Development of a biosensor for urea assay based on amidase inhibition, using an ion-selective electrode

A biosensor for urea has been developed based on the observation that urea is a powerful active-site inhibitor of amidase, which catalyzes the hydrolysis of amides such as acetamide to produce ammonia and the corresponding organic acid. Cell-free extract from Pseudomonas aeruginosa was the source of amidase (acylamide hydrolase, EC 3.5.1.4) which was immobilized on a polyethersulfone membrane in the presence of glutaraldehyde; anion-selective electrode for ammonium ions was used for biosensor development. Analysis of variance was used for optimization of the biosensorresponse and showed that 30 mu L of cell-free extract containing 7.47 mg protein mL(-1), 2 mu L of glutaraldehyde (5%, v/v) and 10 mu L of gelatin (15%, w/v) exhibited the highest response. Optimization of other parameters showed that pH 7.2 and 30 min incubation time were optimum for incubation ofmembranes in urea. The biosensor exhibited a linear response in the range of 4.0-10.0 mu M urea, a detection limit of 2.0 mu M for urea, a response timeof 20 s, a sensitivity of 58.245 % per mu M urea and a storage stability of over 4 months. It was successfully used for quantification of urea in samples such as wine and milk; recovery experiments were carried out which revealed an average substrate recovery of 94.9%. The urea analogs hydroxyurea, methylurea and thiourea inhibited amidase activity by about 90%, 10% and 0%, respectively, compared with urea inhibition

Long-term Safety of Treatment with Autologous Mesenchymal Stem Cells in Patients with Radiation-Induced Xerostomia:Primary Results of the MESRIX Phase I/II Randomized Trial

Purpose: Mesenchymal stem/stromal cell therapy may reduce radiation-induced xerostomia. We investigated the long-term safety of autologous adipose tissue-derived mesenchymal stem/stromal cell (ASC) injections into the submandibular glands. Experimental Design: An investigator-initiated, randomized, single-center, placebo-controlled trial. Previous patients with oropharyngeal squamous cell carcinoma with radiation-induced xerostomia were randomly (1:1) allocated to receive a 2.8 million ASCs/cm3 injection or placebo in both submandibular glands and followed for a minimum of 2 years. The primary endpoint was number of serious adverse events (SAE). Secondary endpoints included whole saliva flow rates and xerostomia-related symptoms. Data analysis was based on the intention-to-treat population using repeated measures mixed-effects linear models. Results: Thirty-three patients were randomized; 30 patients were treated (ASC group, n = 15; placebo group, n = 15). Longterm safety data were collected from all 30 patients. During follow-up, 6 of 15 (40%) of the ASC-treated patients versus 5 of 15 (33%) of the placebo patients experienced an SAE; no SAEs appeared to be treatment related. Unstimulated whole saliva flow rate increased to 0.20 and 0.16 mL/minute in the ASC and placebo group, respectively, yielding a 0.05 mL/minute (95% confidence interval: 0.00-0.10; P = 0.051) difference between groups. Patient-reported xerostomia symptoms diminished according to a decreased xerostomia questionnaire summary score of 35.0 and 45.1, respectively [-10.1 (-18.1 to -2.2); P = 0.013]. Three of the visual analog scale xerostomia measures indicated clinical benefit following use of ASC. Conclusions:Our data show that ASC therapy is safe with a clinically relevant effect on xerostomia-related symptoms. Confirmation in larger randomized controlled trials is warranted. </p

Cleavage of Kininogen and Subsequent Bradykinin Release by the Complement Component: Mannose-Binding Lectin-Associated Serine Protease (MASP)-1

Bradykinin (BK), generated from high-molecular-weight kininogen (HK) is the major mediator of swelling attacks in hereditary angioedema (HAE), a disease associated with C1-inhibitor deficiency. Plasma kallikrein, activated by factor XIIa, is responsible for most of HK cleavage. However other proteases, which activate during episodes of angioedema, might also contribute to BK production. The lectin pathway of the complement system activates after infection and oxidative stress on endothelial cells generating active serine proteases: MASP-1 and MASP-2. Our aim was to study whether activated MASPs are able to digest HK to release BK. Initially we were trying to find potential new substrates of MASP-1 in human plasma by differential gel electrophoresis, and we identified kininogen cleavage products by this proteomic approach. As a control, MASP-2 was included in the study in addition to MASP-1 and kallikrein. The proteolytic cleavage of HK by MASPs was followed by SDS-PAGE, and BK release was detected by HPLC. We showed that MASP-1 was able to cleave HK resulting in BK production. MASP-2 could also cleave HK but could not release BK. The cleavage pattern of MASPs is similar but not strictly identical to that of kallikrein. The catalytic efficiency of HK cleavage by a recombinant version of MASP-1 and MASP-2 was about 4.0×102 and 2.7×102 M−1s−1, respectively. C1-inhibitor, the major inhibitor of factor XIIa and kallikrein, also prevented the cleavage of HK by MASPs. In all, a new factor XII- and kallikrein-independent mechanism of bradykinin production by MASP-1 was demonstrated, which may contribute to the pro-inflammatory effect of the lectin pathway of complement and to the elevated bradykinin levels in HAE patients

Effects of MASP-1 of the Complement System on Activation of Coagulation Factors and Plasma Clot Formation

BACKGROUND: Numerous interactions between the coagulation and complement systems have been shown. Recently, links between coagulation and mannan-binding lectin-associated serine protease-1 (MASP-1) of the complement lectin pathway have been proposed. Our aim was to investigate MASP-1 activation of factor XIII (FXIII), fibrinogen, prothrombin, and thrombin-activatable fibrinolysis inhibitor (TAFI) in plasma-based systems, and to analyse effects of MASP-1 on plasma clot formation, structure and lysis. METHODOLOGY/PRINCIPAL FINDINGS: We used a FXIII incorporation assay and specific assays to measure the activation products prothrombin fragment F1+2, fibrinopeptide A (FPA), and activated TAFI (TAFIa). Clot formation and lysis were assessed by turbidimetric assay. Clot structure was studied by scanning electron microscopy. MASP-1 activated FXIII and, contrary to thrombin, induced FXIII activity faster in the Val34 than the Leu34 variant. MASP-1-dependent generation of F1+2, FPA and TAFIa showed a dose-dependent response in normal citrated plasma (NCP), albeit MASP-1 was much less efficient than FXa or thrombin. MASP-1 activation of prothrombin and TAFI cleavage were confirmed in purified systems. No FPA generation was observed in prothrombin-depleted plasma. MASP-1 induced clot formation in NCP, affected clot structure, and prolonged clot lysis. CONCLUSIONS/SIGNIFICANCE: We show that MASP-1 interacts with plasma clot formation on different levels and influences fibrin structure. Although MASP-1-induced fibrin formation is thrombin-dependent, MASP-1 directly activates prothrombin, FXIII and TAFI. We suggest that MASP-1, in concerted action with other complement and coagulation proteins, may play a role in fibrin clot formation

Effects of pre-operative isolation on postoperative pulmonary complications after elective surgery: an international prospective cohort study

We aimed to determine the impact of pre-operative isolation on postoperative pulmonary complications after elective surgery during the global SARS-CoV-2 pandemic. We performed an international prospective cohort study including patients undergoing elective surgery in October 2020. Isolation was defined as the period before surgery during which patients did not leave their house or receive visitors from outside their household. The primary outcome was postoperative pulmonary complications, adjusted in multivariable models for measured confounders. Pre-defined sub-group analyses were performed for the primary outcome. A total of 96,454 patients from 114 countries were included and overall, 26,948 (27.9%) patients isolated before surgery. Postoperative pulmonary complications were recorded in 1947 (2.0%) patients of which 227 (11.7%) were associated with SARS-CoV-2 infection. Patients who isolated pre-operatively were older, had more respiratory comorbidities and were more commonly from areas of high SARS-CoV-2 incidence and high-income countries. Although the overall rates of postoperative pulmonary complications were similar in those that isolated and those that did not (2.1% vs 2.0%, respectively), isolation was associated with higher rates of postoperative pulmonary complications after adjustment (adjusted OR 1.20, 95%CI 1.05-1.36, p = 0.005). Sensitivity analyses revealed no further differences when patients were categorised by: pre-operative testing; use of COVID-19-free pathways; or community SARS-CoV-2 prevalence. The rate of postoperative pulmonary complications increased with periods of isolation longer than 3 days, with an OR (95%CI) at 4-7 days or ≥ 8 days of 1.25 (1.04-1.48), p = 0.015 and 1.31 (1.11-1.55), p = 0.001, respectively. Isolation before elective surgery might be associated with a small but clinically important increased risk of postoperative pulmonary complications. Longer periods of isolation showed no reduction in the risk of postoperative pulmonary complications. These findings have significant implications for global provision of elective surgical care

Contemporary methods for evaluating complex project proposals

The ability to evaluate project proposals, assessing future success, and organizational value is critical to overall business performance for most enterprises. Yet, predicting project success is difficult and often unreliable. A four-year field study shows that the effectiveness of available methods for evaluating and selecting large, complex project depends on the specific project type, organizational culture, and managerial skills. This paper examines the strength and limitations of various evaluation methods. It also shows that, especially in complex project situations, the decision-making process has to go beyond the application of just analytical methods, but has to incorporate both quantitative and qualitative measures into a combined rational judgmental evaluation process. Equally important, the evaluation process must be effectively linked among functional support groups and with senior management in order to strategically align the project proposal and to unify the evaluation team and stakeholder community behind the mission objectives. All of this requires leadership and managerial skills in planning, organizing, and communicating. The paper suggests specific leadership actions, organizational conditions, and managerial processes for evaluating complex project proposals toward future value and success

Timing of surgery following SARS-CoV-2 infection:an international prospective cohort study

Peri-operative SARS-CoV-2 infection increases postoperative mortality. The aim of this study was to determine the optimal duration of planned delay before surgery in patients who have had SARS-CoV-2 infection. This international, multicentre, prospective cohort study included patients undergoing elective or emergency surgery during October 2020. Surgical patients with pre-operative SARS-CoV-2 infection were compared with those without previous SARS-CoV-2 infection. The primary outcome measure was 30-day postoperative mortality. Logistic regression models were used to calculate adjusted 30-day mortality rates stratified by time from diagnosis of SARS-CoV-2 infection to surgery. From 140,231 patients (116 countries), 3127 patients (2.2%) had a pre-operative SARS-CoV-2 diagnosis. Adjusted 30-day mortality in patients without SARS-CoV-2 infection was 1.5% (95%CI 1.4-1.5). In patients with a pre-operative SARS-CoV-2 diagnosis, mortality was increased in patients having surgery within 0-2 weeks, 3-4 weeks and 5-6 weeks of the diagnosis (odd ratio (95%CI) 4.1 (3.3-4.8), 3.9 (2.6-5.1) and 3.6 (2.0-5.2), respectively). Surgery performed ≥ 7 weeks after SARS-CoV-2 diagnosis was associated with a similar mortality risk to baseline (odd ratio (95%CI) 1.5 (0.9-2.1)). After a ≥ 7 week delay in undertaking surgery following SARS-CoV-2 infection, patients with ongoing symptoms had a higher mortality than patients whose symptoms had resolved or who had been asymptomatic (6.0% (95%CI 3.2-8.7) vs. 2.4% (95%CI 1.4-3.4) vs. 1.3% (95%CI 0.6-2.0), respectively). Where possible, surgery should be delayed for at least 7 weeks following SARS-CoV-2 infection. Patients with ongoing symptoms ≥ 7 weeks from diagnosis may benefit from further delay.</p

Outcomes of elective liver surgery worldwide: a global, prospective, multicenter, cross-sectional study

Background: The outcomes of liver surgery worldwide remain unknown. The true population-based outcomes are likely different to those vastly reported that reflect the activity of highly specialized academic centers. The aim of this study was to measure the true worldwide practice of liver surgery and associated outcomes by recruiting from centers across the globe. The geographic distribution of liver surgery activity and complexity was also evaluated to further understand variations in outcomes. Methods: LiverGroup.org was an international, prospective, multicenter, cross-sectional study following the Global Surgery Collaborative Snapshot Research approach with a 3-month prospective, consecutive patient enrollment within January–December 2019. Each patient was followed up for 90 days postoperatively. All patients undergoing liver surgery at their respective centers were eligible for study inclusion. Basic demographics, patient and operation characteristics were collected. Morbidity was recorded according to the Clavien–Dindo Classification of Surgical Complications. Country-based and hospital-based data were collected, including the Human Development Index (HDI). (NCT03768141). Results: A total of 2159 patients were included from six continents. Surgery was performed for cancer in 1785 (83%) patients. Of all patients, 912 (42%) experienced a postoperative complication of any severity, while the major complication rate was 16% (341/2159). The overall 90-day mortality rate after liver surgery was 3.8% (82/2,159). The overall failure to rescue rate was 11% (82/ 722) ranging from 5 to 35% among the higher and lower HDI groups, respectively. Conclusions: This is the first to our knowledge global surgery study specifically designed and conducted for specialized liver surgery. The authors identified failure to rescue as a significant potentially modifiable factor for mortality after liver surgery, mostly related to lower Human Development Index countries. Members of the LiverGroup.org network could now work together to develop quality improvement collaboratives

Introduction and Validation of a Novel Acute Pancreatitis Digital Tool: Interrogating Large Pooled Data From 2 Prospectively Ascertained Cohorts

Objectives: Acute pancreatitis (AP) is a sudden onset, rapidly evolving inflammatory response with systemic inflammation and multiorgan failure (MOF) in a subset of patients. New highly accurate clinical decision support tools are needed to allow local doctors to provide expert care. Methods: Ariel Dynamic Acute Pancreatitis Tracker (ADAPT) is a digital tool to guide physicians in ordering standard tests, evaluate test results and model progression using available data, propose emergent therapies. The accuracy of the severity score calculators was tested using 2 prospectively ascertained Acute Pancreatitis Patient Registry to Examine Novel Therapies in Clinical Experience cohorts (pilot University of Pittsburgh Medical Center, n = 163; international, n = 1544). Results: The ADAPT and post hoc expert-calculated AP severity scores were 100% concordant in both pilot and international cohorts. High-risk criteria of all 4 severity scores at admission were associated with moderately-severe or severe AP and MOF (both P < 0.0001) and prediction of no MOF was 97.8% to 98.9%. The positive predictive value for MOF was 7.5% to 14.9%. Conclusions: The ADAPT tool showed 100% accuracy with AP predictive metrics. Prospective evaluation of ADAPT features is needed to determine if additional data can accurately predict and mitigate severe AP and MOF