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.

Important forensic diagnostic indicators of sudden death in coronary atherosclerotic heart dis-ease,such as acute or chronic myocardial ischemic changes,sometimes make it difficult to locate the ischemic site due to the short death process,the lack of tissue reaction time.In some cases,the de-ceased died of sudden death on the first-episode,resulting in difficulty for medical examiners to make an accurate diagnosis.However,clinical studies on coronary instability plaque revealed the key role of coronary spasm and thrombosis caused by their lesions in sudden coronary death process.This paper mainly summarizes the pathological characteristics of unstable coronary plaque based on clinical medi-cal research,including plaque rupture,plaque erosion and calcified nodules,as well as the influencing factors leading to plaque instability,and briefly describes the research progress and technique of the atherosclerotic plaques,in order to improve the study on the mechanism of sudden coronary death and improve the accuracy of the forensic diagnosis of sudden coronary death by diagnosing different patho-logic states of coronary atherosclerotic plaques.

Microplastics(MPs)and nanoplastics(NPs)have become hazardous materials due to the massive amount of plastic waste and disposable masks,but their specific health effects remain uncertain.In this study,fluorescence-labeled polystyrene NPs(PS-NPs)were injected into the circulatory systems of mice to determine the distribution and potential toxic effects of NPs in vivo.Interestingly,whole-body imaging found that PS-NPs accumulated in the testes of mice.Therefore,the toxic effects of PS-NPs on the reproduction systems and the spermatocytes cell line of male mice,and their mechanisms,were investigated.After oral exposure to PS-NPs,their spermatogenesis was affected and the spermatogenic cells were damaged.The spermatocyte cell line GC-2 was exposed to PS-NPs and analyzed using RNA sequencing(RNA-seq)to determine the toxic mechanisms;a ferroptosis pathway was found after PS-NP exposure.The phenomena and indicators of ferroptosis were then determined and verified by ferroptosis inhibitor ferrostatin-1(Fer-1),and it was also found that nuclear factor erythroid 2-related factor 2(Nrf2)played an important role in spermatogenic cell ferroptosis induced by PS-NPs.Finally,it was confirmed in vivo that this mechanism of Nrf2 played a protective role in PS-NPs-induced male reproductive toxicity.This study demonstrated that PS-NPs induce male reproductive dysfunction in mice by causing spermatogenic cell ferroptosis dependent on Nrf2.

Objective:To establishment an assay for HIV-1 intact proviral DNA assay through droplet digital PCR (ddPCR).Methods:DNA was extracted by culturing 8E5 cells, a Tlymphocyte cell line containing a single copy of integrated HIV-1 provirus. Serial diluting DNA were prepared by amplified 1-fold, 5-fold, 25-fold, 625-fold, 3 125-fold, and 15 625-fold across the HIV-1 Ψ region, env region, and eukaryotic chromosome 10 RPP30 regions, and the linear relationship was calculated and the minimum detection concentration. DNA solution of 5 μl, 3.1 μl, 2.5 μl was added to the ddPCR mixture respectively, with each dilution undergoing two batches of detection, and each was repeated four times. The intra-batch variation coefficient was detected, while the inter-batch variation coefficient was detected by the same DNA amount and different DNA amounts to determine the stability; 8E5 cell was used to detect the intact proviral content in cells.Results:The linear fitting goodness of Ψ region, env region and RPP30 region are R2≥0.999, R2≥0.993, R2≥0.996 in 6 dilutions of DNA, respectively. At a 3 125-fold dilution, the lowest positive droplets were detected in the Ψ region, env region and RPP30 region were 3, 2 and 2, respectively, the detected concentrations were 2.37 copies/μl, 1.21 copies/μl and 1.58 copies/μl. The ddPCR repeatability experimental detecting DNA showed that the Ψ region of the intra-batch variation coefficients ranged from 0.66% to 3.43%, with the inter-batch variation coefficients of the same DNA at 3.19%, 4.3% and 3.45% respectively, and the inter-batch variation coefficients of the different DNA at only 4.35%. The env region of the intra-batch variation coefficients ranged from 0.7% to 3.20%, with the inter-batch variation coefficients of the same DNA at 3.18%, 4.52% and 3.4% respectively, and the inter-batch variation coefficients of the different DNA at only 4.02%. The RPP30 region of the intra-batch variation coefficients ranged from 0.91% to 2.91%, with the inter-batch variation coefficients of the same DNA at 3%, 4.55% and 3.37% respectively, and the inter-batch variation coefficients of the different DNA at only 3.98%. The proportion of 8E5 cells containing defective provirus and the proportion of intact provirus were calculated to be approximately 90% and 45%, respectively. Conclusions:Droplet digital PCR used to detect HIV-1 intact proviral DNA, showed strong stability and provided a technical means for HIV-1 infection reservoir detection.

Silver nanoparticles(AgNPs)is widely used in biomedicine,daily chemicals,food industry and other fields,and the possible negative health effects of its exposure have attracted widespread attention.Accurate analysis of AgNPs in biological matrices is the basis for biosafety studies of AgNPs.Among the existing analytical techniques,single particle-inductively coupled plasma-mass spectrometry(sp-ICP-MS)has significant advantages such as high sensitivity and simultaneous detection of different forms of silver.However,AgNPs in biological matrices is uniquely highly dynamic and low in content,and the matrix interference is severe,which increases the complexity of the analysis.Although some scholars have reviewed the application of this method for detection of metal nanoparticles in different scenarios,there is a lack of a summary of the quality control and optimization of the whole process from the perspective of AgNPs detection.There is still a lack of reference standards for the sp-ICP-MS analysis of AgNPs in biological matrices,and the existing methods need to be summarized and further optimized to achieve accurate quantification.Therefore,this paper reviewed the recent studies on the analysis of silver-containing nanoparticles in biological matrices based on sp-ICP-MS,mainly included the principles of the technique,the extraction methods of the particles,and the process of data processing,which focused on elaborating and comparing different pre-treatment methods,and explored issues of the current application of sp-ICP-MS for detection of AgNPs in biological tissues and the development of future optimization trends.The current problems of sp-ICP-MS for detection of AgNPs in biological tissues and the future development trend were also discussed.

Objective To investigate the incidence rate,clinical characteristics,and prognosis of neonatal stroke in Shenzhen,China.Methods Led by Shenzhen Children's Hospital,the Shenzhen Neonatal Data Collaboration Network organized 21 institutions to collect 36 cases of neonatal stroke from January 2020 to December 2022.The incidence,clinical characteristics,treatment,and prognosis of neonatal stroke in Shenzhen were analyzed.Results The incidence rate of neonatal stroke in 21 hospitals from 2020 to 2022 was 1/15 137,1/6 060,and 1/7 704,respectively.Ischemic stroke accounted for 75％(27/36);boys accounted for 64％(23/36).Among the 36 neonates,31(86％)had disease onset within 3 days after birth,and 19(53％)had convulsion as the initial presentation.Cerebral MRI showed that 22 neonates(61％)had left cerebral infarction and 13(36％)had basal ganglia infarction.Magnetic resonance angiography was performed for 12 neonates,among whom 9(75％)had involvement of the middle cerebral artery.Electroencephalography was performed for 29 neonates,with sharp waves in 21 neonates(72％)and seizures in 10 neonates(34％).Symptomatic/supportive treatment varied across different hospitals.Neonatal Behavioral Neurological Assessment was performed for 12 neonates(33％,12/36),with a mean score of(32±4)points.The prognosis of 27 neonates was followed up to around 12 months of age,with 44％(12/27)of the neonates having a good prognosis.Conclusions Ischemic stroke is the main type of neonatal stroke,often with convulsions as the initial presentation,involvement of the middle cerebral artery,sharp waves on electroencephalography,and a relatively low neurodevelopment score.Symptomatic/supportive treatment is the main treatment method,and some neonates tend to have a poor prognosis.

Objective To summarize the reasons and management strategies of reoperation after oblique lateral interbody fusion(OLIF),and put forward preventive measures.Methods From October 2015 to December 2019,23 patients who under-went reoperation after OLIF in four spine surgery centers were retrospectively analyzed.There were 9 males and 14 females with an average age of(61.89±8.80)years old ranging from 44 to 81 years old.The index diagnosis was degenerative lumbar intervertebral dics diseases in 3 cases,discogenie low back pain in 1 case,degenerative lumbar spondylolisthesis in 6 cases,lumbar spinal stenosis in 9 cases and degenerative lumbar spinal kyphoscoliosis in 4 cases.Sixteen patients were primarily treated with Stand-alone OLIF procedures and 7 cases were primarily treated with OLIF combined with posterior pedicle screw fixation.There were 17 cases of single fusion segment,2 of 2 fusion segments,4 of 3 fusion segments.All the cases underwent reoperation within 3 months after the initial surgery.The strategies of reoperation included supplementary posterior pedicle screw instrumentation in 16 cases;posterior laminectomy,cage adjustment and neurolysis in 2 cases,arthroplasty and neuroly-sis under endoscope in 1 case,posterior laminectomy and neurolysis in 1 case,pedicle screw adjustment in 1 case,exploration and decompression under percutaneous endoscopic in 1 case,interbody fusion cage and pedicle screw revision in 1 case.Visu-al analogue scale(VAS)and Oswestry disability index(ODI)index were used to evaluate and compare the recovery of low back pain and lumbar function before reoperation and at the last follow-up.During the follow-up process,the phenomenon of fusion cage settlement or re-displacement,as well as the condition of intervertebral fusion,were observed.The changes in in-tervertebral space height before the first operation,after the first operation,before the second operation,3 to 5 days after the second operation,6 months after the second operation,and at the latest follow-up were measured and compared.Results There was no skin necrosis and infection.All patients were followed up from 12 to 48 months with an average of(28.1±7.3)months.Nerve root injury symptoms were relieved within 3 to 6 months.No cage transverse shifting and no dislodgement,loosening or breakage of the instrumentation was observed in any patient during the follow-up period.Though the intervertebral disc height was obviously increased at the first postoperative,there was a rapid loss in the early stage,and still partially lost after reopera-tion.The VAS for back pain recovered from(6.20±1.69)points preoperatively to(1.60±0.71)points postoperatively(P＜0.05).The ODI recovered from(40.60±7.01)％preoperatively to(9.14±2.66)％postoperatively(P＜0.05).Conclusion There is a risk of reoperation due to failure after OLIF surgery.The reasons for reoperation include preoperative bone loss or osteoporosis the initial surgery was performed by Stand-alone,intraoperative endplate injury,significant subsidence of the fusion cage after surgery,postoperative fusion cage displacement,nerve damage,etc.As long as it is discovered in a timely manner and handled properly,further surgery after OLIF surgery can achieve better clinical results,but prevention still needs to be strengthened.

Transcatheter aortic valve replacement(TAVR)has emerged as a promising therapeutic alternative for addressing aortic valve-related pathologies.However,the occurrence of rapid arrhythmias linked to TAVR procedures is progressively drawing scrutiny.Presently,pharmacologic interventions constitute the mainstay of managing atrial arrhythmias related to TAVR,while the potential of ablation as a viable treatment modality remains undefined.Notably,in cases where the arrhythmia's genesis is presumed to be intricately linked to the prosthetic valve,the practicality and safety of ablation procedures remain unverified.Our institution has successfully ventured into radiofrequency ablation for a distinctive patient presenting with this intricate condition,thereby tentatively affirming the efficacy and safety of catheter ablation administered on the surface of prosthetic valves.

Here,5 important pathogens carried by ticks in Maanshan City,Anhui Province,China were identified.In to-tal,642 ticks were collected from 13 villages around Maanshan City and identified by morphological and mitochondrial COI genes.The 16S rRNA gene of Francisella tularensis,ssrA gene of Bartonella,16S rRNA,ompA and ompB genes of Rickett-sia,16S rRNA and gltA genes of Anaplasma,and groEL and rpoB genes of Coxiella were sequenced.Reference sequences were retrieved from a public database.Phylogenetic trees were constructed with MEG A1 1.0 software.In total,36 Rickettsiae isolates were detected in 640 Haemaphysalis longicornis ticks,which included 20 isolates of Rickettsia heilongjian-gensis,16 of Candidatus Rickettsia jingxinensis,2 of Ana-plasma bovis,and 186 of Coxiella-like endosymbiont.R.hei-longjiangensis HY2 detected in this study and Anhui B8 strain,Ca.R.jingxinensis QL3 and those from Shanxi Prov-ince and Jiangsu Province,A.bovis JX4 and those from Shanxi Province were clustered on the same branch.Overall,17 ticks had combined infections and none of the 5 bacteria were detected in two Amblyomma testudinarium ticks.This is the first report of Ca.R.jingxinensis detected in H.longicornis ticks from Anhui Province.It is recommended that the two types of Rickettsia that cause spotted fever and A.bovis should be reported to local health authorities to initiate appropriate prevention and control measures.

Objective To establish an ideal modeling method for diarrhea predominant irritable bowel syndrome(IBS-D)with anxiely and depression in rats,and to provide a basis for the clinical study of IBS-D.Methods 60 rats were used in this study.(1)At first,20 rats were randomly divided into blank,3%acetic acid enema,4%acetic acid enema,and 5%acetic acid enema groups.After the modeling and observation period,the diarrhea status and the degree of colon injury caused by different modeling concentrations were observed by diarrhea related index and colon histopathology.(2)After the optimal modeling concentration was assessed,40 rats were randomly divided into control(a),acetic acid enema(b),acetic acid+binding(c),and acetic acid+binding+tail clip(d)groups and correspondingly treated for 8 days.After the treatments,the general condition,diarrhea-related index,open field test(OFT)score,and colonic histopathology of rats were evaluated.Results(1)Compared with the blank group,the fecal trait score of 4%acetic acid enema group was increased on days 1 to 3 after intervention(P＜0.001),and gradually decreased on days 4 to 7 after intervention.After 1 week,there was no significant difference between the fecal trait score and that of the blank group(P＞0.05).Body weight was lower(P＜0.01),fecal water content was higher(P＜0.001).Compared with blank group,body weight of the 5%acetic acid enema group was decreased(P＜0.001),the fecal trait score and diarrhea index were increased(P＜0.01).No significant difference was found between 3%acetic acid enema and blank groups.The pathological colon tissue showed that,compared with the blank group,the mucosal structure of the 4%acetic acid enema group was complete with a small amount of inflammatory cell infiltration,and the pathological tissue score showed no significant difference(P＞0.05),whereas the 5%acetic acid enema had a medium to large amount of inflammatory cell infiltration,and the pathological tissue score was increased(P＜0.01).(2)Compared with group a,group b had lower body weight(P＜0.001),and higher fecal trait score,fecal water content and diarrhea index(P＜0.01).Compared with a and b groups,the body weight of c and d groups was lower(P＜0.001),the fecal traits score,fecal water content,and diarrhea index were increased(P＜0.01),and the colon running time was decreased(P＜0.01).Compared with group c,Fecal water content in group D was higher(P＜0.001).In the OFT score,compared with a and b groups,the OFT distance,standing times,and upright times in c and d groups were lower(P＜0.05).Compared with c,the OFT distance,standing times,and upright times in d group were lower(P＜0.05).The pathological tissue of colon showed that the mucosal structure of the four groups was complete,and there were different degrees of inflammatory cell infiltration.The pathological tissue scores of groups c and d were higher than those of groups a and b(P＜0.05).Conclusions The 4%acetic acid concentration is appropriate for IBS-D modeling.After superposition and binding,the IBS-D diarrhea and internal hypersensitivity characteristic state can be better simulated.After superposition of a tail clip,the IBS-D model of liver stagnation and spleen deficiency can be established successfully.