Penicillin G acylases (PGAs) are industrially important enzymes primarily used for the synthesis of first- and second-generation cephalosporins or penicillins. This study aims to establish a high-efficiency biosynthetic system for cefradine on the purpose of significantly enhancing its catalytic efficiency in cefradine synthesis and developing its potentials for industrial application. In this study, we identified and engineered penicillin G acylase and obtained a highly active mutant KsPGA M7(M168F/F313G) for the synthesis of cefradine. The mutant achieved a conversion rate over 95% in the scaled-up reaction. To validate its industrial applicability, we immobilized both the wild-type and mutant enzymes and applied them in continuous flow reactions, which achieved a space-time yield of 2 800 g/(L·d). This study lays a foundation for the future applications of penicillin G acylases in the industrial synthesis of cefradine.
Penicillin Amidase/biosynthesis* ; Protein Engineering/methods* ; Cephradine/metabolism* ; Escherichia coli/metabolism* ; Enzymes, Immobilized/metabolism* ; Recombinant Proteins/biosynthesis*

Objective To explore the association between cardiorespiratory fitness and total atherosclerotic burden of brain-and heart-arteries in patients with ischemic stroke.Methods This study retrospectively enrolled consecutive patients with ischemic stroke of the large-artery atherosclerosis or small-artery occlusion subtypes accompanied by cervicocephalic atherosclerosis,who were admitted to the Department of Neurology,Xuanwu Hospital,Capital Medical University,between May 2023 and December 2024.General and clinical data,including age(＜60years,≥60years),sex,admission blood pressure,body mass index,history of hypertension,diabetes mellitus,hyperlipidemia,ischemic stroke,symptomatic coronary artery disease,smoking,alcohol consumption,and medication use(including antiplatelet drugs,anticoagulants,lipid-lowering drugs,antidiabetic drugs,antihypertensive drugs,and β-blockers),characteristics of ischemic stroke(including the duration of onset,ischemic side[left,right,bilateral],and ischemic location[anterior circulation,posterior circulation,bilateral circulation])were collected from all patients enrolled.On the morning after admission,fasting venous blood samples were collected to measure the levels of triglycerides,total cholesterol,low-density lipoprotein cholesterol,high-density lipoprotein cholesterol,fasting blood glucose,glycated hemoglobin,homocysteine,fibrinogen,D-dimer,C-reactive protein,high-sensitivity C-reactive protein,N-terminal pro-B-type natriuretic peptide,creatine kinase-MB,cardiac troponin T,and myoglobin.Transthoracic echocardiography was performed within 7 days after admission to evaluate cardiac function.Left ventricular ejection fraction and cardiac output were recorded,and cardiac index were calculated.Upon admission,neurological impairment,disability,motor function,and balance ability were assessed using the National Institutes of Health stroke scale(NIHSS),modified Rankin scale(mRS),Fugl-Meyer assessment(FMA),and Berg balance scale,respectively.The Saltin-Grimby physical activity level scale was used to evaluate the intensity of habitual physical activity prior to stroke onset.Cardiopulmonary exercise testing was performed within 7 days after admission to assess cardiopulmonary fitness.During the test,peak oxygen uptake(VO2peak),percentage of predicted VO2peak(VO2peak%pred),and metabolic equivalent of task(MET)were recorded.Based on the minimum oxygen uptake required for independent living(15 ml/[kg·min]),VO2peak was classified into low VO2peak(＜15 ml/[kg·min])and high VO2peak(≥15 ml/[kg·min]).With VO2peak%pred＜0.60 as the cutoff,VO2peak%pred was divided into low VO2peak%pred(＜0.60)and high VO2peak%pred(≥0.60).MET were categorized into low MET(＜mean MET)and high MET(≥mean MET)according to the mean value of MET in this study population.Imaging data of cardio-cerebral arteries were obtained by simultaneous CT arteriography within 7 days after admission.Total atherosclerotic burden of brain-and heart-supplying arteries(TAB-BHAs)was used to quantitatively evaluate the overall degree of cardio-cerebral atherosclerosis.And patients were stratified into high TAB-BHAs(≥4 points)and low TAB-BHAs(＜4 points)groups.The differences in general and clinical characteristics,as well as cardiopulmonary fitness indices,were assessed among different groups of TAB-BHAs.Spearman' s correlation was used in the preliminary analysis of the relationship between cardiorespiratory fitness indicators and TAB-BHAs.Multivariate Logistic regression was conducted with VO2peak,low VO2peak%pred,and low METs as independent variables,and high TAB-BHAs as the dependent variable to assess the association between cardiopulmonary fitness and TAB-BHAs.Covariates were selected through regressions stratified by age≥60 years,male,the least absolute shrinkage and selection operator(LASSO),and the variables exhibit statistically significant differences between the low and high TAB-BHAs groups,respectively.Results A total of 104 patients with ischemic stroke were enrolled.Among all patients enrolled,60patients(with ages ranging from 30 to 72years and a mean age of[54±11]years)met the inclusion and exclusion criteria,and were ultimately included.Amid the 60 patients,52 were male and 8 were female.There were 20 patients(33.3%)with low VO2peak,28 patients(46.7%)with low VO2peak%pred,and 31 patients(51.7%)with low MET(MET＜5.0).45 patients were classified into the low TAB-BHAs group and 15 into the high TAB-BHAs group.(1)Compared with the low TAB-BHAs group,patients in the high TAB-BHAs group were significantly older and had a higher proportion of patients aged≥60 years,they also showed significantly higher fibrinogen levels and a history of antihypertensive drug use(all P＜0.05).No significant differences were observed in other general or clinical characteristics between the two groups(all P＞0.05).Regarding cardiopulmonary fitness parameters,patients in the high TAB-BHAs group had lower VO2peak and MET levels,and higher proportions of patients with low VO2peak,low VO2peak%pred,and low MET compared with those in the low TAB-BHAs group(all P＜0.05).(2)Based on the Spearman's correlation analysis,VO2peak(r=-0.298,P=0.021),VO2peak%pred(r=-0.305,P=0.018),MET(r=-0.303,P=0.018)all exhibited negative correlations between and TAB-BHAs.(3)LASSO regression was used to identify the most pertinent variables for high TAB-BHAs,including low VO2peak,low VO2peak%pred,and fibrinogen.Multivariate Logistic regression analysis was then performed with low VO2peak,low VO2peak%pred,and low MET as independent variables and high TAB-BHAs as the dependent variable.The results showed that after adjusting for age≥60 years,male,and fibrinogen,both low VO2peak(adjusted OR,4.420,95%CI 1.052-18.573,P=0.042)and low VO2peak%pred(adjusted OR,7.423,95%CI 1.411-39.046,P=0.018)were independently associated with high TAB-BHAs in ischemic stroke patients.After further adjusting for variables that differed among high and low TAB-BHAs groups(including age≥60 years,male,fibrinogen,and history of using antihypertensive drugs),multivariate Logistic regression showed that low VO2peak%pred remained independently associated with high TAB-BHAs in ischemic stroke patients(adjusted OR,6.347,95%CI 1.170-34.418,P=0.032).Conclusion Poor cardiopulmonary fitness might be associated with high degree of overall cardio-cerebral atherosclerosis,suggesting cardiorespiratory fitness as a potential correlated indicator that could provide clues for improving early screening of patients with heavy cardio-cerebral atherosclerotic burden and for exploring the underlying mechanisms.

Objective To investigate the current status and barriers to implementing non-accompanied care services in medical institutions in Zhejiang Province,and to provide a basis for the improvement of standardized management of such services.Methods A self-designed questionnaire was conducted from September to October 2024 among all secondary and tertiary medical institutions in Zhejiang Province to assess the implementation status and barriers to non-accompanied care services.Results A total of 397 questionnaires were distributed,with 389 valid responses,yielding a valid response rate of 97.98％.Non-accompanied care services were implemented in 118 institutions(30.33％).Among these,90 institutions(76.27％)had established management systems for non-accompanied wards;71 institutions(60.17％)had a medical nursing assistant-to-bed ratio lower than 1∶5;41 institu-tions(34.75％)provided tiered training for medical nursing assistants;93 institutions(78.81％)required patients to bear the full cost of the service.Compared with secondary medical institutions,tertiary medical institutions have more complete management system for non-accompanied care services.The main obstacles hindering the development of non-accompanied care services include an imperfect management system for non-accompanied wards,a shortage of medical nursing assistants,a lack of standardized training for such assistants,inconsistent charging standards,and low acceptance among patients and their families.Conclusion The promotion of non-accompanied care services in medical institutions in Zhejiang Province has achieved initial success.However,challenges persist,including incomplete management systems,uneven development across hospital tiers,and imperfect charging mechanisms.It is recommended that relevant authorities strengthen policy support,enhance standardized training for healthcare nursing assistants,refine cost-sharing mechanisms,and improve the quality and sustainability of non-accompanied care services through multi-party collaboration.

Objective To investigate the current status and barriers to implementing non-accompanied care services in medical institutions in Zhejiang Province,and to provide a basis for the improvement of standardized management of such services.Methods A self-designed questionnaire was conducted from September to October 2024 among all secondary and tertiary medical institutions in Zhejiang Province to assess the implementation status and barriers to non-accompanied care services.Results A total of 397 questionnaires were distributed,with 389 valid responses,yielding a valid response rate of 97.98％.Non-accompanied care services were implemented in 118 institutions(30.33％).Among these,90 institutions(76.27％)had established management systems for non-accompanied wards;71 institutions(60.17％)had a medical nursing assistant-to-bed ratio lower than 1∶5;41 institu-tions(34.75％)provided tiered training for medical nursing assistants;93 institutions(78.81％)required patients to bear the full cost of the service.Compared with secondary medical institutions,tertiary medical institutions have more complete management system for non-accompanied care services.The main obstacles hindering the development of non-accompanied care services include an imperfect management system for non-accompanied wards,a shortage of medical nursing assistants,a lack of standardized training for such assistants,inconsistent charging standards,and low acceptance among patients and their families.Conclusion The promotion of non-accompanied care services in medical institutions in Zhejiang Province has achieved initial success.However,challenges persist,including incomplete management systems,uneven development across hospital tiers,and imperfect charging mechanisms.It is recommended that relevant authorities strengthen policy support,enhance standardized training for healthcare nursing assistants,refine cost-sharing mechanisms,and improve the quality and sustainability of non-accompanied care services through multi-party collaboration.

Objective To explore the association between cardiorespiratory fitness and total atherosclerotic burden of brain-and heart-arteries in patients with ischemic stroke.Methods This study retrospectively enrolled consecutive patients with ischemic stroke of the large-artery atherosclerosis or small-artery occlusion subtypes accompanied by cervicocephalic atherosclerosis,who were admitted to the Department of Neurology,Xuanwu Hospital,Capital Medical University,between May 2023 and December 2024.General and clinical data,including age(＜60years,≥60years),sex,admission blood pressure,body mass index,history of hypertension,diabetes mellitus,hyperlipidemia,ischemic stroke,symptomatic coronary artery disease,smoking,alcohol consumption,and medication use(including antiplatelet drugs,anticoagulants,lipid-lowering drugs,antidiabetic drugs,antihypertensive drugs,and β-blockers),characteristics of ischemic stroke(including the duration of onset,ischemic side[left,right,bilateral],and ischemic location[anterior circulation,posterior circulation,bilateral circulation])were collected from all patients enrolled.On the morning after admission,fasting venous blood samples were collected to measure the levels of triglycerides,total cholesterol,low-density lipoprotein cholesterol,high-density lipoprotein cholesterol,fasting blood glucose,glycated hemoglobin,homocysteine,fibrinogen,D-dimer,C-reactive protein,high-sensitivity C-reactive protein,N-terminal pro-B-type natriuretic peptide,creatine kinase-MB,cardiac troponin T,and myoglobin.Transthoracic echocardiography was performed within 7 days after admission to evaluate cardiac function.Left ventricular ejection fraction and cardiac output were recorded,and cardiac index were calculated.Upon admission,neurological impairment,disability,motor function,and balance ability were assessed using the National Institutes of Health stroke scale(NIHSS),modified Rankin scale(mRS),Fugl-Meyer assessment(FMA),and Berg balance scale,respectively.The Saltin-Grimby physical activity level scale was used to evaluate the intensity of habitual physical activity prior to stroke onset.Cardiopulmonary exercise testing was performed within 7 days after admission to assess cardiopulmonary fitness.During the test,peak oxygen uptake(VO2peak),percentage of predicted VO2peak(VO2peak%pred),and metabolic equivalent of task(MET)were recorded.Based on the minimum oxygen uptake required for independent living(15 ml/[kg·min]),VO2peak was classified into low VO2peak(＜15 ml/[kg·min])and high VO2peak(≥15 ml/[kg·min]).With VO2peak%pred＜0.60 as the cutoff,VO2peak%pred was divided into low VO2peak%pred(＜0.60)and high VO2peak%pred(≥0.60).MET were categorized into low MET(＜mean MET)and high MET(≥mean MET)according to the mean value of MET in this study population.Imaging data of cardio-cerebral arteries were obtained by simultaneous CT arteriography within 7 days after admission.Total atherosclerotic burden of brain-and heart-supplying arteries(TAB-BHAs)was used to quantitatively evaluate the overall degree of cardio-cerebral atherosclerosis.And patients were stratified into high TAB-BHAs(≥4 points)and low TAB-BHAs(＜4 points)groups.The differences in general and clinical characteristics,as well as cardiopulmonary fitness indices,were assessed among different groups of TAB-BHAs.Spearman' s correlation was used in the preliminary analysis of the relationship between cardiorespiratory fitness indicators and TAB-BHAs.Multivariate Logistic regression was conducted with VO2peak,low VO2peak%pred,and low METs as independent variables,and high TAB-BHAs as the dependent variable to assess the association between cardiopulmonary fitness and TAB-BHAs.Covariates were selected through regressions stratified by age≥60 years,male,the least absolute shrinkage and selection operator(LASSO),and the variables exhibit statistically significant differences between the low and high TAB-BHAs groups,respectively.Results A total of 104 patients with ischemic stroke were enrolled.Among all patients enrolled,60patients(with ages ranging from 30 to 72years and a mean age of[54±11]years)met the inclusion and exclusion criteria,and were ultimately included.Amid the 60 patients,52 were male and 8 were female.There were 20 patients(33.3%)with low VO2peak,28 patients(46.7%)with low VO2peak%pred,and 31 patients(51.7%)with low MET(MET＜5.0).45 patients were classified into the low TAB-BHAs group and 15 into the high TAB-BHAs group.(1)Compared with the low TAB-BHAs group,patients in the high TAB-BHAs group were significantly older and had a higher proportion of patients aged≥60 years,they also showed significantly higher fibrinogen levels and a history of antihypertensive drug use(all P＜0.05).No significant differences were observed in other general or clinical characteristics between the two groups(all P＞0.05).Regarding cardiopulmonary fitness parameters,patients in the high TAB-BHAs group had lower VO2peak and MET levels,and higher proportions of patients with low VO2peak,low VO2peak%pred,and low MET compared with those in the low TAB-BHAs group(all P＜0.05).(2)Based on the Spearman's correlation analysis,VO2peak(r=-0.298,P=0.021),VO2peak%pred(r=-0.305,P=0.018),MET(r=-0.303,P=0.018)all exhibited negative correlations between and TAB-BHAs.(3)LASSO regression was used to identify the most pertinent variables for high TAB-BHAs,including low VO2peak,low VO2peak%pred,and fibrinogen.Multivariate Logistic regression analysis was then performed with low VO2peak,low VO2peak%pred,and low MET as independent variables and high TAB-BHAs as the dependent variable.The results showed that after adjusting for age≥60 years,male,and fibrinogen,both low VO2peak(adjusted OR,4.420,95%CI 1.052-18.573,P=0.042)and low VO2peak%pred(adjusted OR,7.423,95%CI 1.411-39.046,P=0.018)were independently associated with high TAB-BHAs in ischemic stroke patients.After further adjusting for variables that differed among high and low TAB-BHAs groups(including age≥60 years,male,fibrinogen,and history of using antihypertensive drugs),multivariate Logistic regression showed that low VO2peak%pred remained independently associated with high TAB-BHAs in ischemic stroke patients(adjusted OR,6.347,95%CI 1.170-34.418,P=0.032).Conclusion Poor cardiopulmonary fitness might be associated with high degree of overall cardio-cerebral atherosclerosis,suggesting cardiorespiratory fitness as a potential correlated indicator that could provide clues for improving early screening of patients with heavy cardio-cerebral atherosclerotic burden and for exploring the underlying mechanisms.

ObjectiveTo systematically and objectively characterize the pharmacological effects of Fufang Biejia Ruangan pills （FBRP） in the intervention of alcoholic liver disease （ALD） using acute and chronic ALD mouse models and to elucidate its molecular mechanisms. MethodFifty SPF-grade male BALB/c mice were randomly divided into the normal group， model group， and FBRP low-， medium-， and high-dose groups （9.6， 19.2， 38.4 mg·kg^-1）. Except for the normal group， the remaining groups were given 56° white wine by gavage to establish the acute ALD model， with samples collected after 4 weeks. Thirty SPF-grade male C57BL/6N mice were randomly divided into the normal group， model group， and FBRP medium-dose group （19.2 mg·kg^-1）. The chronic ALD mouse model was established using the Lieber-DeCarli method over a 10-week period. Inflammatory markers in liver tissues were assessed using hematoxylin-eosin （HE）， Sirius Red， oil red O staining， and enzyme-linked immunosorbent assay （ELISA）. Intoxication behaviors of each group were objectively evaluated through sobering-up time， net-catching， and pole-climbing tests. Further bioinformatics analyses based on clinical transcriptomic data were conducted to identify key targets and molecular mechanisms of FBRP in alleviating ALD through liver-brain dialogue， with experimental validation by ELISA， Western blot， and immunohistochemical staining. ResultCompared with the normal group, the levels of aspartate aminotransferase （AST） and alanine aminotransferase （ALT） in liver tissues of mice in the acute and chronic ALD model groups were significantly increased （P<0.05）. Compared with the model group， the levels of AST and ALT in liver tissue of mice in FBRP groups were significantly decreased （P<0.05）. Compared with the normal group， the time of grasping the net and climbing the pole in the acute ALD model group was significantly decreased within 4 weeks （P<0.01）. Compared with the model group， the grasping and climbing time of FBRP high dose groups increased significantly within 4 weeks （P<0.05）. Compared with the normal group， the expression of high mobility group protein B1 （HMGB1） protein in liver tissue and prefrontal lobe tissue of mice in the chronic ALD model group was significantly increased （P<0.01）. Compared with the model group， the expression of HMGB1 protein in FBRP medium dose group was significantly decreased （P<0.05，P<0.01）. Compared with the normal group， the expression of brain-derived neurotrophic factor （BDNF） protein and the release of γ-aminobutyric acid （GABA） in the prefrontal cortex of the model group were significantly decreased （P<0.01）. Compared with the model group, the expression of BDNF protein and the release of GABA in the FBRP medium dose group were significantly increased （P<0.05）. ConclusionThis study revealed that FBRP improved key pathological changes in ALD by modulating liver-brain dialogue mediated by the HMGB1-BDNF axis. These findings provide experimental evidence for the clinical use of FBRP in treating ALD and offer new insights for the development of ALD therapeutic agents.

Objective:To analyze the research hotspots and trends in the field of pressure injury support surfaces both domestically and abroad and to provide references for future studies in this domain.Methods:Relevant literature on pressure injury support surfaces indexed in the Web of Science Core Collection, CNKI, Wanfang, and VIP database were retrieved, with the search spanning from January 1, 2010, to February 28, 2023. CiteSpace was utilized to perform a visualization analysis of the amassed data.Results:A total of 307 Chinese articles and 434 English articles were included in the analysis. The United States emerged as the country with the highest number of publications. The General Hospital of Eastern Military Command topped the list in China in terms of the number of publications. Chinese Journal of Modern Nursing and Journal of Tissue Viability were the journals with the highest publication frequencies in China and abroad, respectively. Jiang Qixia was the author with the highest number of publications in China, while Gefen secured this position internationally. The Chinese literature formed 12 clusters and the English literature formed 17, culminating in the identification of 8 meaningful categories (population, location, research type, risk factors, static support surfaces, dynamic support surfaces, intelligent monitoring, and effectiveness evaluation), among which intelligent monitoring was seen as a future research trend. Conclusions:Pressure injury support surfaces have been a research hotspot in recent years both domestically and internationally. It is recommended to promote the development of domestic research on pressure injury preventive care through strategies such as expanding the range of research populations, developing intelligent support surface devices conducive to promotion in collaboration with computer and artificial intelligence disciplines, and carrying out large-scale high-quality original research and corresponding quality improvement projects.

Objective:To study the value of shear wave elastography (SWE) combined with contrast-enhanced ultrasound (CEUS) in diagnosing the invasiveness of papillary thyroid microcarcinoma (PTMC), and analyze its risk factors.Methods:This study included 200 patients with pathologically confirmed PTMC who underwent surgery in Fenyang Hospital from January 2019 and June 2021. All were diagnosed with SWE and CEUS before surgery. The value of the two methods in diagnosing the invasiveness of PTMC was explored. The patients′ data were collected to screen the risk factors for the invasiveness of PTMC.Results:It was pathologically confirmed that among the 200 patients with PTMC, there were 112 cases with malignant nodules, 88 cases with benign nodules, 75 cases with cervical lymph node metastasis (including 71 cases with capsular invasion) and 125 cases without lymph node metastasis. CEUS parameters of malignant nodules were significantly higher than those of benign nodules ( P<0.05). The sensitivity, specificity, accuracy, positive predictive value and negative predictive value of SWE combined with CEUS to diagnose capsular invasion were 94.66%, 85.60%, 89.00%, 79.77% and 96.39%, with high consistency with the pathological results ( Kappa>0.75). Multivariate Logistic regression analysis showed that multiple foci, irregular shape, breakthrough capsule and small calcification were independent risk factors for the invasiveness of PTMC (VIF<3). The ROC curve results showed that the AUC of SWE combined with CEUS to diagnose capsular invasion was 0.772, and the diagnostic sensitivity and specificity were 73.91% and 80.56%. Conclusions:SWE combined with CEUS can significantly improve the diagnostic accuracy for the invasiveness of PTMC.

Objective:To develop a diagnostic analysis software for determining the type of acid-base balance disorder.Methods:Mathematical models were built based on Henderson-Hasselbalch equations and compensation formulas, to determine the important parameters of acid-base balance disorder, and to develope acid-base balance disorder analysis process. The software was compiled using the Visual Basic.NET programming language, and the installation package was generated after debugging. Acid-base balance disorder cases were searched by PubMed, Wanfang and CNKI databases from 1980 to 2015, and the blood gas parameters [pH, arterial partial pressure of carbon dioxide (PaCO 2), HCO 3- and anion gap (AG)] and the types of acid-base imbalance (literature results) were recorded. All cases were reanalyzed by software and the type of acid-base balance disorder was determined (software diagnostic type). Kappa-test and McNemar-test were performed for the two diagnostic results. Results:The "four parameters-four steps" analysis method was used as the analysis process to judge the types of acid-base balance disorder. "Four parameters" included pH, PaCO 2, HCO 3- and AG. "Four steps" were outlined by following aspects:①according to the pH, combined with PaCO 2 and HCO 3-, the primary types of acid-base balance disorder was determined; ② according to the compensation situation, double mixed acid-base balance disorder (DABD) was determined; ③according to AG value, three mixed acid-base disorders (TABD) were determined; ④ the ratio of ΔAG↑/ΔHCO 3-↓ was also calculated to determine whether there was normal AG metabolic acidosis or metabolic alkalosis. The software had the characteristics of simple interface, convenient operation, rapid judgment, and comprehensive analysis. It could judge all acid-base balance disorder types excepted "AG normal metabolic acidosis combined metabolic alkalosis". The software was used to reanalyze 112 cases of acid-base balance disorder reported in the literature, with a consistent rate of 87.50% and better consistency of the diagnostic results (Kappa test: κ = 0.84, P < 0.01; McNemar test: χ2 = 0.87, P = 0.65). Conclusion:The software can be used as an important tool to judge the type of acid-base balance disorder, and provide clinicians with diagnostic reference, which have practical value and application prospect.

A 69-year-old female patient with diabetes mellitus was scheduled to undergo angiography of head because of acute cerebral infarction. When iopromide injection-370 was injected intravenously with a high-pressure syringe before operation, the contrast medium extravasated at the injection site of her right hand, resulting in severe subcutaneous hematoma, tissue necrosis, and infection. The maximum area of extravasation wound extended to 6.0 cm × 7.0 cm. Therapies such as local wet compress with cold 50% magnesium sulfate and dexamethasone, daily debridement and dressing change, and external application of hydrogel, silver containing hydrophilic foam, and hydrocolloid were given. On day 14 after extravasation, the necrotic tissue of the wound gradually fell off and the wound began to heal. Then the wound healed on day 35 after extravasation.