Sandwich ELISA for quantitative detection of human collagen prolyl 4-hydroxylase

Background: We describe a method for specific, quantitative and quick detection of human collagen prolyl 4- hydroxylase (C-P4H), the key enzyme for collagen prolyl-4 hydroxylation, in crude samples based on a sandwich ELISA principle. The method is relevant to active C-P4H level monitoring during recombinant C-P4H and collagen production in different expression systems. The assay proves to be specific for the active C-P4H α2β2 tetramer due to the use of antibodies against its both subunits. Thus in keeping with the method C-P4H is captured by coupled to an anti-α subunit antibody magnetic beads and an anti-β subunit antibody binds to the PDI/β subunit of the protein. Then the following holoenzyme detection is accomplished by a goat anti-rabbit IgG labeled with alkaline phosphatase which AP catalyzes the reaction of a substrate transformation with fluorescent signal generation. Results: We applied an experimental design approach for the optimization of the antibody concentrations used in the sandwich ELISA. The assay sensitivity was 0.1 ng of C-P4H. The method was utilized for the analysis of C-P4H accumulation in crude cell extracts of E. coli overexpressing C-P4H. The sandwich ELISA signals obtained demonstrated a very good correlation with the detected protein activity levels measured with the standard radioactive assay. The developed assay was applied to optimize C-P4H production in E. coli Origami in a system where the C-P4H subunits expression acted under control by different promoters. The experiments performed in a shake flask fed-batch system (EnBase®) verified earlier observations that cell density and oxygen supply are critical factors for the use of the inducer anhydrotetracycline and thus for the soluble C-P4H yield. Conclusions: Here we show an example of sandwich ELISA usage for quantifying multimeric proteins. The method was developed for monitoring the amount of recombinant C-P4H tetramer in crude E. coli extracts. Due to the specificity of the antibodies used in the assay against the different C-P4H subunits, the method detects the entire holoenzyme, and the signal is not disturbed by background expression of the separate subunits

Safety of enzymatic debridement in extensive burns larger than 15% total body surface area

OBJECTIVES Bromelain-based enzymatic debridement has emerged as an alternative to surgical eschar removal. Indications include partial thickness, mixed pattern, and full-thickness burns. Enzymatic debridement has been approved by the European Medicines Agency for treating burn wounds affecting 15% TBSA in one session is scarce. The aim of this retrospective study was to retrospectively analyze off-label use of enzymatic debridement in a single burn center for large TBSA burns. METHODS Between 01/2017 and 12/2018, 59 patients with partial- to full-thickness burns underwent enzymatic debridement in a single center study. Patients were categorized into two groups: the regular use group with a treated area less than 15% TBSA and the off-label group (OG) with larger TBSA debrided in one session. Treatment was evaluated for systemic inflammatory reaction, bleeding, hemodynamic instability and electrolyte shifts. RESULTS In total, 49 patients were treated in the regular use group with a median application area of 6% (IQR 2.5-9.5) and 10 patients were treated in the off-label group with a median application area of 18% (IQR 15-19) TBSA. We found no significant differences regarding blood pressure, body temperature or hemodynamic stability during and after enzymatic debridement. No treatment-related serious adverse events were observed in either group. Catecholamine use was similar in both groups. No differences in leukocyte counts, CRP, PCT and lactate prior to application and during the following three days were observed. Sodium, potassium, chloride and phosphate levels did not differ. We found no evidence of an electrolyte shift. Survival was 49 of 49 patients (100%) in the RG and 7 of 10 patients (70%) in the OG (p = 0.004). CONCLUSION Enzymatic debridement did not result in any expected or unexpected side effects in the patient groups investigated. These preliminary results indicate the potential safety of bromelain-based enzymatic debridementin the treatment of burns greater than 15% TBSA

The doctor's point of view: eye-tracking as an investigative tool in the extubationprocess in intensive care units. A pilot study

BACKGROUND Patient safety is a top priority in healthcare. Little is known about the visual behaviour of professionals during high-risk procedures. The aim of this study was to assess feasibility, usability and safety of eye-tracking to analyse gaze patterns during the extubation-process in the intensive care unit. METHODS Eye-tracking was used in this observational study to analyse the extubationprocess in 22 participants. Independent variables were average fixation time, dwell time, fixation count, hit ratio and revisit count for eighteen areas of interest. Primary outcome was dwell time for all areas of interest. Secondary outcomes were average fixation time, fixation count and revisits. In subgroup analyses, experienced and non-experienced physicians were compared. RESULTS The most important area of interest was the patient, as analyzed by dwell time. Fixation of other areas of interest varied significantly among participants. Only 54% checked ventilator respiratory rate, despite declaring it as important in questionnaires. Other neglected areas of interest included tidal volume (59%), peak pressure (63.6%), CO2 (63.6%), temperature (18.2%), blood pressure (59%) and heart rate (68%). Experienced physicians gazed more frequently and longer at the patient while spending less time on monitor and ventilator parameters. CONCLUSIONS Eye-tracking can demonstrate that there is a mismatch between physicians' subjective evaluations and corresponding objective real-life measurements. Structured and standardized extubation-processes should be performed to improve patient safety. In the immediate post-extubation phase, long dwell time on the patient shows that clinical observation remains the most important cornerstone beyond monitoring devices

ICG-liver test versus new biomarkers as prognostic markers for prolonged length of stay in critically ill patients - a prospective study of accuracy for prediction of length of stay in the ICU

BACKGROUND: Prognostic abilities of medical parameters, which are scoring systems, measurements and biomarkers, are important for stratifying critically ill patients. Indocyanine green plasma disappearance (ICG-PDR) is an established clinical tool for the assessment of liver perfusion and function. Copeptin, MR-proANP and pro-ADM are biomarkers whose prognostic value is still unclear. The goal of this prospective study was to evaluate ICG-PDR, copeptin, MR-proANP and pro-ADM to predict prolonged length of stay (pLOS) in the ICU. METHODS: This study was conducted as a prospective single center study including 110 consecutively admitted ICU patients. Primary endpoint was prolonged length of stay (pLOS) in the ICU, defined as more than three days of stay there. RESULTS: ROC analysis showed an AUC of 0.73 for ICG-PDR, 0.70 for SAPS II, 0.65 for MR-proANP, 0.64 for pro-ADM and 0.54 for copeptin for pLOS in the ICU. CONCLUSIONS: The prediction of pLOS in the ICU might be better by means of ICG-PDR than with the new biomarkers copeptin, MR-proANP or pro-ADM. Nevertheless, there is more need for research to evaluate whether ICG-PDR is an overall prognostic marker for pLOS. TRIAL REGISTRATION: (ClinicalTrials.gov number, NCT01126554)

Role of arginine vasopressin and terlipressin as first-line vasopressor agents in fulminant ovine septic shock

SCOPUS: ar.jinfo:eu-repo/semantics/publishe