α, ERRα, NRF-1, NRF-2, PPARα), mRNA expression of TFAM, COX1, and ND1, ND2, and protein expression of Na~+-K~+-ATPase in mitochondrial DNA, were also reduced. In the TSG and CoQ10 groups, ATP content and CK activity increased, and apoptosis rates decreased compared with those in the model group. The TSG group showed decreased protein expression of apoptosis-related proteins Bax, caspase-3, caspase-8, and caspase-9, increased protein and mRNA expression of mitochondrial biogenesis factors PGC-1α, ERRα, NRF-1, and PPARα, and increased NRF-2 protein expression and TFAM mRNA expression in mitochondrial DNA. Conversely, in the AMA group, ATP content and CK activity decreased, the apoptosis rate increased, Bcl-2 expression decreased, and Bax, caspase-3, caspase-8, and caspase-9 expression increased, alongside reductions in PGC-1α, ERRα, NRF-1, NRF-2, PPARα protein and mRNA expression, as well as TFAM, COX1, ND1, ND2 mRNA expression and Na~+-K~+-ATPase protein expression. Compared with the TSG group, the TSG+AMA group exhibited decreased ATP content and CK activity, increased apoptosis rates, decreased Bcl-2 expression, and increased Bax, caspase-3, caspase-8, and caspase-9 expression, along with decreased PGC-1α, ERRα, NRF-1, NRF-2, and PPARα protein and mRNA expression and TFAM, COX1, and ND1, ND2 mRNA expression. Compared with the AMA group, the TSG+AMA group showed increased CK activity, decreased apoptosis rate, increased Bcl-2 expression, and decreased Bax, caspase-8, and caspase-9 expression. Additionally, the protein and mRNA expression of PGC-1α, ERRα, NRF-1, PPARα, mRNA expression of TFAM, COX1, ND1, ND2, and Na~+-K~+-ATPase protein expression increased. In conclusion, TSG enhance ATP content and CK activity and inhibit apoptosis in H9c2 cells under hypoxia, and the mechanisms may be related to the regulation of PGC-1α, ERRα, NRF-1, NRF-2, PPARα, and TFAM expression, thus promoting mitochondrial biogenesis.
Apoptosis/drug effects* ; Ginsenosides/pharmacology* ; Energy Metabolism/drug effects* ; Mitochondria/metabolism* ; Animals ; Rats ; Cell Line ; Cell Hypoxia/drug effects* ; Organelle Biogenesis ; Adenosine Triphosphate/metabolism* ; Humans ; Cell Survival/drug effects*

Objective To explore the regulatory effect of moderate intensity aerobic exercise on glu-cose and lipid metabolism disordersafter immune stimulation in high-fat diet induced insulin resistance(IR)mice by inducing tolerance of trained immunity.Methods Eighteen of 70 male C57BL/6 male mice were randomly selected as the control group(CON group),while the remaining mice were subjected to high-fat diet intervention(HFD group)for 8 weeks.After modeling,six of the CON group and HFD group were measured their glucose and lipid metabolism and inflammation levels.The remaining mice in the HFD group were randomly divided into HFD sedentary group(HS group)and HFD exer-cise group(HE group),each of 16.The HE groups underwent a daily 60-minute running on a hori-zontal treadmill at 12 m/min,5 days a week for 8 weeks.After that,the CON,HS and HE groups were randomly divided into an immune stimulation group(lipopolysaccharides,LPS group)and a con-trol group(phosphate buffered saline,PBS group),with 6 in CON-PBS and CON-LPS groups,and 8 in HS-PBS,HS-LPS,HE-PBS and HE-LPS groups.The LPS group received intraperitoneal injec-tion of LPS as an immune stimulus,and were taken samples 24 hours after intervention.During the intervention period,the body weight,food intake,fasting blood glucose(FBG),glucose tolerance,and insulin tolerance were tested for all groups.Then,the serum blood lipid level was tested.More-over,the levels of tumor necrosis factor-α(TNF-α),interleukin-1 β(IL-1β)and interleukin-10(IL-10)in serum were detected by using the enzyme-linked immunosorbent assay,while the protein expression of IL-1β,IL-10,induciblenitricoxidesynthase(iNOS)andarginase-1(Arg-1)were measured by using Western Blot.Results(1)After 8 weeks of high-fat diet intervention,compared with the CON group,there was a significant increase in the body weight,food intake,and FBG,the level of TNF-α and IL-1β in serum and expressions of IL-1β and iNOS in liver,skeletal muscle and adipose tissue,but a significant decrease in glucose and insulin tolerance,the level of anti-in-flammatory factor IL-10,as well as IL-10 and Arg-1 in liver and Arg-1 in adipose tissues in group HFD(P<0.01,P<0.05).(2)After 8 weeks of diet intervention,the body weight of group HS-PBS was still significantly higher than group CON-PBS(P<0.01).However,no significant differences were found between group CON-PBS and HS-PBS in other indicators(P>0.05).After LPS intervention,the food intake of both CON-LPS and HS-LPS groups was significantly lower than the corresponding PBS group(P<0.05).However,the levels of FBG,TG,TC,LDL-C,TNF-α and IL-1β in serum,IL-1β and iNOS in liver and adipose tissues,and iNOS in skeletal muscles of group HS-LPS were signif-icantly higher compared with group HS-PBS and CON-LPS(P<0.05).Meanwhile,the levels of IL-10 in serum,skeletal muscles and adipose tissues,and Arg-1 in skeletal muscles and adipose tissues of group HS-LPS decreased significantly compared with group HS-PBS and CON-LPS(P<0.05).(3)Af-ter simultaneous aerobic exercise intervention,there were no significant differences between group HE-LPS and HE-PBS in FBG,TC,LDL-C,TNF-α,IL-1β and IL-10 in serum,as well as IL-1β,iN-OS and IL-10 in the liver,skeletal muscles and adipose tissues(P>0.05).Moreover,thelevels of FBG,TG,TC,TNF-α,IL-1β in serum,and IL-1β and iNOS in the liver,skeletal muscle and adi-pose tissue decreased significantly in group HE-LPS compared with group HS-LPS(P<0.05).Howev-er,the levels of IL-10 in the serum and liver,and IL-10 and Arg-1 in skeletal muscles and adi-pose tissues increased significantly in group HE-LPS compared with group HS-LPS(P<0.05).Conclu-sion Eight-week aerobic exercise can effectively relieve the glucose and lipid metabolism disorder after immune stimulation in IR mice,which may be related to the stimulation of immunologic tolerance in innate immune cells,thereby reducing the excessive inflammatory response caused by secondary im-mune stimulation.

To evaluate the immunogenicity and immunoprotective effects of a tRNA thiouridylase TgMnmA deletion strain of Toxoplasma gondii(T.gondii)on ICR mice,we constructed a mouse model immunized with RH△MnmA.Mice were immunized with 10 RH△MnmA tachyzoites by in-traperitoneal injection.After 30 d,indirect ELISA was used to detect the specific IgG antibody and its subtypes of immunized mice.Spleen lymphocyte suspension was prepared,and the splenic lym-phocyte subsets were analyzed by flow cytometry.Moreover,the relative expression level of cyto-kine mRNA was detected by real-time fluorescence quantitative PCR.After 30 d of immunization,mice were intraperitoneally inoculated with RH△ku80 tachyzoites.At 5 d post infection,the para-site load in the ascites,heart,liver and brain of mice was measured,and the survival of mice within 30 d after infection was observed and recorded.The results showed that compared with the control PBS group,RH△MnmA immunized group produced higher level of IgG and IgG2a antibodies,higher mRNA relative expression level of cytokines IL-2,IL-4,IL-6,IL-10,IL-12 and IFN-γ,and the number of CD4+and CD8a+in spleen lymphocytes also increased significantly.Mean-while,for the attack of RH△ku80 strain,the immune group can effectively reduce the parasite load in the ascites and some tissues,inhibit the reproduction of parasites In vivo,and significantly improve the survival rate of mice.The results of this study showed that the TgMnmA deletion strain of T.gondii can induce strong humoral and cellular immune responses in mice,and provide good immune protection against the infection of RHΔku80 strain,which has the potential to be-come a potentially promising live attenuated vaccine candidate against T.gondii.

Objective To investigate the distribution and antimicrobial resistance profiles of common pathogens isolated from cerebrospinal fluid(CSF)in CHINET program from 2015 to 2021.Methods The bacterial strains isolated from CSF were identified in accordance with clinical microbiology practice standards.Antimicrobial susceptibility test was conducted using Kirby-Bauer method and automated systems per the unified CHINET protocol.Results A total of 14 014 bacterial strains were isolated from CSF samples from 2015 to 2021,including the strains isolated from inpatients(95.3％)and from outpatient and emergency care patients(4.7％).Overall,19.6％of the isolates were from children and 80.4％were from adults.Gram-positive and Gram-negative bacteria accounted for 68.0％and 32.0％,respectively.Coagulase negative Staphylococcus accounted for 73.0％of the total Gram-positive bacterial isolates.The prevalence of MRSA was 38.2％in children and 45.6％in adults.The prevalence of MRCNS was 67.6％in adults and 69.5％in children.A small number of vancomycin-resistant Enterococcus faecium(2.2％)and linezolid-resistant Enterococcus faecalis(3.1％)were isolated from adult patients.The resistance rates of Escherichia coli and Klebsiella pneumoniae to ceftriaxone were 52.2％and 76.4％in children,70.5％and 63.5％in adults.The prevalence of carbapenem-resistant E.coli and K.pneumoniae(CRKP)was 1.3％and 47.7％in children,6.4％and 47.9％in adults.The prevalence of carbapenem-resistant Acinetobacter baumannii(CRAB)and Pseudomonas aeruginosa(CRPA)was 74.0％and 37.1％in children,81.7％and 39.9％in adults.Conclusions The data derived from antimicrobial resistance surveillance are crucial for clinicians to make evidence-based decisions regarding antibiotic therapy.Attention should be paid to the Gram-negative bacteria,especially CRKP and CRAB in central nervous system(CNS)infections.Ongoing antimicrobial resistance surveillance is helpful for optimizing antibiotic use in CNS infections.

Objective To investigate the antimicrobial resistance of clinical isolates from elderly patients(≥65 years)in major medical institutions across China.Methods Bacterial strains were isolated from elderly patients in 52 hospitals participating in the CHINET Antimicrobial Resistance Surveillance Program during the period from 2015 to 2021.Antimicrobial susceptibility test was carried out by disk diffusion method and automated systems according to the same CHINET protocol.The data were interpreted in accordance with the breakpoints recommended by the Clinical and Laboratory Standards Institute(CLSI)in 2021.Results A total of 514 715 nonduplicate clinical isolates were collected from elderly patients in 52 hospitals from January 1,2015 to December 31,2021.The number of isolates accounted for 34.3％of the total number of clinical isolates from all patients.Overall,21.8％of the 514 715 strains were gram-positive bacteria,and 78.2％were gram-negative bacteria.Majority(90.9％)of the strains were isolated from inpatients.About 42.9％of the strains were isolated from respiratory specimens,and 22.9％were isolated from urine.More than half(60.7％)of the strains were isolated from male patients,and 39.3％isolated from females.About 51.1％of the strains were isolated from patients aged 65-＜75 years.The prevalence of methicillin-resistant strains(MRSA)was 38.8％in 32 190 strains of Staphylococcus aureus.No vancomycin-or linezolid-resistant strains were found.The resistance rate of E.faecalis to most antibiotics was significantly lower than that of Enterococcus faecium,but a few vancomycin-resistant strains(0.2％,1.5％)and linezolid-resistant strains(3.4％,0.3％)were found in E.faecalis and E.faecium.The prevalence of penicillin-susceptible S.pneumoniae(PSSP),penicillin-intermediate S.pneumoniae(PISP),and penicillin-resistant S.pneumoniae(PRSP)was 94.3％,4.0％,and 1.7％in nonmeningitis S.pneumoniae isolates.The resistance rates of Klebsiella spp.(Klebsiella pneumoniae 93.2％)to imipenem and meropenem were 20.9％and 22.3％,respectively.Other Enterobacterales species were highly sensitive to carbapenem antibiotics.Only 1.7％-7.8％of other Enterobacterales strains were resistant to carbapenems.The resistance rates of Acinetobacter spp.(Acinetobacter baumannii 90.6％)to imipenem and meropenem were 68.4％and 70.6％respectively,while 28.5％and 24.3％of P.aeruginosa strains were resistant to imipenem and meropenem,respectively.Conclusions The number of clinical isolates from elderly patients is increasing year by year,especially in the 65-＜75 age group.Respiratory tract isolates were more prevalent in male elderly patients,and urinary tract isolates were more prevalent in female elderly patients.Klebsiella isolates were increasingly resistant to multiple antimicrobial agents,especially carbapenems.Antimicrobial resistance surveillance is helpful for accurate empirical antimicrobial therapy in elderly patients.

BACKGROUND:Spinal cord injury often leads to neurogenic bladder with hyperreflexia of the forced urethral muscle,but there is a lack of clear understanding of its pathogenesis and treatment,and establishing a stable and reliable animal model has an important impact on revealing its pathomechanisms and exploring therapeutic approaches.OBJECTIVE:To investigate the effect of the number of times to urinate on neurogenic model rats after complete spinal cord transection in order to improve the postoperative survival and modeling rate of neurogenic model rats.METHODS:Out of 46 female Sprague-Dawley rats,6 were selected as the sham-operated group using the random number table method,and the remaining 40 rats were randomly divided into 0,1,3,and 5 times daily urination groups after complete spinal cord transection modeling,with 10 rats in each group.The residual urine volume was measured every 3 days within 19 postoperative days,and the survival and modeling were observed on the 19th day after the operation,and urodynamics tests and contraction experiments of isolated forced urethra muscle strips were performed.RESULTS AND CONCLUSION:(1)Survival and modeling rate:there was 10%survival rate and 10%modeling rate in the group of 0 times daily urination;20%survival rate and 10%modeling rate in the group of 1 time daily urination;70%survival rate and 70%modeling rate in the groups of 3 and 5 times daily urination.(2)Residual urine volume:compared with the sham-operated group,the residual urine volume of the groups of 3 and 5 times daily urination was significant increased on postoperative days 3,6,9,12,and 15(P<0.01);the residual urine volume of the groups of 3 and 5 times daily urination was increased on the 18th day after surgery(P<0.05).Compared with the 3 times daily urination group,the residual urine volume was decreased in the 5 times daily urination group on the 6th day after surgery(P<0.05),while there was no significant difference in the residual urine volume between the 3 and 5 times daily urination groups on the 3rd,9th,12th,15th,and 18th days after surgery.(3)Urodynamics:Compared with the sham-operated group,the differential pressure at the point of leakage was significantly reduced(P<0.01)and the maximal volume was significantly increased(P<0.01)in the 3 and 5 times daily urination groups.There was no statistical difference in the differential pressure at the point of leakage and the maximal volume between the 3 and 5 times daily urination groups.(4)Muscle-strip contraction test of isolated detrusor muscles:Compared with the sham-operated group,the contraction amplitude and frequency of detrusor muscle strips were significantly reduced in the 3 and 5 times daily urination groups(P<0.01).There was no statistically significant difference in the contraction amplitude and frequency of detrusor muscle strips between the 3 and 5 times daily-urination groups.In conclusion,assisted urination is one of the keys to establish a successful neurogenic model of urethral reflexes,and there is no significant difference in the effects of urinating 3 or 5 times a day on the neurogenic model.It is recommended to urinate at least 3 times a day based on the actual workload and the modeling rate.