Recognition Marker Research Articles

132. Acid phosphatase 5 removes the mannose 6-phosphate recognition marker from lysosomal proteins

132. Acid phosphatase 5 removes the mannose 6-phosphate recognition marker from lysosomal proteins

“The Relation of the Nurse to the Working World”: Professionalization, Citizenship, and Class in Germany, Great Britain, and the United States before World War I

Campaigns for state nursing registration in the United States and Great Britain have a prominent place in the historical scholarship on nursing professionalization; the closely related German campaign has received less scholarly attention. Applying a transnational perspective to these three national movements highlights the collaborative and interrelated nature of nursing reform prior to World War I and recognizes the important contribution of German nurses to this dialogue and agenda. Focusing particularly on the years 1909-12, this article depicts a generation of German, American, and British nurses who organized national and international nursing associations to realize state registration as a stepping stone to other markers of professional recognition, such as collegiate education, full political citizenship, social welfare, and labor legislation. However, the consequent reliance of these strategies on nation-states as arbiters of citizenship and professional status undermined the shared ideological foundation of international and national nursing leaders. This article contributes to a more multinational understanding of how these international nursing leaders transcended and were confined by the limits of their nation-states in the years leading up to World War I.

COMPARING THE PROTEOME OF SQUAMOUS CELL CARCINOMA VERSUS NORMAL ESOPHAGUS TO FIND MOLECULAR MARKERS FOR RECOGNITION OF THE DISEASE

Since esophagus cancer is among the most fatal cancers all over the world and our country has the most frequent number of patients, identification of squamous cell carcinoma associated proteins would be essential. Finding molecular markers for recognition of the disease could be beneficial for efficient and early treatment of the disease. As protein expression in cancerous cells is different from normal ones, identification of protein expression pattern could be helpful for recognition of the disease. Proteomics is a novel method for recognition of protein collection in a specific tissue. In this method 2D gel electrophoresis is performed to separate all proteins, and then spot analysis will be done through mass spectrometry and bioinformatics software would help in the recognition of proteins. In comparison to normal tissue in cancerous cells, we would have up-regulation, down-regulation, appearance or disappearance of a specific protein. So, protein extraction was performed from healthy and cancerous mucosal tissue of esophagus and all peptides were separated through 2D gel electrophoresis. After spot analysis, proteins with different expression were identified with mass spectrometry and bioinformatics. In comparison to normal tissue, 14 proteins were found to over expressed or have no expression in tumors. The main objective of this study is that proteomics is an ideal method to find the molecular basis of squamous cell carcinoma in esophageal cancer.

Functions of the α, β, and γ Subunits of UDP-GlcNAc:Lysosomal Enzyme N-Acetylglucosamine-1-phosphotransferase

UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase is an alpha(2)beta(2)gamma(2) hexamer that mediates the first step in the synthesis of the mannose 6-phosphate recognition marker on lysosomal acid hydrolases. Using a multifaceted approach, including analysis of acid hydrolase phosphorylation in mice and fibroblasts lacking the gamma subunit along with kinetic studies of recombinant alpha(2)beta(2)gamma(2) and alpha(2)beta(2) forms of the transferase, we have explored the function of the alpha/beta and gamma subunits. The findings demonstrate that the alpha/beta subunits recognize the protein determinant of acid hydrolases in addition to mediating the catalytic function of the transferase. In mouse brain, the alpha/beta subunits phosphorylate about one-third of the acid hydrolases at close to wild-type levels but require the gamma subunit for optimal phosphorylation of the rest of the acid hydrolases. In addition to enhancing the activity of the alpha/beta subunits toward a subset of the acid hydrolases, the gamma subunit facilitates the addition of the second GlcNAc-P to high mannose oligosaccharides of these substrates. We postulate that the mannose 6-phosphate receptor homology domain of the gamma subunit binds and presents the high mannose glycans of the acceptor to the alpha/beta catalytic site in a favorable manner.

Loss of N‐acetylglucosamine‐1‐phosphotransferase gamma subunit due to intronic mutation in GNPTG causes mucolipidosis type III gamma: Implications for molecular and cellular diagnostics

Mucolipidosis type III gamma (MLIII, pseudo-Hurler polydystrophy) is a rare autosomal recessive disorder where the activity of the multimeric GlcNAc-1-phosphotransferase is reduced and formation of the mannose 6-phosphate (M6P) recognition marker on lysosomal enzymes is impaired. In this disease, the targeting of lysosomal enzymes is affected resulting in their hypersecretion, and an intracellular deficiency of multiple hydrolases. We report the biochemical and molecular diagnosis of MLIII in three siblings, aged 17, 15, and 14 years, who presented with joint pain and progressive joint stiffness. In addition to missorting of newly synthesized lysosomal protease cathepsin D, there were low levels of M6P-containing proteins in cell extracts and media of cultured fibroblasts of the Patients. Direct sequencing identified a novel homozygous mutation in intron 7, IVS7-10G>A, of the GNPTG gene, which encodes the gamma-subunit of the GlcNAc-1-phosphotransferase. This mutation created a cryptic 3'-splice site resulting in a frameshift and premature translational termination (p.V176GfsX18). The GNPTG mRNA levels were markedly reduced in Patients' fibroblasts indicating that the intronic mutation mediates mRNA decay, which was confirmed by absence of the gamma-subunit protein. These data contribute to an efficient diagnostic strategy to identify Patients with MLIII gamma and characterize their biochemical defect in fibroblasts.

Mannose phosphorylation in health and disease

Lysosomal hydrolases catalyze the degradation of a variety of macromolecules including proteins, carbohydrates, nucleic acids and lipids. The biogenesis of lysosomes or lysosome-related organelles requires a continuous substitution of soluble acid hydrolases and lysosomal membrane proteins. The targeting of lysosomal hydrolases depends on mannose 6-phosphate residues (M6P) that are recognized by specific receptors mediating their transport to an endosomal/prelysosomal compartment. The key role in the formation of M6P residues plays the GlcNAc-1-phosphotransferase localized in the Golgi apparatus. Two genes have been identified recently encoding the type III α/β-subunit precursor membrane protein and the soluble γ-subunit of GlcNAc-1-phosphotransferase. Mutations in these genes result in two severe diseases, mucolipidosis type II (MLII) and III (MLIII), biochemically characterized by the missorting of multiple lysosomal hydrolases due to impaired formation of the M6P recognition marker, and general lysosomal dysfunction. This review gives an update on structural properties, localization and functions of the GlcNAc-1-phosphotransferase subunits and improvements of pre- and postnatal diagnosis of ML patients. Further, the generation of recombinant single-chain antibody fragments against M6P residues and of new mouse models of MLII and MLIII will have considerable impact to provide deeper insight into the cell biology of lysosomal dysfunctions and the pathomechanisms underlying these lysosomal disorders.

An essential glycobiology resource for developmental biologists

![Figure][1] Essentials of Glycobiology, Second Edition Edited by Ajit Varki, Richard D. Cummings, Jeffrey D. Esko, Hudson H. Freeze, Pamela Stanley, Carolyn R. Bertozzi, Gerald W. Hart and Marilynn E. Etzler Cold Spring Harbor Laboratory Press (2009) 784 pages, ISBN 978-087969770-9, $158

A carbon-isotope perturbation at the Pliensbachian–Toarcian boundary: evidence from the Lias Group, NE England

AbstractA perturbation in the carbon-isotope record at the time of the Pliensbachian–Toarcian boundary (~ 184 Ma) in the Early Jurassic is reported, based on new data from Yorkshire, England. Two sharp δ13Corgnegative excursions, each with a magnitude of ~ −2.5 ‰ and reaching minimum values of −28.5 ‰, are recorded in the bulk organic-matter record in sediments of latest Pliensbachian to earliest Toarcian age. A similar pattern of negative carbon-isotope excursions has been observed at the stage boundary in the SW European section at Peniche, Portugal in δ13Ccarbonate, δ13Cwoodand δ13Cbrachiopodrecords. The isotopic excursion is of interest when considering the genesis and development of the later Toarcian Oceanic Anoxic Event (OAE), as well as the second-order global extinction event that spans the stage boundary. Furthermore, the isotope excursion potentially provides a chemostratigraphic marker for recognition of the stage boundary, which is currently achieved on the basis of different ammonite faunas in the NW European and Tethyan realms.

ON THE EVOLUTION OF HARMING AND RECOGNITION IN FINITE PANMICTIC AND INFINITE STRUCTURED POPULATIONS

Natural selection may favor two very different types of social behaviors that have costs in vital rates (fecundity and/or survival) to the actor: helping behaviors, which increase the vital rates of recipients, and harming behaviors, which reduce the vital rates of recipients. Although social evolutionary theory has mainly dealt with helping behaviors, competition for limited resources creates ecological conditions in which an actor may benefit from expressing behaviors that reduce the vital rates of neighbors. This may occur if the reduction in vital rates decreases the intensity of competition experienced by the actor or that experienced by its offspring. Here, we explore the joint evolution of neutral recognition markers and marker-based costly conditional harming whereby actors express harming, conditional on actor and recipient bearing different conspicuous markers. We do so for two complementary demographic scenarios: finite panmictic and infinite structured populations. We find that marker-based conditional harming can evolve under a large range of recombination rates and group sizes under both finite panmictic and infinite structured populations. A direct comparison with results for the evolution of marker-based conditional helping reveals that, if everything else is equal, marker-based conditional harming is often more likely to evolve than marker-based conditional helping.

Phenotype and genotype in mucolipidoses II and III alpha/beta: a study of 61 probands

BackgroundMucolipidoses II and III alpha/beta (ML II and ML III) are lysosomal disorders in which the essential mannose 6-phosphate recognition marker is not synthesised on to lysosomal hydrolases and other...

Comparative pathology of murine mucolipidosis types II and IIIC.

UDP-GlcNAc: lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase) is an alpha(2)beta(2)gamma(2) hexameric enzyme that catalyzes the first step in the synthesis of the mannose 6-phosphate targeting signal on lysosomal hydrolases. In humans, mutations in the gene encoding the alpha/beta subunit precursor give rise to mucolipidosis II (MLII), whereas mutations in the gene encoding the gamma subunit cause the less severe mucolipidosis IIIC (MLIIIC). In this study we describe the phenotypic, histologic, and serum lysosomal enzyme abnormalities in knockout mice lacking the gamma subunit and compare these findings to those of mice lacking the alpha/beta subunits and humans with MLII and MLIIIC. We found that both lines of mutant mice had elevated levels of serum lysosomal enzymes and cytoplasmic alterations in secretory cells of several exocrine glands; however, lesions in gamma-subunit deficient (Gnptg(-/-)) mice were milder and more restricted in distribution than in alpha/beta-subunit deficient (Gnptab(-/-)) mice. We found that onset, extent, and severity of lesions that developed in these two different knockouts correlated with measured lysosomal enzyme activity; with a more rapid, widespread, and severe storage disease phenotype developing in Gnptab(-/-) mice. In contrast to mice deficient in the alpha/beta subunits, the mice lacking the gamma subunits were of normal size, lacked cartilage defects, and did not develop retinal degeneration. The milder disease in the gamma-subunit deficient mice correlated with residual synthesis of the mannose 6-phosphate recognition marker. Of significance, neither strain of mutant mice developed cytoplasmic vacuolar inclusions in fibrocytes or mesenchymal cells (I-cells), the characteristic lesion associated with the prominent skeletal and connective tissue abnormalities in humans with MLII and MLIII. Instead, the predominant lesions in both lines of mice were found in the secretory epithelial cells of several exocrine glands, including the pancreas, and the parotid, submandibular salivary, nasal, lacrimal, bulbourethral, and gastric glands. The absence of retinal and chondrocyte lesions in Gnptg(-/-) mice might be attributed to residual beta-glucuronidase activity. We conclude that mice lacking either alpha/beta or gamma subunits displayed clinical and pathologic features that differed substantially from those reported in humans having mutations in orthologous genes.

Characterization of regulatory T cells identified as CD4+CD25highCD39+ in patients with active tuberculosis

Forkhead box P3 (FoxP3) is a transcription factor whose expression characterizes regulatory T cells (T(reg)), but it is also present on activated T cells, thus hindering correct T(reg) identification. Using classical markers for T(reg) recognition, discordant results were found in terms of T(reg) expansion during active tuberculosis (TB) disease. Recently CD39 has been shown to be an accurate marker for T(reg) detection. The objectives of this study were: (i) to identify T(reg) expressing CD39 in patients with TB and to compare the results with those obtained by the standard phenotypic markers; (ii) to evaluate if T(reg) are expanded in vitro by exogenous interleukin (IL)-2 or by antigen-specific stimulation; and (iii) to characterize T(reg) function on the modulation of antigen-specific responses. We enrolled 13 patients with pulmonary TB and 12 healthy controls. T(reg) were evaluated by flow cytometry ex vivo and after antigen-specific in vitro stimulation using CD25, FoxP3, CD127 and CD39 markers. Results indicate that CD39(+) cells within the CD4(+)CD25(high) cells have T(reg) properties (absence of interferon-gamma production and transforming growth factor-beta1 release upon stimulation). Ex vivo analysis did not show significant differences between TB patients and controls of T(reg) by classical or novel markers. In contrast, a significantly higher percentage of T(reg) was found in TB patients after antigen-specific stimulation both in the presence or absence of IL-2. Depletion of CD39(+) T(reg) increased RD1-specific responses significantly. In conclusion, CD39 is an appropriate marker for T(reg) identification in TB. These results can be useful for future studies to monitor Mycobacterium tuberculosis-specific response during TB.

Compensatory expression of human N-Acetylglucosaminyl-1-phosphotransferase subunits in mucolipidosis type III gamma

The N-Acetylglucosaminyl-1-phosphotransferase plays a key role in the generation of mannose 6-phosphate (M6P) recognition markers essential for efficient transport of lysosomal hydrolases to lysosomes. The phosphotransferase is composed of six subunits (α 2, β 2, γ 2). The α- and β-subunits are catalytically active and encoded by a single gene, GNPTAB, whereas the γ-subunit encoded by GNPTG is proposed to recognize conformational structures common to lysosomal enzymes. Defects in GNPTG cause mucolipidosis type III gamma, which is characterized by missorting and cellular loss of lysosomal enzymes leading to lysosomal accumulation of storage material. Using plasmon resonance spectrometry, we showed that recombinant γ-subunit failed to bind the lysosomal enzyme arylsulfatase A. Additionally, the overexpression of the γ-subunit in COS7 cells did not result in hypersecretion of newly synthesized lysosomal enzymes expected for competition for binding sites of the endogenous phosphotransferase complex. Analysis of fibroblasts exhibiting a novel mutation in GNPTG (c.619insT, p.K207IfsX7) revealed that the expression of GNPTAB was increased whereas in γ-subunit overexpressing cells the GNPTAB mRNA was reduced. The data suggest that the γ-subunit is important for the balance of phosphotransferase subunits rather for general binding of lysosomal enzymes.

Comparative Pathology of Murine Mucolipidosis Types II and IIIC

UDP-GlcNAc: lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase) is an α2β2γ2 hexameric enzyme that catalyzes the first step in the synthesis of the mannose 6-phosphate targeting signal on lysosomal hydrolases. In humans, mutations in the gene encoding the α/β subunit precursor give rise to mucolipidosis II (MLII), whereas mutations in the gene encoding the γ subunit cause the less severe mucolipidosis IIIC (MLIIIC). In this study we describe the phenotypic, histologic, and serum lysosomal enzyme abnormalities in knockout mice lacking the γ subunit and compare these findings to those of mice lacking the α/β subunits and humans with MLII and MLIIIC. We found that both lines of mutant mice had elevated levels of serum lysosomal enzymes and cytoplasmic alterations in secretory cells of several exocrine glands; however, lesions in γ-subunit deficient (Gnptab-/-) mice were milder and more restricted in distribution than in α/β-subunit deficient (Gnptab-/-) mice. We found that onset, extent, and severity of lesions that developed in these two different knockouts correlated with measured lysosomal enzyme activity; with a more rapid, widespread, and severe storage disease phenotype developing in Gnptab-/- mice. In contrast to mice deficient in the α/β subunits, the mice lacking the γ subunits were of normal size, lacked cartilage defects, and did not develop retinal degeneration. The milder disease in the γ-subunit deficient mice correlated with residual synthesis of the mannose 6-phosphate recognition marker. Of significance, neither strain of mutant mice developed cytoplasmic vacuolar inclusions in fibrocytes or mesenchymal cells (I-cells), the characteristic lesion associated with the prominent skeletal and connective tissue abnormalities in humans with MLII and MLIII. Instead, the predominant lesions in both lines of mice were found in the secretory epithelial cells of several exocrine glands, including the pancreas, and the parotid, submandibular salivary, nasal, lacrimal, bulbourethral, and gastric glands. The absence of retinal and chondrocyte lesions in Gnptab-/- mice might be attributed to residual β-glucuronidase activity. We conclude that mice lacking either α/β or γ subunits displayed clinical and pathologic features that differed substantially from those reported in humans having mutations in orthologous genes.

One-step DNA-programmed growth of luminescent and biofunctionalized nanocrystals

Colloidal semiconductor nanocrystals are widely used as lumiphores in biological imaging because their luminescence is both strong and stable, and because they can be biofunctionalized. During synthesis, nanocrystals are typically passivated with hydrophobic organic ligands, so it is then necessary either to replace these ligands or encapsulate the nanocrystals with hydrophilic moieties to make the lumiphores soluble in water. Finally, biological labels must be added to allow the detection of nucleic acids, proteins and specific cell types. This multistep process is time- and labour-intensive and thus out of reach of many researchers who want to use luminescent nanocrystals as customized lumiphores. Here, we show that a single designer ligand--a chimeric DNA molecule--can controllably program both the growth and the biofunctionalization of the nanocrystals. One part of the DNA sequence controls the nanocrystal passivation and serves as a ligand, while another part controls the biorecognition. The synthetic protocol reported here is straightforward and produces a homogeneous dispersion of nanocrystal lumiphores functionalized with a single biomolecular receptor. The nanocrystals exhibit strong optical emission in the visible region, minimal toxicity and have hydrodynamic diameters of approximately 6 nm, which makes them suitable for bioimaging. We show that the nanocrystals can specifically bind DNA, proteins or cells that have unique surface recognition markers.

The syndrome of cardiac failure in adults with congenitally corrected transposition

To assess neurohormonal activation of cardiac failure in adults with congenitally corrected transposition, and to determine the most sensitive marker for recognition of the cardiac failure. The onset of morphologically right ventricular dysfunction is unpredictable in patients with congenitally corrected transposition, the combination of discordant atrioventricular and ventriculo-arterial connections, and its markers are unknown. We measured amino terminal pro brain natriuretic peptide in 19 patients, aged 35 plus or minus 13.1 years, and in 19 control subjects. Morhologically right ventricular function was assessed by echocardiography, including tissue Doppler echocardiography and magnetic resonance imaging or multislice computed tomography. The patients showed a highly significant increase in the levels of amino terminal pro brain natriuretic peptide, the levels being significantly elevated even in asymptomatic patients. Left atrial dimensions were larger in patients, and significantly lower tissue Doppler echocardiographic velocities were measured at the lateral site of the tricuspid annulus and at the basal segment of the interventricular septum. The ejection fraction of the morphologically right ventricle correlated significantly with the levels of brain natriuretic peptide, and with left atrial dimensions. Neurohormonal activation is present in patients with congenitally corrected transposition even when they are asymptomatic. It is correlated with left atrial dimensions and tissue Doppler echocardiographic parameters. Levels of brain natriuretic peptide, and peak tricuspid early diastolic annular velocity, are the earliest and most sensitive markers of morphologically right ventricular dysfunction.

Acid phosphatase 5 is responsible for removing the mannose 6-phosphate recognition marker from lysosomal proteins

Most newly synthesized proteins destined for the lysosome reach this location via a specific intracellular pathway. In the Golgi, a phosphotransferase specifically labels lysosomal proteins with mannose 6-phosphate (Man-6-P). This modification is recognized by receptors that target the lysosomal proteins to the lysosome where, in most cell types, the Man-6-P recognition marker is rapidly removed. Despite extensive characterization of this pathway, the enzyme responsible for the removal of the targeting modification has remained elusive. In this study, we have identified this activity. Preliminary investigations using a cell-based bioassay were used to follow a dephosphorylation activity that was associated with the lysosomal fraction. This activity was high in the liver, where endogenous lysosomal proteins are efficiently dephosphorylated, but present at a much lower level in the brain, where the modification persists. This observation, combined with an analysis of the expression of lysosomal proteins in different tissues, led us to identify acid phosphatase 5 (ACP5) as a candidate for the enzyme that removes Man-6-P. Expression of ACP5 in N1E-115 neuroblastoma cells, which do not efficiently dephosphorylate lysosomal proteins, significantly decreased the steady state levels of Man6-P glycoproteins. Analysis of ACP5-deficient mice revealed that levels of Man-6-P glycoproteins were highly elevated in tissues that normally express ACP5, and this resulted from a failure to dephosphorylate lysosomal proteins. These results indicate a central role for ACP5 in removal of the Man-6-P recognition marker and open up new avenues to investigate the importance of this process in cell biology and medicine.

Synthesis of new sulfonate and phosphonate derivatives for cation-independent mannose 6-phosphate receptor targeting

The cation-independent mannose 6-phosphate receptor (CI-M6PR) is essential for the endocytosis of proteins bearing the mannose 6-phosphate (M6P) recognition marker. This study described the synthesis of M6P and M6S analogs presenting greater affinity for CI-M6PR than their natural compounds. Moreover, the finding of their lack of cytotoxicity for human cells and of their increased stability in human serum supports the high potential of these isosteric derivatives in therapies requiring CI-M6PR targeting.

Geoarchaeological sea-level proxies from a silted up harbour: A case study of the Roman colony of Luni (northern Tyrrhenian Sea, Italy)

The Luni Plain, which faces the northern Tyrrhenian Sea, underwent major landscape changes over the last 3000 years. The coastline shifted south and west, separating the ruins of the ancient Roman colony of Luni (founded in 177 BC) from the sea by means of a new coastal plain. The precise location of the coastline during the main settlement phase as well as during the city's decline has never been established, although partial and qualitative scenarios have been suggested by various authors. According to these authors, an indentation of the coastline west of the city formed a major water basin, along the shores of which traces of pre-Roman settlements exist; near the city, two minor basins were present, suitable for hosting harbour structures. The city of Luni was famed for its harbour (Portus Lunae), from which the marbles quarried in the Apuan Alps, used to build many of the monuments in Rome, were delivered. The traces of its port, however, are scattered and uncertain. Newly available subsurface data are presented, associated with a revision of unpublished archaeological evidence of the ancient city topography. Progress in palaeoenvironmental reconstructions at Luni has been possible thanks to the collection and analysis of ancient and recent cores, supported by radiocarbon dates, as well as the interpretation of archaeological findings suitable as markers for sea-level recognition. The main environmental changes in the area have been refined: since a few centuries before the colony was founded, the Luni plain had been characterized by a complex architecture of swamps and marshes limited by dune ridges and fluvial sand bars. The positions of these landforms were not fixed, but shifted, mainly depending on the spatial relationship between the coastline and the river mouths. Determining the precise position of the Roman coastline is useful to constrain the area in which archaeological surveys should be concentrated in order to identify the harbour location. Moreover, some of these results have been used to infer altitude constraints on the sea-level position in Roman times: preliminary data are shown for constructing a Late Holocene local sea-level curve in the area.

Differences and evolution of the methods for the assessment of microsatellite instability

Microsatellite instability (MSI) originates from the systematic accumulation of uncorrected deletion/insertion in repetitive DNA tracts in cancer cells with a deficient mismatch repair system. Among colorectal cancers, the MSI signature identifies hereditary cases arising in patients with germline mutations in hMLH1, hMSH2, PMS2 and a fraction of those with hMSH6 mutations, as well as sporadic cancers with epigenetic hMLH1 promoter hypermethylation. Considering the specific pathogenesis, pathological features, natural history and response to 5-fluoro-uracil-based chemotherapy of the MSI cancers, confusion about the genetic markers for MSI recognition seems surprising. In this clinically relevant field, an agreement has not been reached concerning the use of di- or mononucleotide markers for MSI assessment. The Revised Bethesda Guidelines still recommend a panel of markers consisting of mono- and dinucleotides, despite being questioned whether it is congruous to continue to use dinucleotide markers for MSI identification. In any event, no single marker is accurate enough for MSI testing, and an awareness of their pros and cons is required for proper interpretation of results. In recent years, several papers have reported different prevalence of MSI in unrelated series, largely depending on the detection and classification method, suggesting that MSI test interpretation also requires the understanding of the phenomenon rather than simply the crude satisfaction of panel recommendations. Inaccuracies can otherwise lead to under- or overdiagnosis and inaccurate disease classification, which always have a negative impact on the clinical practice of medicine.