Albumin determined by bromocresol green leads to erroneous results in routine evaluation of patients with chronic kidney disease.

OBJECTIVES: Measurement of plasma albumin is pivotal for clinical decision-making in patients with chronic kidney disease (CKD). Routinely used methods as bromocresol green (BCG) and bromocresol purple (BCP) can suffer from aselectivity, but the impact of aselectivity on the accuracy of plasma albumin results of CKD-patients is still unknown. Therefore, we evaluated the performance of BCG-, BCP- and JCTLM-endorsed immunological methods in patients with various stages of CKD. METHODS: We evaluated the performance of commonly used albumin methods in patients with CKD stages G1 through G5, the latter divided in two groups based on whether they received hemodialysis treatment. In total, 163 patient plasma samples were measured at 14 laboratories, on six different BCG and BCP-platforms, and four different immunological platforms. The results were compared with an ERM-DA-470k-corrected nephelometric assay. The implications on outcome is evaluated by the proportion of patient results <38 g/L for the diagnosis of protein energy wasting. RESULTS: Albumin results determined with BCP- and immunological methods showed the best agreement with the target value (92.7 and 86.2 %, respectively vs. 66.7 % for BCG, namely due to overestimation). The relative agreement of each method with the target value was platform-dependent, with larger variability in agreement between platforms noted for BCG and immunological methods (3.2-4.6 and 2.6-5.3 %) as opposed to BCP (0.7-1.5 %). The stage of CKD had similar effects on the variability in agreement for the three method-groups (0.6-1.8 % vs. 0.7-1.5 % vs. 0.4-1.6 %). The differences between methods cause discrepancies in clinical decision-making, as structurally fewer patients were diagnosed with protein energy wasting upon using BCG-based albumin results. CONCLUSIONS: Our study shows that BCP is fit for the intended use to measure plasma albumin levels in CKD patients from all stages, including patients on hemodialysis. In contrast, most BCG-based platforms falsely overestimate the plasma albumin concentration.

Automated prediction of low ferritin concentrations using a machine learning algorithm.

OBJECTIVES: Computational algorithms for the interpretation of laboratory test results can support physicians and specialists in laboratory medicine. The aim of this study was to develop, implement and evaluate a machine learning algorithm that automatically assesses the risk of low body iron storage, reflected by low ferritin plasma levels, in anemic primary care patients using a minimal set of basic laboratory tests, namely complete blood count and C-reactive protein (CRP). METHODS: Laboratory measurements of anemic primary care patients were used to develop and validate a machine learning algorithm. The performance of the algorithm was compared to twelve specialists in laboratory medicine from three large teaching hospitals, who predicted if patients with anemia have low ferritin levels based on laboratory test reports (complete blood count and CRP). In a second round of assessments the algorithm outcome was provided to the specialists in laboratory medicine as a decision support tool. RESULTS: Two separate algorithms to predict low ferritin concentrations were developed based on two different chemistry analyzers, with an area under the curve of the ROC of 0.92 (Siemens) and 0.90 (Roche). The specialists in laboratory medicine were less accurate in predicting low ferritin concentrations compared to the algorithms, even when knowing the output of the algorithms as support tool. Implementation of the algorithm in the laboratory system resulted in one new iron deficiency diagnosis on average per day. CONCLUSIONS: Low ferritin levels in anemic patients can be accurately predicted using a machine learning algorithm based on routine laboratory test results. Moreover, implementation of the algorithm in the laboratory system reduces the number of otherwise unrecognized iron deficiencies.

Rapid identification of SARS-CoV-2-infected patients at the emergency department using routine testing.

Objectives: The novel coronavirus disease 19 (COVID-19), caused by SARS-CoV-2, spreads rapidly across the world. The exponential increase in the number of cases has resulted in overcrowding of emergency departments (ED). Detection of SARS-CoV-2 is based on an RT-PCR of nasopharyngeal swab material. However, RT-PCR testing is time-consuming and many hospitals deal with a shortage of testing materials. Therefore, we aimed to develop an algorithm to rapidly evaluate an individual's risk of SARS-CoV-2 infection at the ED. Methods: In this multicenter retrospective study, routine laboratory parameters (C-reactive protein, lactate dehydrogenase, ferritin, absolute neutrophil and lymphocyte counts), demographic data and the chest X-ray/CT result from 967 patients entering the ED with respiratory symptoms were collected. Using these parameters, an easy-to-use point-based algorithm, called the corona-score, was developed to discriminate between patients that tested positive for SARS-CoV-2 by RT-PCR and those testing negative. Computational sampling was used to optimize the corona-score. Validation of the model was performed using data from 592 patients. Results: The corona-score model yielded an area under the receiver operating characteristic curve of 0.91 in the validation population. Patients testing negative for SARS-CoV-2 showed a median corona-score of 3 vs. 11 (scale 0-14) in patients testing positive for SARS-CoV-2 (p<0.001). Using cut-off values of 4 and 11 the model has a sensitivity and specificity of 96 and 95%, respectively. Conclusions: The corona-score effectively predicts SARS-CoV-2 RT-PCR outcome based on routine parameters. This algorithm provides the means for medical professionals to rapidly evaluate SARS-CoV-2 infection status of patients presenting at the ED with respiratory symptoms.

Improved prospective risk analysis for clinical laboratories compensated for the throughput in processes.

BACKGROUND: Practical application of prospective risk analysis (PRA) in clinical laboratories should reflect processes as they are carried out, while making the PRA results obtained from different processes comparable. This means that not only STAT and standard testing and testing for critical and less critical parameters should be distinguished (as published), but also that the throughput in processes and process steps should be taken into account. METHODS: Building on our previously published PRA, a method was developed to compensate for the throughput in processes and process steps. A factor T, related to the actually observed throughput, was introduced in the risk score calculation. Introduction of this compensation factor leads to different overall risk scores. The criteria by which the risk scores are evaluated were modified accordingly. RESULTS: Introduction of a factor in the PRA to compensate for throughput leads to a change in the risk score for various conceivable failures in process steps. As compared to the PRA in which no compensation for throughput is made, in a process with low throughput the risk score for various conceivable failures in process steps comes out higher after introduction of the compensation factor, while in a process with high throughput various risk scores come out lower. CONCLUSIONS: Introduction of a factor to account for the throughput in a process (and process steps) leads to an improved, more realistic PRA, the results of which makes the risk scores of different processes (and process steps) better comparable to each other.

Practical motives are prominent in test-ordering in the Emergency Department.

BACKGROUND: Laboratory test ordering under time pressure may impact test-ordering behavior. METHODS: To investigate the test-ordering behavior of doctors working under such pressure, we designed a questionnaire for trainees and staff in the Emergency Department (ED). This questionnaire addressed topics such as necessity of requested tests, time spent on ordering, costs and availability of tests, and the time of the day. We hypothesized that ordering behavior would be guided predominantly by the medical need of tests and aimed at identifying practical motives that also have an effect. RESULTS: Remarkably, two-third of the respondents (67%) admitted that tests were ordered that would not influence treatment policy directly and 48% of the doctors stated that tests were ordered that do not impact treatment at all. The frequency of such orders was "sometimes" and "frequent" in a 50:50 ratio. Interestingly, tests that could prove relevant at a later stage are often ordered simultaneously to reduce burden on the patient. None of the respondents spent more than 3 min on the ordering process and very few (8%) desired more time for ordering. Most respondents (81%) declared to have limited knowledge of the costs of laboratory tests. A random survey covering four tests confirmed this. Generally, turnaround time did influence ordering behavior while time of the day did not. CONCLUSIONS: In conclusion, doctors in an ED - besides first of all medical motives - heavily exploit practical (non-medical) reasoning for laboratory test ordering, e.g. taking availability of tests into account and ordering non-immediate tests.

Cell division: control of the chromosomal passenger complex in time and space.

The ultimate goal of cell division is equal transmission of the duplicated genome to two new daughter cells. Multiple surveillance systems exist that monitor proper execution of the cell division program and as such ensure stability of our genome. One widely studied protein complex essential for proper chromosome segregation and execution of cytoplasmic division (cytokinesis) is the chromosomal passenger complex (CPC). This highly conserved complex consists of Borealin, Survivin, INCENP, and Aurora B kinase, and has a dynamic localization pattern during mitosis and cytokinesis. Not surprisingly, it also performs various functions during these phases of the cell cycle. In this review, we will give an overview of the latest insights into the regulation of CPC localization and discuss if and how specific localization impacts its diverse functions in the dividing cell.

FOXO4 transcriptional activity is regulated by monoubiquitination and USP7/HAUSP.

FOXO (Forkhead box O) transcription factors are important regulators of cellular metabolism, cell-cycle progression and cell death. FOXO activity is regulated by multiple post-translational modifications, including phosphorylation, acetylation and polyubiquitination. Here, we show that FOXO becomes monoubiquitinated in response to increased cellular oxidative stress, resulting in its re-localization to the nucleus and an increase in its transcriptional activity. Deubiquitination of FOXO requires the deubiquitinating enzyme USP7/HAUSP (herpesvirus-associated ubiquitin-specific protease), which interacts with and deubiquitinates FOXO in response to oxidative stress. Oxidative stress-induced ubiquitination and deubiquitination by USP7 do not influence FOXO protein half-life. However, USP7 does negatively regulate FOXO transcriptional activity towards endogenous promoters. Our results demonstrate a novel mechanism of FOXO regulation and indicate that USP7 has an important role in regulating FOXO-mediated stress responses.

Cell division control by the Chromosomal Passenger Complex.

The Chromosomal Passenger Complex (CPC) consisting of Aurora B kinase, INCENP, Survivin and Borealin, is essential for genomic stability by controlling multiple processes during both nuclear and cytoplasmic division. In mitosis it ensures accurate segregation of the duplicated chromosomes by regulating the mitotic checkpoint, destabilizing incorrectly attached spindle microtubules and by promoting the axial shortening of chromosomal arms in anaphase. During cytokinesis the CPC most likely prevents chromosome damage by imposing an abscission delay when a chromosome bridge connects the two daughter cells. Moreover, by controlling proper cytoplasmic division, the CPC averts tetraploidization. This review describes recent insights on how the CPC is capable of conducting its various functions in the dividing cell to ensure chromosomal stability.

Arginine methylation increases the stability of human immunodeficiency virus type 1 Tat.

Arginine methylation of human immunodeficiency virus type 1 (HIV-1) Tat protein downregulates its key function in viral-gene transactivation. The fate of methylated Tat is unknown, so it is unclear whether methylated Tat is degraded or persists in the cell for additional functions. Here we show that the arginine methyltransferase PRMT6 increases Tat protein half-life by 4.7-fold. Tat stabilization depends on the catalytic activity of PRMT6 and requires arginine methylation within the Tat basic domain. In contrast, HIV-1 Rev, which is also methylated by PRMT6, is completely refractory to the stabilizing effect. Proteasome inhibition and silencing experiments demonstrated that Tat can be degraded by a REGgamma-independent proteasome, against which PRMT6 appears to act to increase Tat half-life. Our data reveal a proteasome-dependent Tat degradation pathway that is inhibited by arginine methylation. The stabilizing action of PRMT6 could allow Tat to persist within the cell and the extracellular environment and thereby enable functions implicated in AIDS-related cancer, neurodegeneration, and T-cell death.

The Caenorhabditis elegans nicotinamidase PNC-1 enhances survival.

In yeast, increasing the copy number of the nicotinamide adenine dinucleotide (NAD)-dependent deacetylase Sir2 extends lifespan, which can be inhibited by nicotinamide (Nam), the end-product of Sir2-mediated NAD-breakdown. Furthermore, the yeast pyrazinamidase/nicotinamidase PNC-1 can extend yeast lifespan by converting Nam. In Caenorhabditis elegans (C. elegans), increased dosage of the gene encoding SIR-2.1 also increases lifespan. Here, we report that knockdown of the C. elegans homologue of yeast PNC-1 as well as growing worms on Nam-containing medium significantly decreases adult lifespan. Accordingly, increased gene dosage of pnc-1 increases adult survival under conditions of oxidative stress. These data show for the first time the involvement of PNC-1/Nam in the survival of a multicellular organism and may also contribute to our understanding of lifespan regulation in mammals.

Cell cycle inhibition by FoxO forkhead transcription factors involves downregulation of cyclin D.

The FoxO forkhead transcription factors FoxO4 (AFX), FoxO3a (FKHR.L1), and FoxO1a (FKHR) represent important physiological targets of phosphatidylinositol-3 kinase (PI3K)/protein kinase B (PKB) signaling. Overexpression or conditional activation of FoxO factors is able to antagonize many responses to constitutive PI3K/PKB activation including its effect on cellular proliferation. It was previously shown that the FoxO-induced cell cycle arrest is partially mediated by enhanced transcription and protein expression of the cyclin-dependent kinase inhibitor p27(kip1) (R. H. Medema, G. J. Kops, J. L. Bos, and B. M. Burgering, Nature 404:782-787, 2000). Here we have identified a p27(kip1)-independent mechanism that plays an important role in the antiproliferative effect of FoxO factors. Forced expression or conditional activation of FoxO factors leads to reduced protein expression of the D-type cyclins D1 and D2 and is associated with an impaired capacity of CDK4 to phosphorylate and inactivate the S-phase repressor pRb. Downregulation of D-type cyclins involves a transcriptional repression mechanism and does not require p27(kip1) function. Ectopic expression of cyclin D1 can partially overcome FoxO factor-induced cell cycle arrest, demonstrating that downregulation of D-type cyclins represents a physiologically relevant mechanism of FoxO-induced cell cycle inhibition.

Activation of FoxO transcription factors contributes to the antiproliferative effect of cAMP.

cAMP is a potent inhibitor of cell proliferation in a variety of cell lines. Downregulation of cyclin D1 and upregulation of the cell cycle inhibitor p27Kip1 are two mechanisms by which cAMP may induce a G1-arrest. Here we show that cAMP inhibits proliferation of cells that constitutively express cyclin D1 or are deficient for Rb, demonstrating that changes in these cell cycle regulators do not account for the cAMP-induced growth effects in mouse embryo fibroblasts (MEFs). Interestingly, the antiproliferative effect of cAMP mimics the effect previously observed for FoxO transcription factors. These transcription factors are under negative control of protein kinase B (PKB). We show that in MEFs cAMP strongly induces transcriptional activation of FoxO4 through the inhibition of PKB. Accordingly, not only p27Kip1 but also the FoxO target MnSOD is upregulated by cAMP. Importantly, introduction of dominant-negative FoxO partially rescues cAMP-induced inhibition of proliferation. From these results we conclude that inhibition of PKB and subsequent activation of FoxO transcription factors mediates an antiproliferative effect of cAMP.

Inter-domain Cooperation in INCENP Promotes Aurora B Relocation from Centromeres to Microtubules.

The chromosomal passenger complex is essential for error-free chromosome segregation and proper execution of cytokinesis. To coordinate nuclear division with cytoplasmic division, its enzymatic subunit, Aurora B, relocalizes from centromeres in metaphase to the spindle midzone in anaphase. In budding yeast, this requires dephosphorylation of the microtubule-binding (MTB) domain of the INCENP analog Sli15. The mechanistic basis for this relocalization in metazoans is incompletely understood. We demonstrate that the putative coiled-coil domain within INCENP drives midzone localization of Aurora B via a direct, electrostatic interaction with microtubules. Furthermore, we provide evidence that the CPC multimerizes via INCENP's centromere-targeting domain (CEN box), which increases the MTB affinity of INCENP. In (pro)metaphase, the MTB affinity of INCENP is outcompeted by the affinity of its CEN box for centromeres, while at anaphase onset­when the histone mark H2AT120 is dephosphorylated­INCENP and Aurora B switch from centromere to microtubule localization.

No association between transforming growth factor beta gene polymorphism and acute allograft rejection after cardiac transplantation.

Transforming growth factor-beta1 (TGF-beta1) is a multifunctional cytokine, which inhibits both development of Th1 and Th2 subsets and the Th1 proinflammatory response. TGF-beta1 production is influenced through several single nucleotide polymorphisms (SNP) in the structural gene and promoter region. Acute rejection of transplants depends on the Th1/Th2 balance within the graft, high levels of TGF-beta1 shift this balance towards Th2. We investigated whether genotypes of 4 SNP (-800 and -509 in the promoter region, codon 10 and codon 25 in the first exon) were correlated with cardiac disease or with incidence of rejection after heart transplantation (HTX). Genotypes were determined for 70 HTX patients and 61 donors by sequencing or oligonucleotide ligation assay. No association between SNP genotypes and heart disease or acute transplant rejection was observed. We conclude that genetic variation in the TGF-beta1 gene neither influences the existence of cardiomyopathy nor the incidence of rejection upon HTX.

Centrobin regulates centrosome function in interphase cells by limiting pericentriolar matrix recruitment.

The amount of pericentriolar matrix at the centrosome is tightly linked to both microtubule nucleation and centriole duplication, although the exact mechanism by which pericentriolar matrix levels are regulated is unclear. Here we show that Centrobin, a centrosomal protein, is involved in regulating these levels. Interphase microtubule arrays in Centrobin-depleted cells are more focused around the centrosome and are less stable than the arrays in control cells. Centrobin-depleted cells initiate microtubule nucleation more rapidly than control cells and exhibit an increase in the number of growing microtubule ends emanating from the centrosome, while the parameters of microtubule plus end dynamics around the centrosome are not significantly altered. Finally, we show that Centrobin depletion results in the increased recruitment of pericentriolar matrix proteins to the centrosome, including γ-tubulin, AKAP450, Kendrin and PCM-1. We propose that Centrobin might regulate microtubule nucleation and organization by controlling the amount of pericentriolar matrix.

The peptidyl-prolyl isomerase Pin1 regulates cytokinesis through Cep55.

Failure of cytokinesis results in tetraploidy and can increase the genomic instability frequently observed in cancer. The peptidyl-prolyl isomerase Pin1, which is deregulated in many tumors, regulates several processes, including cell cycle progression. Here, we show a novel role for Pin1 in cytokinesis. Pin1 knockout mouse embryonic fibroblasts show a cytokinesis delay, and depletion of Pin1 from HeLa cells also causes a cytokinesis defect. Furthermore, we provide evidence that Pin1 localizes to the midbody ring and regulates the final stages of cytokinesis by binding to centrosome protein 55 kDa (Cep55), an essential component of this ring. This interaction induces Polo-like kinase 1-mediated phosphorylation of Cep55, which is critical for the function of Cep55 during cytokinesis. Importantly, Pin1 knockdown does not enhance the cytokinesis defect in Cep55-depleted cells, indicating that Pin1 and Cep55 act in the same pathway. These data are the first evidence that Pin1 regulates cytokinesis and may provide a mechanistic explanation as to how pathologic levels of Pin1 can stimulate tumorigenesis.

Cep55 stabilization is required for normal execution of cytokinesis.

Cep55 is a mitotic phosphoprotein that plays an important role in cytokinesis, the final stage of cell division during which physical separation of the two daughter cells is accomplished. We recently demonstrated that the peptidyl-prolyl isomerase Pin1 regulates this cell cycle event by enhancing the Plk1-dependent phosphorylation of Cep55. We show here that Cep55 is stabilized post-translationally during mitosis and that siRNA-mediated knockdown of Pin1 prevents this stabilization. Consistent with this, Cep55 is unstable in Pin1 knockout mouse embryonic fibroblasts. Moreover, mutation of the Pin1 binding sites in Cep55 reduces its stability during mitosis. Mutation of the Plk1 phosphorylation site also lowers Cep55 stability, whereas overexpression of Plk1 increases Cep55 levels, in keeping with Pin1 regulating Plk1-mediated phosphorylation of Cep55. Importantly, expression of wild-type Cep55 at levels similar to that of the phosphorylation mutants only partially reverts the cytokinesis defect induced by depletion of Cep55, indicating that inadequate levels of Cep55 prevent proper execution of cytokinesis. Taken together, these data provide more insight into the regulation of the final stages of cell division. As cytokinesis defects can cause chromosomal instability, knowledge about the processes that regulate normal cytokinesis adds to our understanding of events that lead to tumorigenesis.

The peptidyl-isomerase Pin1 regulates p27kip1 expression through inhibition of Forkhead box O tumor suppressors.

The Forkhead box O (FOXO) protein family is an evolutionarily conserved subclass of transcription factors recently identified as bona fide tumor suppressors. Preventing the accumulation of cellular damage due to oxidative stress is thought to underlie its tumor-suppressive role. Oxidative stress, in turn, also feedback controls FOXO4 function. Regulation of this process, however, is poorly understood but may be relevant to the ability of FOXO to control tumor suppression. Here, we characterize novel FOXO4 phosphorylation sites after increased cellular oxidative stress and identify the isomerase Pin1, a protein frequently found to be overexpressed in cancer, as a critical regulator of p27(kip1) through FOXO4 inhibition. We show that Pin1 requires these phosphorylation events to act negatively on FOXO4 transcriptional activity. Consistent with this, oxidative stress induces binding of Pin1 to FOXO, thereby attenuating its monoubiquitination, a yet uncharacterized mode of substrate modulation by Pin1. We have previously shown that monoubiquitination is involved in controlling nuclear translocation in response to cellular stress, and indeed, Pin1 prevents nuclear FOXO4 accumulation. Interestingly, Pin1 acts on FOXO through stimulation of the activity of the deubiquitinating enzyme HAUSP/USP7. Ultimately, this results in decreased transcriptional activity towards target genes, including the cell cycle arrest gene p27(kip1). Notably, in a primary human breast cancer panel, low p27(kip1) levels inversely correlated with Pin1 expression. Thus, Pin1 is identified as a novel negative FOXO regulator, interconnecting FOXO phosphorylation and monoubiquitination in response to cellular stress to regulate p27(kip1).

Stressing the role of FoxO proteins in lifespan and disease.

Members of the class O of forkhead box transcription factors (FoxO) have important roles in metabolism, cellular proliferation, stress tolerance and probably lifespan. The activity of FoxOs is tightly regulated by post-translational modifications, including phosphorylation, acetylation and ubiquitylation. Several of the enzymes that regulate the turnover of these post-translational modifications are shared between FoxO and p53. These regulatory enzymes affect FoxO and p53 function in an opposite manner. This shared yet opposing regulatory network between FoxOs and p53 may underlie a 'trade-off' between disease and lifespan.