Does Intraoperative Fibrinogen Affect Blood Loss or Transfusion Practice After Aortic Arch Surgery: A Prematurely Ended Randomized Trial.

Cardiovascular surgery is often complicated by significant bleeding due to perioperative coagulopathy. The effectiveness of treatment with fibrinogen concentrate to reduce the perioperative blood transfusion rate after thoracic aortic replacement surgery in prior studies has shown conflicting results. Therefore, we conducted a double-blind randomized controlled trial to investigate if a single dose of intraoperative fibrinogen administration reduced blood loss and allogeneic transfusion rate after elective surgery for thoracic arch aneurysm with deep hypothermic circulatory arrest. Twenty patients were randomized to fibrinogen concentrate (N = 10) or placebo (N = 10). The recruitment of study patients was prematurely ended due to a low inclusion rate. Perioperative transfusion, 5-minute bleeding mass after study medication and postoperative blood loss were not different between the groups with fibrinogen concentrate or placebo. Due to small volumes of postoperative blood loss and premature study termination, a beneficial effect of fibrinogen concentrate on the number of blood transfusions could not be established. However, treatment with fibrinogen efficiently restored fibrinogen levels and clot strength to preoperative values with a more effective preserved postoperative thrombin generation capacity. This result might serve as a pilot for further multicenter studies to assess the prospective significance of automated and standardized thrombin generation as a routine assay for monitoring perioperative coagulopathy and its impact on short- and long-term operative results.

[Supportive treatment and decision-making in a patient with severe anaemia when blood transfusion is not an option]. / Ernstige anemie terwijl bloedtransfusie geen optie is.

In severely anaemic patients, blood transfusions remain the standard of care when haemoglobin levels become dangerously low. However, in some situations blood transfusion is not an option. In this clinical lesson, we present a case of a young Jehovah's Witness who developed a life-threatening anaemia due to a gastro-intestinal bleeding. The patient did not want to receive blood products. Although blood transfusions seemed crucial, we successfully treated our patient with only supportive measures. This articles gives an overview of supportive treatment options in severely anaemic patients in the absence of blood transfusions. These measures include monitoring and optimization of hemodynamics, prevention of further blood loss, correction of the haemostatic balance, enhancing haemostasis, improving oxygen delivery and optimizing haematopoiesis.

Evaluation of the Atellica® UAS 800: a new member of the automated urine sediment analyzer family.

BACKGROUND: In 2017 the Atellica® UAS 800 urine sediment analyzer was introduced by Siemens Healthineers. We investigated its applicability in the standardization and automation of the laboratory urinalysis workflow, including the prediction of urine culture outcome and glomerular pathology. METHODS: We evaluated the performance characteristics of the Atellica® UAS 800 and its correlation with the iQ200 (Beckman Coulter). In addition, we studied the agreement between Atellica® UAS 800 and CLINITEK Novus® and determined the predictive value of bacteria and leukocyte counts for urine culture outcome. Furthermore, we investigated the ability of Atellica® UAS 800 to identify pathological casts and dysmorphic erythrocytes in comparison to manual microscopy. RESULTS: Erythrocyte and leukocyte analyses indicated high intra- and inter-run precisions and good correlations with the iQ200. We found that the Atellica® UAS 800 detects bacteria with higher sensitivity than the iQ200. The Atellica® UAS 800 and CLINITEK Novus® showed a high degree of conformity. We determined seven combinations of clinical cut-off values of bacteria and leukocytes for predicting urine culture outcome with sensitivity, specificity, and negative predictive values of 95%, 52%, and 93%, respectively. Using the Atellica® UAS 800, hyaline casts, erythrocyte casts, leukocyte casts, and dysmorphic erythrocytes were correctly recognized in 76%, 22%, 2%, and 39% of the samples, respectively. CONCLUSIONS: The Atellica® UAS 800 is a robust, fast, and user-friendly analyzer, which accurately quantifies erythrocytes, leukocytes, bacteria and squamous epithelial cells, and may be utilized for predicting positive urine cultures. The detection of clinically important pathological casts and dysmorphic erythrocytes proved insufficient.

Protease-Activatable Scaffold Proteins as Versatile Molecular Hubs in Synthetic Signaling Networks.

Protease signaling and scaffold-induced control of protein-protein interactions represent two important mechanisms for intracellular signaling. Here we report a generic and modular approach to control the activity of scaffolding proteins by protease activity, creating versatile molecular platforms to construct synthetic signaling networks. Using 14-3-3 proteins as a structurally well-characterized and important class of scaffold proteins, three different architectures were explored to achieve optimal protease-mediated control of scaffold activity, fusing either one or two monovalent inhibitory ExoS peptides or a single bivalent ExoS peptide to T14-3-3 using protease-cleavable linkers. Analysis of scaffolding activity before and after protease-induced cleavage revealed optimal control of 14-3-3 activity for the system that contained monovalent ExoS peptides fused to both the N-and C-terminus, each blocking a single T14-3-3 binding site. The protease-activatable 14-3-3 scaffolds were successfully applied to construct a three-step signaling cascade in which dimerization and activation of FGG-caspase-9 on an orthogonal supramolecular platform resulted in activation of a 14-3-3 scaffold, which in turn allowed 14-3-3-templated complementation of a split-luciferase. In addition, by combining 14-3-3-templated activation of caspase-9 with a caspase-9-activatable 14-3-3 scaffold, the first example of a synthetic self-activating protease signaling network was created. Protease-activatable 14-3-3 proteins thus represent a modular platform whose properties can be rationally engineered to fit different applications, both to create artificial in vitro synthetic molecular networks and as a novel signaling hub to re-engineer intracellular signaling pathways.

Engineering BRET-Sensor Proteins.

FRET-sensors have become important tools for intracellular imaging, but their dependence on external illumination presents some limitations, such as photobleaching and phototoxicity, which limit measurements over extended periods of time. Fluorescence measurements also suffer from autofluorescence and light scattering, which hampers in vivo imaging and measurements in strongly absorbing and scattering media such as blood. In principle, these issues can be resolved by using sensors based on bioluminescence resonance energy transfer (BRET). The recent development of brighter and more stable luciferases and the concomitant improvement in luciferase substrates have substantially decreased the sensitivity gap between fluorescence and bioluminescence. As a result, the application of BRET-sensors is no longer restricted to measurements on cell populations, but they can also be used for imaging of single living cells, and BRET has started to emerge as an attractive sensor format for point-of-care diagnostics. The aim of this chapter is to first provide a brief overview of the basic design principles for BRET-sensors. Next, important design considerations will be discussed in more detail by describing the development of three different classes of BRET-sensors, both from our own work and that of others. These examples are all based on the NanoLuc luciferase, a bright and very stable blue light-emitting luciferase developed by Promega that has quickly risen to prominence in the bioluminescence field.

A Novel Organ Culture Model to Quantify Collagen Remodeling in Tree Shrew Sclera.

Increasing evidence suggests that unknown collagen remodeling mechanisms in the sclera underlie myopia development. We are proposing a novel organ culture system in combination with two-photon fluorescence imaging to quantify collagen remodeling at the tissue- and lamella-level. Tree shrew scleral shells were cultured up to 7 days in serum-free media and cellular viability was investigated under: (i) minimal tissue manipulations; (ii) removal of intraocular tissues; gluing the eye to a washer using (iii) 50 µL and (iv) 200 µL of cyanoacrylate adhesive; (v) supplementing media with Ham's F-12 Nutrient Mixture; and (vi) culturing eyes subjected to 15 mmHg intraocular pressure in our new bioreactor. Two scleral shells of normal juvenile tree shrews were fluorescently labeled using a collagen specific protein and cultured in our bioreactor. Using two-photon microscopy, grid patterns were photobleached into and across multiple scleral lamellae. These patterns were imaged daily for 3 days, and tissue-/lamella-level strains were calculated from the deformed patterns. No significant reduction in cell viability was observed under conditions (i) and (v). Compared to condition (i), cell viability was significantly reduced starting at day 0 (condition (ii)) and day 3 (conditions (iii, iv, vi)). Tissue-level strain and intralamellar shear angel increased significantly during the culture period. Some scleral lamellae elongated while others shortened. Findings suggest that tree shrew sclera can be cultured in serum-free media for 7 days with no significant reduction in cell viability. Scleral fibroblasts are sensitive to tissue manipulations and tissue gluing. However, Ham's F-12 Nutrient Mixture has a protective effect on cell viability and can offset the cytotoxic effect of cyanoacrylate adhesive. This is the first study to quantify collagen micro-deformations over a prolonged period in organ culture providing a new methodology to study scleral remodeling in myopia.

Dual Readout BRET/FRET Sensors for Measuring Intracellular Zinc.

Genetically encoded FRET-based sensor proteins have significantly contributed to our current understanding of the intracellular functions of Zn2+. However, the external excitation required for these fluorescent sensors can give rise to photobleaching and phototoxicity during long-term imaging, limits applications that suffer from autofluorescence and light scattering, and is not compatible with light-sensitive cells. For these applications, sensor proteins based on Bioluminescence Resonance Energy Transfer (BRET) would provide an attractive alternative. In this work, we used the bright and stable luciferase NanoLuc to create the first genetically encoded BRET sensors for measuring intracellular Zn2+. Using a new sensor approach, the NanoLuc domain was fused to the Cerulean donor domain of two previously developed FRET sensors, eCALWY and eZinCh-2. In addition to preserving the excellent Zn2+ affinity and specificity of their predecessors, these newly developed sensors enable both BRET- and FRET-based detection. While the dynamic range of the BRET signal for the eCALWY-based BLCALWY-1 sensor was limited by the presence of two competing BRET pathways, BRET/FRET sensors based on the eZinCh-2 scaffold (BLZinCh-1 and -2) yielded robust 25-30% changes in BRET ratio. In addition, introduction of a chromophore-silencing mutation resulted in a BRET-only sensor (BLZinCh-3) with increased BRET response (50%) and an unexpected 10-fold increase in Zn2+ affinity. The combination of robust ratiometric response, physiologically relevant Zn2+ affinities, and stable and bright luminescence signal offered by the BLZinCh sensors allowed monitoring of intracellular Zn2+ in plate-based assays as well as intracellular BRET-based imaging in single living cells in real time.

Rewiring Multidomain Protein Switches: Transforming a Fluorescent Zn(2+) Sensor into a Light-Responsive Zn(2+) Binding Protein.

Protein-based sensors and switches provide attractive tools for the real-time monitoring and control of molecular processes in complex biological environments. Fluorescent sensor proteins have been developed for a wide variety of small molecules, but the construction of genetically encoded light-responsive ligand binding proteins remains mostly unexplored. Here we present a generic approach to reengineer a previously developed FRET-based Zn(2+) sensor into a light-activatable Zn(2+) binding protein using a design strategy based on mutually exclusive domain interactions. These so-called VividZn proteins consist of two light-responsive Vivid domains that homodimerize upon illumination with blue light, thus preventing the binding of Zn(2+) between two Zn(2+) binding domains, Atox1 and WD4. Following optimization of the linker between WD4 and the N-terminus of one of the Vivid domains, VividZn variants were obtained that show a 9- to 55-fold decrease in Zn(2+) affinity upon illumination, which is fully reversible following dark adaptation. The Zn(2+) affinities of the switch could be rationally tuned between 1 pM and 2 nM by systematic variation of linker length and mutation of one of the Zn(2+) binding residues. Similarly, introduction of mutations in the Vivid domains allowed tuning of the switching kinetics between 10 min and 7 h. Low expression levels in mammalian cells precluded the demonstration of light-induced perturbation of cytosolic Zn(2+) levels. Nonetheless, our results firmly establish the use of intramolecular Vivid dimerization as an attractive light-sensitive input module to rationally engineer light-responsive protein switches based on mutually exclusive domain interactions.

Colorful protein-based fluorescent probes for collagen imaging.

Real-time visualization of collagen is important in studies on tissue formation and remodeling in the research fields of developmental biology and tissue engineering. Our group has previously reported on a fluorescent probe for the specific imaging of collagen in live tissue in situ, consisting of the native collagen binding protein CNA35 labeled with fluorescent dye Oregon Green 488 (CNA35-OG488). The CNA35-OG488 probe has become widely used for collagen imaging. To allow for the use of CNA35-based probes in a broader range of applications, we here present a toolbox of six genetically-encoded collagen probes which are fusions of CNA35 to fluorescent proteins that span the visible spectrum: mTurquoise2, EGFP, mAmetrine, LSSmOrange, tdTomato and mCherry. While CNA35-OG488 requires a chemical conjugation step for labeling with the fluorescent dye, these protein-based probes can be easily produced in high yields by expression in E. coli and purified in one step using Ni2+-affinity chromatography. The probes all bind specifically to collagen, both in vitro and in porcine pericardial tissue. Some first applications of the probes are shown in multicolor imaging of engineered tissue and two-photon imaging of collagen in human skin. The fully-genetic encoding of the new probes makes them easily accessible to all scientists interested in collagen formation and remodeling.

MagFRET: the first genetically encoded fluorescent Mg2+ sensor.

Magnesium has important structural, catalytic and signaling roles in cells, yet few tools exist to image this metal ion in real time and at subcellular resolution. Here we report the first genetically encoded sensor for Mg(2+), MagFRET-1. This sensor is based on the high-affinity Mg(2+) binding domain of human centrin 3 (HsCen3), which undergoes a transition from a molten-globular apo form to a compactly-folded Mg(2+)-bound state. Fusion of Cerulean and Citrine fluorescent domains to the ends of HsCen3, yielded MagFRET-1, which combines a physiologically relevant Mg(2+) affinity (K d = 148 µM) with a 50% increase in emission ratio upon Mg(2+) binding due to a change in FRET efficiency between Cerulean and Citrine. Mutations in the metal binding sites yielded MagFRET variants whose Mg(2+) affinities were attenuated 2- to 100-fold relative to MagFRET-1, thus covering a broad range of Mg(2+) concentrations. In situ experiments in HEK293 cells showed that MagFRET-1 can be targeted to the cytosol and the nucleus. Clear responses to changes in extracellular Mg(2+) concentration were observed for MagFRET-1-expressing HEK293 cells when they were permeabilized with digitonin, whereas similar changes were not observed for intact cells. Although MagFRET-1 is also sensitive to Ca(2+), this affinity is sufficiently attenuated (K d of 10 µM) to make the sensor insensitive to known Ca(2+) stimuli in HEK293 cells. While the potential and limitations of the MagFRET sensors for intracellular Mg(2+) imaging need to be further established, we expect that these genetically encoded and ratiometric fluorescent Mg(2+) sensors could prove very useful in understanding intracellular Mg(2+) homeostasis and signaling.

No washing, less waiting: engineering biomolecular reporters for single-step antibody detection in solution.

Detection of antibodies is essential for the diagnosis of many disease states, including infectious diseases, autoimmune diseases and allergies. Most current antibody detection assays involve multistep detection schemes in which molecular recognition and signal generation are separate processes. A well-known example is the enzyme-linked immunosorbent assay (ELISA), which combines high sensitivity and specificity with strong signal amplification. However, ELISA and other heterogeneous methods require multiple, time-consuming washing and incubation steps, which limits their applicability in point-of-care diagnostics and high-throughput applications. In recent years, several new antibody detection strategies have been developed in which antibody binding and signal generation are integrated within a single biomolecular reporter. These strategies aim to rival ELISA in terms of sensitivity and specificity, while decreasing the time and effort required to perform an assay. Here, we review recent developments in this field according to their mechanism of action and discuss their advantages and limitations.

Switchable reporter enzymes based on mutually exclusive domain interactions allow antibody detection directly in solution.

Detection of antibodies is essential for the diagnosis of many diseases including infections, allergies, and autoimmune diseases. Current heterogeneous immunoassays require multiple time-consuming binding and washing steps, which limits their application in point-of-care diagnostics and high-throughput screening. Here, we report switchable reporter enzymes that allow simple colorimetric detection of antibodies directly in solution. Our approach is based on the antibody-induced disruption of an intramolecular interaction between TEM1 ß-lactamase and its inhibitor protein BLIP. Using the HIV1-p17 antibody as an initial target, the interaction between enzyme and inhibitor was carefully tuned to yield a reporter enzyme whose activity increased 10-fold in the presence of pM antibody concentrations. Reporter enzymes for two other antibodies (HA-tag and Dengue virus type I) were obtained by simply replacing the epitope sequences. This new sensor design represents a modular and generic approach to construct antibody reporter enzymes without the cumbersome optimization required by previous engineering strategies.