Small-Volume Blood Collection Tubes to Reduce Transfusions in Intensive Care: The STRATUS Randomized Clinical Trial.

Importance: Blood collection for laboratory testing in intensive care unit (ICU) patients is a modifiable contributor to anemia and red blood cell (RBC) transfusion. Most blood withdrawn is not required for analysis and is discarded. Objective: To determine whether transitioning from standard-volume to small-volume vacuum tubes for blood collection in ICUs reduces RBC transfusion without compromising laboratory testing procedures. Design, Setting, and Participants: Stepped-wedge cluster randomized trial in 25 adult medical-surgical ICUs in Canada (February 5, 2019 to January 21, 2021). Interventions: ICUs were randomized to transition from standard-volume (n = 10 940) to small-volume tubes (n = 10 261) for laboratory testing. Main Outcomes and Measures: The primary outcome was RBC transfusion (units per patient per ICU stay). Secondary outcomes were patients receiving at least 1 RBC transfusion, hemoglobin decrease during ICU stay (adjusted for RBC transfusion), specimens with insufficient volume for testing, length of stay in the ICU and hospital, and mortality in the ICU and hospital. The primary analysis included patients admitted for 48 hours or more, excluding those admitted during a 5.5-month COVID-19-related trial hiatus. Results: In the primary analysis of 21 201 patients (mean age, 63.5 years; 39.9% female), which excluded 6210 patients admitted during the early COVID-19 pandemic, there was no significant difference in RBC units per patient per ICU stay (relative risk [RR], 0.91 [95% CI, 0.79 to 1.05]; P = .19; absolute reduction of 7.24 RBC units/100 patients per ICU stay [95% CI, -3.28 to 19.44]). In a prespecified secondary analysis (n = 27 411 patients), RBC units per patient per ICU stay decreased after transition from standard-volume to small-volume tubes (RR, 0.88 [95% CI, 0.77 to 1.00]; P = .04; absolute reduction of 9.84 RBC units/100 patients per ICU stay [95% CI, 0.24 to 20.76]). Median decrease in transfusion-adjusted hemoglobin was not statistically different in the primary population (mean difference, 0.10 g/dL [95% CI, -0.04 to 0.23]) and lower in the secondary population (mean difference, 0.17 g/dL [95% CI, 0.05 to 0.29]). Specimens with insufficient quantity for analysis were rare (≤0.03%) before and after transition. Conclusions and Relevance: Use of small-volume blood collection tubes in the ICU may decrease RBC transfusions without affecting laboratory analysis. Trial Registration: ClinicalTrials.gov Identifier: NCT03578419.

Using Proteins As Markers for Anabolic Steroid Abuse: A New Perspective in Doping Control?

Drug toxicity is a major concern and has motivated numerous studies to elucidate specific adverse mechanisms, with acetaminophen being the favorite candidate in toxicology studies. Conversely, androgenic anabolic steroids (AASs) also represent a severe public health issue in sports for elite and non-elite athletes. Supraphysiological dosages of AASs are associated with various adverse effects, from cardiovascular to neurological repercussions including liver dysfunction. Yet, few studies have addressed the toxicity of anabolic steroids, and a significant amount of work will be needed to elucidate and understand steroid toxicity properly. This Perspective suggests ideas on how proteomics and liquid chromatography coupled with high-resolution tandem mass spectrometry (LC-HRMS/MS) can contribute to (1) pinpoint serum proteins affected by substantial doses of anabolic steroids that would represent interesting novel candidates for routine testing and (2) provide additional knowledge on androgenic anabolic steroid toxicity to help raise awareness on the harmful effects.

Detection of activin receptor type IIA and IIB-Fc fusion proteins by automated capillary immunoassay.

Activin receptor type IIA and type IIB fusion protein have been designed to sequester circulating molecules of the transforming growth factor-ß (TGF-ß) superfamily and inactivate their actions. Members of this superfamily have been reported as essential regulators of erythropoiesis by triggering the formation of activated ternary complexes containing different combinations of type I and type II receptors, which can limit RBC production by accelerating erythroid differentiation and inhibiting erythroid progenitor expansion. The recent approval of Luspatercept for the treatment of anemia associated to transfusion-dependent MDS and Beta-thalassemia in afflicted patients means that it can now pose a real threat of being abused in sport for its ability to stimulate erythropoiesis. Several methods for the detection of these molecules in blood have been proposed for the purpose of sport antidoping control. Here we propose the detection of the ActRIIA-Fc and ActRIIB-Fc fusion proteins by automated capillary Western immunoassay (Simple Western). The use of these immunoassays for the detection of protein targets has become widespread in the recent years. The work presented here demonstrates that this methodology enables a versatile, rapid, and sensitive detection of activin ligand traps in blood samples: plasma, serum, or dried blood spots (DBS). Preliminary results indicate that detection in urine samples is also possible. The option to use different antibodies allows the possibility to use this method as an initial testing procedure as well as a confirmation procedure. Finally, results coming from an administration study confirm that the method is suitable for routine analysis.

Standardization of reticulocyte counts in the athlete biological passport.

INTRODUCTION: The percentage of circulating reticulocytes (RET%) is a useful marker of blood doping in the context of the Athlete Biological Passport (ABP). The viability of the ABP depends on the comparability of sample data obtained across multiple laboratories for a given athlete. With the recent introduction of a different technology for the measurement of reticulocytes, the goal of this study was to compare currently employed Sysmex XT/XE analyzers to the recently introduced Sysmex XN analyzer. METHODS: RET% differences were searched in two independent data sets, the first consisting of 95 369 RET% values coming from 29 laboratories located in five continents as part of routine testing for the ABP, the second from a targeted study involving 510 samples analyzed on both a Sysmex XT and XN analyzers by two different laboratories. RESULTS: A relatively small but significant bias of 0.27 ([0.22-0.35] 95% CI) for the first data set and 0.19% ([0.16-0.22] 95% CI) for the second data set was observed with Sysmex XN analyzers returning higher values than Sysmex XT/XE analyzers. This bias appears constant over most of the range of RET% measured in elite athletes. CONCLUSION: When RET% values are obtained for the same athlete with different technologies (XT/XE vs XN), an adjustment of RET% emanating from the XT/XE instruments through a decrease of 0.22% within the ABP calculated ranges appears to be sufficient to integrate the results from the two technologies.

Detection of erythropoiesis stimulating agents in urine samples using a capillary Western system.

The presence of erythropoiesis stimulating agents (ESAs) in the urine samples collected from athletes is detected using traditional Western blotting following either size-based separation (SDS/SAR-PAGE) or isoelectric focusing (IEF). Although there is an important testing effort, there is little doubt that ESAs are still abused in sports and that reducing the costs of the tests might increase the number of tests and improve deterrence. The capillary electrophoresis system developed by Protein Simple may be useful to this end. This platform is fully automated and could be easily implemented in anti-doping laboratories, which would contribute to the improvement of the overall assay performance and standardization of the method. Such an automated system could be of interest during major sports events, such as the Olympic Games, where a high number of samples needs to be analyzed in a short period of time. From the experiments conducted so far, we conclude that the technique is promising, with the sensitivity and reproducibility needed to screen ESAs in human urine samples.

Drainage of Cerebrospinal Fluid and Blood Pressure Augmentation as Rescue Therapies for Ischemic Myelitis After Bronchial Embolization: A Case Report.

A 62-year-old man presented to the emergency department with massive hemoptysis. After bronchial artery embolization, he developed ischemic myelitis, a rare complication in this setting for which no specific therapy is currently recommended. The symptoms were managed with lumbar drainage of cerebrospinal fluid and blood pressure augmentation therapy. To our knowledge, this is the first report of using lumbar drainage of cerebrospinal fluid and blood pressure augmentation in the treatment of anterior medullary ischemia after bronchial artery embolization for massive hemoptysis. The treatment was associated with neurological recovery.

Immunomagnetic beads-based isolation of erythropoietins from urine and blood for sports anti-doping control.

According to the World Anti-Doping Agency (WADA) technical document for erythropoiesis stimulating agents (ESA) analysis (TD2014EPO), double-blotting of serum/plasma samples is mandatory for all analysis by isoelectric focusing (IEF) and for the confirmation procedures (CP) performed by SDS-PAGE or SAR-PAGE. The goal is to prevent potential cross-reactions of the secondary antibody with remaining proteins in the purified samples. To this end, we have developed an immunopurification method of ESA in serum/plasma samples using a combination of streptavidin-coated immunomagnetic beads and biotinylated anti-EPO polyclonal antibodies. Here we report that this immunomagnetic bead-based purification allows the analysis of serum/plasma samples by single-blotting. Serum and plasma samples, either intact or spiked with different ESAs, were immunopurified and analyzed by single-blotting, after SAR-PAGE or IEF using a cross-reaction minimized secondary antibody coupled to HRP. The results show that when samples are immunopurified according to this strategy, there is no non-specific binding when single-blotting is performed after SAR-PAGE. With IEF, we observe a faint smearing, however, in the pH gradient outside the ESA detection region. These interferences did not alter ESA profiles of spiked urinary samples or of samples received for routine testing. This approach was compared to the MAIIA monoliths purification or to the isolation of ESAs with other combinations of immunomagnetic reagents (ie, anti-Mouse IgG-coated magnetic beads and anti-EPO mAb). The recovery of ESAs was shown to be significant for serum/plasma samples. Our results suggest that single-blotting could be performed on serum/plasma samples without non-specific interferences. Copyright © 2017 John Wiley & Sons, Ltd.

Changes in Urinary and Serum Levels of Novel Biomarkers after Administration of Gadolinium-based Contrast Agents.

OBJECTIVE: The aim of our study is to describe the changes in urinary and serum levels of novel biomarkers after gadolinium contrast administration in patients with normal renal function. METHODS: We measured four biomarkers in 28 volunteers: interleukin-18 (IL-18), N-acetyl-glucosaminidase (NAG), neutrophil gelatinase-associated lipocalin, and cystatin C. Urinary and serum samples were collected at 0, 3, and 24 hours following gadolinium administration. RESULTS: Baseline serum creatinine was 57.8 ± 34.5 µmol/L and remained stable. Urinary IL-18 levels increased significantly at three hours (10.7 vs. 7.3 ng/mg creatinine; P < 0.05). Similarly, urinary NAG levels increased significantly at three hours (3.9 vs. 2.2 IU/mg creatinine; P < 0.001). For both these markers, the difference was no longer significant at 24 hours. No statistically significant differences were observed for urinary and serum neutrophil gelatinase-associated lipocalin levels and for serum cystatin C levels. CONCLUSIONS: Urinary IL-18 and NAG levels increased transiently after administration of gadolinium-based contrast agents in patients with normal renal function.

Desialylation improves the detection of recombinant erythropoietins in urine samples analyzed by SDS-PAGE.

Recombinant erythropoietin (rhEPO) has been misused for over two decades by athletes, mainly but not only in endurance sports. A direct rhEPO detection method in urine by isoelectric focusing (IEF) was introduced in 2000, but the emergence of third-generation erythropoiesis-stimulating agents and so-called biosimilar rhEPOs, together with the sensitivity of human endogenous EPO (huEPO) pattern to enzymatic activities and its modification following short strenuous exercise, prompted the development of a complementary test based on SDS-PAGE analysis. While Mircera and NESP are easily detected with the existing IEF and SDS-PAGE methods, some samples containing both epoetin-α/ß and huEPO present profiles that are still difficult to interpret. As doping practices have moved to micro-dosing, these mixed patterns are more frequently observed. We investigated the impact of enzymatic desialylation on the urinary and serum EPO profiles obtained by SDS-PAGE with the aim of improving the separation of the bands in these mixed EPO populations. We observed that the removal with neuraminidase of the sialic acid moieties from the different EPOs studied reduced their apparent molecular weight (MW) and increased the migration distance between huEPO and rhEPO centroids, therefore eliminating the size overlaps between them and improving the detection of rhEPO.

New structural determinants for c-Myc specific heterodimerization with Max and development of a novel homodimeric c-Myc b-HLH-LZ.

c-Myc must heterodimerize with Max to accomplish its functions as a transcription factor. This specific heterodimerization occurs through the b-HLH-LZ (basic region, helix 1-loop-helix 2-leucine zipper) domains. In fact, many studies have shown that the c-Myc b-HLH-LZ (c-Myc'SH) preferentially forms a heterodimer with the Max b-HLH-LZ (Max'SH). The primary mechanism underlying the specific heterodimerization lies on the destabilization of both homodimers and the formation of a more stable heterodimer. In this regard, it has been widely reported that c-Myc'SH has low solubility and homodimerizes poorly and that repulsions within the LZ domain account for the homodimer instability. Here, we show that replacing one residue in the basic region and one residue in Helix 1 (H(1)) of c-Myc'SH with corresponding residues conserved in b-HLH proteins confers to c-Myc'SH a higher propensity to form a stable homodimer in solution. In stark contrast to the wild-type protein, this double mutant (L362R, R367L) of the c-Myc b-HLH-LZ (c-Myc'RL) shows limited heterodimerization with Max'SH in vitro. In addition, c-Myc'RL forms highly stable and soluble complexes with canonical as well as non-canonical E-box probes. Altogether, our results demonstrate for the first time that structural determinants driving the specific heterodimerization of c-Myc and Max are embedded in the basic region and H(1) of c-Myc and that these can be exploited to engineer a novel homodimeric c-Myc b-HLH-LZ with the ability of binding the E-box sequence autonomously and with high affinity.

The Arf tumor suppressor protein inhibits Miz1 to suppress cell adhesion and induce apoptosis.

Oncogenic stress induces expression of the alternate reading frame (Arf) tumor suppressor protein. Arf then stabilizes p53, which leads to cell cycle arrest or apoptosis. The mechanisms that distinguish both outcomes are incompletely understood. In this study, we show that Arf interacts with the Myc-associated zinc finger protein Miz1. Binding of Arf disrupts the interaction of Miz1 with its coactivator, nucleophosmin, induces the sumoylation of Miz1, and facilitates the assembly of a heterochromatic complex that contains Myc and trimethylated H3K9 in addition to Miz1. Arf-dependent assembly of this complex leads to the repression of multiple genes involved in cell adhesion and signal transduction and induces apoptosis. Our data point to a tumor-suppressive pathway that weakens cell-cell and cell-matrix interactions in response to expression of Arf and that may thereby facilitate the elimination of cells harboring an oncogenic mutation.

The Max homodimeric b-HLH-LZ significantly interferes with the specific heterodimerization between the c-Myc and Max b-HLH-LZ in absence of DNA: a quantitative analysis.

Specific heterodimerization plays a crucial role in the regulation of the biology of the cell. For example, the specific heterodimerization between the b-HLH-LZ transcription factors c-Myc and Max is a prerequisite for c-Myc transcriptional activity that leads to cell growth, proliferation and tumorigenesis. On the other hand, the Mad proteins can compete with c-Myc for Max. The Mad/Max heterodimer antagonizes the effect of the c-Myc/Max heterodimer. In this contribution, we have focused on the specific heterodimerization between the b-HLH-LZ domains of c-Myc and Max using CD and NMR. While the c-Myc and Max b-HLH-LZ domains are found to preferentially form a heterodimer; we demonstrate for the first time that a significant population of the Max homodimeric b-HLH-LZ can also form and hence interferes significantly with the specific heterodimerization. This indicates that the Max/Max homodimer can also interfere with c-Myc/Max functions, therefore adding to the complexity of the regulation of transcription by the Myc/Max/Mad network. The demonstration of the existence of the homodimeric population was made possible by the application of numerical routines that enable the simulation of composite spectroscopic signal (e.g. CD) as a function of temperature and total concentration of proteins. From a systems biology perspective, our routines may be of general interest as they offer the opportunity to treat many competing equilibriums in order to predict the probability of existence of protein complexes.

Elucidation of the structural determinants responsible for the specific formation of heterodimeric Mxd1/Max b-HLH-LZ and its binding to E-box sequences.

The proteins of the Mxd family (formally known as Mad) are antagonists of the oncoprotein c-Myc. They compete with c-Myc for their obligate partner Max to prevent the c-Myc/Max heterodimer from binding to E-box sequences in the target gene promoters. In cancer cells, where Myc is overexpressed, the expression of Mxd proteins is usually insufficient or abrogated. However, the reintroduction of Mxd1 expression in these cells prevents growth and proliferation. While the antagonism of c-Myc functions by Mxd proteins is of potential relevance for the development of cancer treatment strategies, the structural determinants responsible for the specific heterodimerization between the Mxd and the Max b-helix-loop-helix-leucine zippers are not fully understood. Moreover, whether the heterodimer is assembled on DNA or in the nucleoplasm prior to DNA binding is under debate. In this article, we demonstrate that Mxd1 D112a and Max N78a and H81d, which are located in the leucine zippers of the proteins, can dictate the specificity of heterodimerization and whether or not the Mxd1/Max/DNA complex forms. Our results also indicate that additional specific determinants exist in the helix-loop-helix domains of Max and Mxd1. Finally, we provide evidence that heterodimerization must precede DNA binding in vivo.

Permanent renal failure induced by pentastarch.

Background. Controversy exists with volume resuscitation using crystalloids or colloids. Renal dysfunction has been reported with some colloids and osmotic agents, but remains poorly defined. Patient. We report the case of a 67-year-old male who had normal kidney function at baseline and who developed anuric ARF in relation to the administration of >10 litres of 10% pentastarch. A renal biopsy confirmed hydropic changes in tubular cells compatible with colloid-induced damage. Conclusion. This case demonstrates that hydroxyethyl starch preparations may be associated with acute kidney injury, and one should carefully consider their use, especially in patients with pre-existing renal dysfunction. Osmotic tubular cell lesions may be long lasting and irreversible.

Thermodynamics of b-HLH-LZ protein binding to DNA: the energetic importance of protein-DNA contacts in site-specific E-box recognition by the complete gene product of the Max p21 transcription factor.

The Myc/Mad/Max network of dimeric basic region-helix-loop-helix-leucine zipper (b-HLH-LZ) transcription factors bind to enhancer box sequences (E-box) in the promotors of a large set of genes that control cell metabolism, proliferation, and differentiation. Max (Myc-associated factor X) is the obligate heterodimerization partner of Myc and Mad proteins. On the other hand, Max is the only member of the family capable of forming a stable homodimer. As part of the transcriptional regulation mechanism, Myc/Max and Mad/Max heterodimers and Max homodimers are thought to compete for binding to the E-box target sequences. E-box recognition is structurally supported by the b-HLH-LZ structural motif, which also promotes dimerization. However, the actual dimerization and heterodimerization constants of the complete gene products and their affinities for E-box sequences are not known. Also, the detailed thermodynamic characterization of DNA binding by these transcription factors has not been done yet. Such knowledge is necessary for complete understanding of the transcriptional regulation carried out by the Myc/Mad/Max network. Here, we report the first in-depth thermodynamic characterization of the stability and specific DNA binding of a full length gene product of the Myc/Mad/Max family, namely, Max protein isoform p21 (Max p21). Using calorimetric methods (DSC and ITC) we have determined the dimerization constant of Max p21 in the low micromolar range, and the Max p21/E-box complex dissociation constant in the low nanomolar range at 37 degrees C. The association is driven by a large exothermic effect, which is partly compensated by entropic factors. The energetic contribution to binding affinity of seven highly conserved residues that contact the DNA was probed by X-to-Ala mutagenesis. The results demonstrate that high binding affinity critically relies on the side chain of Arg 26. Furthermore, the mutational analysis points to the important role of the persistent helical turn that comprises this residue at the junction of the basic region and helix H1. Altogether, the study supports the idea that Max p21 can bind E-box sequences in vivo and likely participates directly in the regulation of transcription as homodimer.

The mechanism of discrimination between cognate and non-specific DNA by dimeric b/HLH/LZ transcription factors.

The Myc/Max/Mad proteins are basic region-helix-loop-helix-leucine zipper (b/HLH/LZ) transcription factors that regulate the transcription of numerous genes involved in cell growth and proliferation. The Max protein is the obligate heterodimeric partner of the Myc and Mad proteins. Heterodimerization and DNA binding to target gene promoters are mediated by the b/HLH/LZ domains. Max can also form a homodimeric b/HLH/LZ. The enhanced expression of Myc and binding to promoters of target genes contribute to almost every aspect of tumor biology. However, the detailed mechanism by which dimeric and heterodimeric b/HLH/LZs discriminate cognate DNA (E-Box: CACGTG) from non-specific sequences in the target gene promoters is still unknown. Here, we use the Max b/HLH/LZ homodimer as a model for this class of transcription factors in the characterization and understanding of the mechanism of discrimination between the E-Box and non-specific DNA sequences. We report the characterization of a cognate and a non-specific Max b/HLH/LZ/DNA complex by EMSA, CD and NMR. Our results support a detailed mechanism by which dimeric b/HLH/LZ transcription factors can discriminate E-Box sequences from non-specific DNA. The mechanism proceeds via the conformational selection of fitting b/HLH/LZ homodimers with the basic region only partially helical. Next, the basic region undergoes a DNA-assisted folding or induced-fit. It is this step that provides the discrimination by stabilizing and destabilizing the alpha-helical conformation of the basic region in the cognate and non-specific complexes, respectively. This leads to a low affinity complex with a higher probability of being dissociated and hence to discrimination. A description of the side-chains and nucleotides proposed to be involved in the discrimination process is provided.

Structural and thermodynamical characterization of the complete p21 gene product of Max.

The b-HLH-LZ family of transcription factors contains numerous proteins including the Myc and Mad families of proteins. Max heterodimerizes with other members to bind the E-Box DNA sequence in target gene promoters. Max is the only protein in this network that recognizes and binds E-Box DNA sequences as a homodimer in vitro and represses transcription of Myc target genes in vivo. Key information such as the structure of p21 Max, the complete gene product, and its KD in the absence of DNA are still unknown. Here, we report the characterization of the secondary and quaternary structures, the dimerization and DNA binding of p21 Max and a thermodynamically stable mutant. The helical content of p21 Max indicates that its N-terminal and C-terminal regions are unstructured in the absence of DNA. NMR experiments further support the location of folded and unfolded domains. We also show that p21 Max has an apparent KD (37 degrees C) of 7 x 10(-6), a value 10-100 times smaller than the b-HLH-LZ itself. We demonstrate that electrostatic repulsions are responsible for the higher KD of the b-HLH-LZ. Finally, we show that a p21 Max double mutant forms a very stable dimer with a KD (37 degrees C) of 3 x 10(-10) and that the protein/DNA complex depicts a higher temperature of denaturation than p21 Max/DNA complex. Our results indicate that Max could homodimerize, bind DNA, and repress transcription in vivo and that its mutant could be more efficient at repressing the expression of c-Myc target genes.

Toward the elucidation of the structural determinants responsible for the molecular recognition between Mad1 and Max.

Mad1 is a member of the Mad family. This family is part of the larger Myc/Max/Mad b-HLH-LZ eukaryotic transcription-factor network. Mad1 forms a specific heterodimer with Max and acts as a transcriptional repressor when bound to an E-box sequence (CACGTG) found in the promoter of c-Myc target genes. Mad1 cannot form a complex with DNA by itself under physiological conditions. A global model for the molecular recognition has emerged in which the Mad1 b-HLH-LZ homodimer is destabilized and the Mad/Max b-HLH-LZ heterodimer is favored. The detailed structural determinants responsible for the molecular recognition remain largely unknown. In this study, we focus on the elucidation of the structural determinants responsible for the destabilization of the Mad1 b-HLH-LZ homodimer. Conserved acidic residues at the dimerization interface (position a) of the LZ of all Max-interacting proteins have been hypothesized to be involved in the destabilization of the homodimeric states. In Mad1, this position corresponds to residue Asp 112. As reported for the complete gene product of Mad1, we show that wild-type b-HLH-LZ does not homodimerize or bind DNA under physiological conditions. On the other hand, the single mutation of Asp 112 to an Asn enables the b-HLH-LZ to dimerize and bind DNA. Our results suggest that Asp 112 is implicated in the destabilization of Mad1 b-HLH-LZ homodimer. Interestingly, this side chain is observed to form a salt bridge at the interface of the LZ domain in the crystal structure of Mad1/Max heterodimeric b-HLH-LZ bound to DNA [Nair, S. K., and Burley, S. K. (2003) Cell 112, 193-205]. This clearly suggests that Asp 112 plays a crucial role in the molecular recognition between Max and Mad1.