Understanding the cell: Future views of structural biology.

Determining the structure and mechanisms of all individual functional modules of cells at high molecular detail has often been seen as equal to understanding how cells work. Recent technical advances have led to a flush of high-resolution structures of various macromolecular machines, but despite this wealth of detailed information, our understanding of cellular function remains incomplete. Here, we discuss present-day limitations of structural biology and highlight novel technologies that may enable us to analyze molecular functions directly inside cells. We predict that the progression toward structural cell biology will involve a shift toward conceptualizing a 4D virtual reality of cells using digital twins. These will capture cellular segments in a highly enriched molecular detail, include dynamic changes, and facilitate simulations of molecular processes, leading to novel and experimentally testable predictions. Transferring biological questions into algorithms that learn from the existing wealth of data and explore novel solutions may ultimately unveil how cells work.

Cone-shaped HIV-1 capsids are transported through intact nuclear pores.

Human immunodeficiency virus (HIV-1) remains a major health threat. Viral capsid uncoating and nuclear import of the viral genome are critical for productive infection. The size of the HIV-1 capsid is generally believed to exceed the diameter of the nuclear pore complex (NPC), indicating that capsid uncoating has to occur prior to nuclear import. Here, we combined correlative light and electron microscopy with subtomogram averaging to capture the structural status of reverse transcription-competent HIV-1 complexes in infected T cells. We demonstrated that the diameter of the NPC in cellulo is sufficient for the import of apparently intact, cone-shaped capsids. Subsequent to nuclear import, we detected disrupted and empty capsid fragments, indicating that uncoating of the replication complex occurs by breaking the capsid open, and not by disassembly into individual subunits. Our data directly visualize a key step in HIV-1 replication and enhance our mechanistic understanding of the viral life cycle.

Disentangling Genetic and Environmental Effects on the Proteotypes of Individuals.

Proteotypes, like genotypes, have been found to vary between individuals in several studies, but consistent molecular functional traits across studies remain to be quantified. In a meta-analysis of 11 proteomics datasets from humans and mice, we use co-variation of proteins in known functional modules across datasets and individuals to obtain a consensus landscape of proteotype variation. We find that individuals differ considerably in both protein complex abundances and stoichiometry. We disentangle genetic and environmental factors impacting these metrics, with genetic sex and specific diets together explaining 13.5% and 11.6% of the observed variation of complex abundance and stoichiometry, respectively. Sex-specific differences, for example, include various proteins and complexes, where the respective genes are not located on sex-specific chromosomes. Diet-specific differences, added to the individual genetic backgrounds, might become a starting point for personalized proteotype modulation toward desired features.

Nuclear Pores Assemble from Nucleoporin Condensates During Oogenesis.

The molecular events that direct nuclear pore complex (NPC) assembly toward nuclear envelopes have been conceptualized in two pathways that occur during mitosis or interphase, respectively. In gametes and embryonic cells, NPCs also occur within stacked cytoplasmic membrane sheets, termed annulate lamellae (AL), which serve as NPC storage for early development. The mechanism of NPC biogenesis at cytoplasmic membranes remains unknown. Here, we show that during Drosophila oogenesis, Nucleoporins condense into different precursor granules that interact and progress into NPCs. Nup358 is a key player that condenses into NPC assembly platforms while its mRNA localizes to their surface in a translation-dependent manner. In concert, Microtubule-dependent transport, the small GTPase Ran and nuclear transport receptors regulate NPC biogenesis in oocytes. We delineate a non-canonical NPC assembly mechanism that relies on Nucleoporin condensates and occurs away from the nucleus under conditions of cell cycle arrest.

Pervasive Protein Thermal Stability Variation during the Cell Cycle.

Quantitative mass spectrometry has established proteome-wide regulation of protein abundance and post-translational modifications in various biological processes. Here, we used quantitative mass spectrometry to systematically analyze the thermal stability and solubility of proteins on a proteome-wide scale during the eukaryotic cell cycle. We demonstrate pervasive variation of these biophysical parameters with most changes occurring in mitosis and G1. Various cellular pathways and components vary in thermal stability, such as cell-cycle factors, polymerases, and chromatin remodelers. We demonstrate that protein thermal stability serves as a proxy for enzyme activity, DNA binding, and complex formation in situ. Strikingly, a large cohort of intrinsically disordered and mitotically phosphorylated proteins is stabilized and solubilized in mitosis, suggesting a fundamental remodeling of the biophysical environment of the mitotic cell. Our data represent a rich resource for cell, structural, and systems biologists interested in proteome regulation during biological transitions.

AlphaFold: Research accelerator and hypothesis generator.

To celebrate the 50th anniversary of Cell Press and the Cell focus issue on structural biology, we discussed with scientists working across diverse fields how AlphaFold has changed their research and brought structural biology to the masses.

Pre-assembled Nuclear Pores Insert into the Nuclear Envelope during Early Development.

Nuclear pore complexes (NPCs) span the nuclear envelope (NE) and mediate nucleocytoplasmic transport. In metazoan oocytes and early embryos, NPCs reside not only within the NE, but also at some endoplasmic reticulum (ER) membrane sheets, termed annulate lamellae (AL). Although a role for AL as NPC storage pools has been discussed, it remains controversial whether and how they contribute to the NPC density at the NE. Here, we show that AL insert into the NE as the ER feeds rapid nuclear expansion in Drosophila blastoderm embryos. We demonstrate that NPCs within AL resemble pore scaffolds that mature only upon insertion into the NE. We delineate a topological model in which NE openings are critical for AL uptake that nevertheless occurs without compromising the permeability barrier of the NE. We finally show that this unanticipated mode of pore insertion is developmentally regulated and operates prior to gastrulation.

Structural basis of RNA-induced autoregulation of the DExH-type RNA helicase maleless.

RNA unwinding by DExH-type helicases underlies most RNA metabolism and function. It remains unresolved if and how the basic unwinding reaction of helicases is regulated by auxiliary domains. We explored the interplay between the RecA and auxiliary domains of the RNA helicase maleless (MLE) from Drosophila using structural and functional studies. We discovered that MLE exists in a dsRNA-bound open conformation and that the auxiliary dsRBD2 domain aligns the substrate RNA with the accessible helicase tunnel. In an ATP-dependent manner, dsRBD2 associates with the helicase module, leading to tunnel closure around ssRNA. Furthermore, our structures provide a rationale for blunt-ended dsRNA unwinding and 3'-5' translocation by MLE. Structure-based MLE mutations confirm the functional relevance of our model for RNA unwinding. Our findings contribute to our understanding of the fundamental mechanics of auxiliary domains in DExH helicase MLE, which serves as a model for its human ortholog and potential therapeutic target, DHX9/RHA.

The nuclear pore complex: understanding its function through structural insight.

Nuclear pore complexes (NPCs) fuse the inner and outer nuclear membranes to form channels across the nuclear envelope. They are large macromolecular assemblies with a complex composition and diverse functions. Apart from facilitating nucleocytoplasmic transport, NPCs are involved in chromatin organization, the regulation of gene expression and DNA repair. Understanding the molecular mechanisms underlying these functions has been hampered by a lack of structural knowledge about the NPC. The recent convergence of crystallographic and biochemical in vitro analysis of nucleoporins (NUPs), the components of the NPC, with cryo-electron microscopic imaging of the entire NPC in situ has provided first pseudo-atomic view of its central core and revealed that an unexpected network of short linear motifs is an important spatial organization principle. These breakthroughs have transformed the way we understand NPC structure, and they provide an important base for functional investigations, including the elucidation of the molecular mechanisms underlying clinically manifested mutations of the nucleocytoplasmic transport system.

Visualizing the disordered nuclear transport machinery in situ.

The approximately 120 MDa mammalian nuclear pore complex (NPC) acts as a gatekeeper for the transport between the nucleus and cytosol1. The central channel of the NPC is filled with hundreds of intrinsically disordered proteins (IDPs) called FG-nucleoporins (FG-NUPs)2,3. Although the structure of the NPC scaffold has been resolved in remarkable detail, the actual transport machinery built up by FG-NUPs-about 50 MDa-is depicted as an approximately 60-nm hole in even highly resolved tomograms and/or structures computed with artificial intelligence4-11. Here we directly probed conformations of the vital FG-NUP98 inside NPCs in live cells and in permeabilized cells with an intact transport machinery by using a synthetic biology-enabled site-specific small-molecule labelling approach paired with highly time-resolved fluorescence microscopy. Single permeabilized cell measurements of the distance distribution of FG-NUP98 segments combined with coarse-grained molecular simulations of the NPC allowed us to map the uncharted molecular environment inside the nanosized transport channel. We determined that the channel provides-in the terminology of the Flory polymer theory12-a 'good solvent' environment. This enables the FG domain to adopt expanded conformations and thus control transport between the nucleus and cytoplasm. With more than 30% of the proteome being formed from IDPs, our study opens a window into resolving disorder-function relationships of IDPs in situ, which are important in various processes, such as cellular signalling, phase separation, ageing and viral entry.

Integrated structural analysis of the human nuclear pore complex scaffold.

The nuclear pore complex (NPC) is a fundamental component of all eukaryotic cells that facilitates nucleocytoplasmic exchange of macromolecules. It is assembled from multiple copies of about 30 nucleoporins. Due to its size and complex composition, determining the structure of the NPC is an enormous challenge, and the overall architecture of the NPC scaffold remains elusive. In this study, we have used an integrated approach based on electron tomography, single-particle electron microscopy, and crosslinking mass spectrometry to determine the structure of a major scaffold motif of the human NPC, the Nup107 subcomplex, in both isolation and integrated into the NPC. We show that 32 copies of the Nup107 subcomplex assemble into two reticulated rings, one each at the cytoplasmic and nuclear face of the NPC. This arrangement may explain how changes of the diameter are realized that would accommodate transport of huge cargoes.

Bioaccumulation of therapeutic drugs by human gut bacteria.

Bacteria in the gut can modulate the availability and efficacy of therapeutic drugs. However, the systematic mapping of the interactions between drugs and bacteria has only started recently1 and the main underlying mechanism proposed is the chemical transformation of drugs by microorganisms (biotransformation). Here we investigated the depletion of 15 structurally diverse drugs by 25 representative strains of gut bacteria. This revealed 70 bacteria-drug interactions, 29 of which had not to our knowledge been reported before. Over half of the new interactions can be ascribed to bioaccumulation; that is, bacteria storing the drug intracellularly without chemically modifying it, and in most cases without the growth of the bacteria being affected. As a case in point, we studied the molecular basis of bioaccumulation of the widely used antidepressant duloxetine by using click chemistry, thermal proteome profiling and metabolomics. We find that duloxetine binds to several metabolic enzymes and changes the metabolite secretion of the respective bacteria. When tested in a defined microbial community of accumulators and non-accumulators, duloxetine markedly altered the composition of the community through metabolic cross-feeding. We further validated our findings in an animal model, showing that bioaccumulating bacteria attenuate the behavioural response of Caenorhabditis elegans to duloxetine. Together, our results show that bioaccumulation by gut bacteria may be a common mechanism that alters drug availability and bacterial metabolism, with implications for microbiota composition, pharmacokinetics, side effects and drug responses, probably in an individual manner.

Convolutional networks for supervised mining of molecular patterns within cellular context.

Cryo-electron tomograms capture a wealth of structural information on the molecular constituents of cells and tissues. We present DeePiCt (deep picker in context), an open-source deep-learning framework for supervised segmentation and macromolecular complex localization in cryo-electron tomography. To train and benchmark DeePiCt on experimental data, we comprehensively annotated 20 tomograms of Schizosaccharomyces pombe for ribosomes, fatty acid synthases, membranes, nuclear pore complexes, organelles, and cytosol. By comparing DeePiCt to state-of-the-art approaches on this dataset, we show its unique ability to identify low-abundance and low-density complexes. We use DeePiCt to study compositionally distinct subpopulations of cellular ribosomes, with emphasis on their contextual association with mitochondria and the endoplasmic reticulum. Finally, applying pre-trained networks to a HeLa cell tomogram demonstrates that DeePiCt achieves high-quality predictions in unseen datasets from different biological species in a matter of minutes. The comprehensively annotated experimental data and pre-trained networks are provided for immediate use by the community.

In-cell architecture of the nuclear pore and snapshots of its turnover.

Nuclear pore complexes (NPCs) fuse the inner and outer membranes of the nuclear envelope. They comprise hundreds of nucleoporins (Nups) that assemble into multiple subcomplexes and form large central channels for nucleocytoplasmic exchange1,2. How this architecture facilitates messenger RNA export, NPC biogenesis and turnover remains poorly understood. Here we combine in situ structural biology and integrative modelling with correlative light and electron microscopy and molecular perturbation to structurally analyse NPCs in intact Saccharomyces cerevisiae cells within the context of nuclear envelope remodelling. We find an in situ conformation and configuration of the Nup subcomplexes that was unexpected from the results of previous in vitro analyses. The configuration of the Nup159 complex appears critical to spatially accommodate its function as an mRNA export platform, and as a mediator of NPC turnover. The omega-shaped nuclear envelope herniae that accumulate in nup116Δ cells3 conceal partially assembled NPCs lacking multiple subcomplexes, including the Nup159 complex. Under conditions of starvation, herniae of a second type are formed that cytoplasmically expose NPCs. These results point to a model of NPC turnover in which NPC-containing vesicles bud off from the nuclear envelope before degradation by the autophagy machinery. Our study emphasizes the importance of investigating the structure-function relationship of macromolecular complexes in their cellular context.

Influenza Polymerase Can Adopt an Alternative Configuration Involving a Radical Repacking of PB2 Domains.

Influenza virus polymerase transcribes or replicates the segmented RNA genome (vRNA) into respectively viral mRNA or full-length copies and initiates RNA synthesis by binding the conserved 3' and 5' vRNA ends (the promoter). In recent structures of promoter-bound polymerase, the cap-binding and endonuclease domains are configured for cap snatching, which generates capped transcription primers. Here, we present a FluB polymerase structure with a bound complementary cRNA 5' end that exhibits a major rearrangement of the subdomains within the C-terminal two-thirds of PB2 (PB2-C). Notably, the PB2 nuclear localization signal (NLS)-containing domain translocates ∼90 Što bind to the endonuclease domain. FluA PB2-C alone and RNA-free FluC polymerase are similarly arranged. Biophysical and cap-dependent endonuclease assays show that in solution the polymerase explores different conformational distributions depending on which RNA is bound. The inherent flexibility of the polymerase allows it to adopt alternative conformations that are likely important during polymerase maturation into active progeny RNPs.

Structural impact of K63 ubiquitin on yeast translocating ribosomes under oxidative stress.

Subpopulations of ribosomes are responsible for fine tuning the control of protein synthesis in dynamic environments. K63 ubiquitination of ribosomes has emerged as a new posttranslational modification that regulates protein synthesis during cellular response to oxidative stress. K63 ubiquitin, a type of ubiquitin chain that functions independently of the proteasome, modifies several sites at the surface of the ribosome, however, we lack a molecular understanding on how this modification affects ribosome structure and function. Using cryoelectron microscopy (cryo-EM), we resolved the first three-dimensional (3D) structures of K63 ubiquitinated ribosomes from oxidatively stressed yeast cells at 3.5-3.2 Å resolution. We found that K63 ubiquitinated ribosomes are also present in a polysome arrangement, similar to that observed in yeast polysomes, which we determined using cryoelectron tomography (cryo-ET). We further showed that K63 ubiquitinated ribosomes are captured uniquely at the rotated pretranslocation stage of translation elongation. In contrast, cryo-EM structures of ribosomes from mutant cells lacking K63 ubiquitin resolved at 4.4-2.7 Å showed 80S ribosomes represented in multiple states of translation, suggesting that K63 ubiquitin regulates protein synthesis at a selective stage of elongation. Among the observed structural changes, ubiquitin mediates the destabilization of proteins in the 60S P-stalk and in the 40S beak, two binding regions of the eukaryotic elongation factor eEF2. These changes would impact eEF2 function, thus, inhibiting translocation. Our findings help uncover the molecular effects of K63 ubiquitination on ribosomes, providing a model of translation control during oxidative stress, which supports elongation halt at pretranslocation.

Structural insights into transcription initiation by yeast RNA polymerase I.

In eukaryotic cells, RNA polymerase I (Pol I) synthesizes precursor ribosomal RNA (pre-rRNA) that is subsequently processed into mature rRNA. To initiate transcription, Pol I requires the assembly of a multi-subunit pre-initiation complex (PIC) at the ribosomal RNA promoter. In yeast, the minimal PIC includes Pol I, the transcription factor Rrn3, and Core Factor (CF) composed of subunits Rrn6, Rrn7, and Rrn11. Here, we present the cryo-EM structure of the 18-subunit yeast Pol I PIC bound to a transcription scaffold. The cryo-EM map reveals an unexpected arrangement of the DNA and CF subunits relative to Pol I. The upstream DNA is positioned differently than in any previous structures of the Pol II PIC. Furthermore, the TFIIB-related subunit Rrn7 also occupies a different location compared to the Pol II PIC although it uses similar interfaces as TFIIB to contact DNA. Our results show that although general features of eukaryotic transcription initiation are conserved, Pol I and Pol II use them differently in their respective transcription initiation complexes.

In situ structural analysis of the human nuclear pore complex.

Nuclear pore complexes are fundamental components of all eukaryotic cells that mediate nucleocytoplasmic exchange. Determining their 110-megadalton structure imposes a formidable challenge and requires in situ structural biology approaches. Of approximately 30 nucleoporins (Nups), 15 are structured and form the Y and inner-ring complexes. These two major scaffolding modules assemble in multiple copies into an eight-fold rotationally symmetric structure that fuses the inner and outer nuclear membranes to form a central channel of ~60 nm in diameter. The scaffold is decorated with transport-channel Nups that often contain phenylalanine-repeat sequences and mediate the interaction with cargo complexes. Although the architectural arrangement of parts of the Y complex has been elucidated, it is unclear how exactly it oligomerizes in situ. Here we combine cryo-electron tomography with mass spectrometry, biochemical analysis, perturbation experiments and structural modelling to generate, to our knowledge, the most comprehensive architectural model of the human nuclear pore complex to date. Our data suggest previously unknown protein interfaces across Y complexes and to inner-ring complex members. We show that the transport-channel Nup358 (also known as Ranbp2) has a previously unanticipated role in Y-complex oligomerization. Our findings blur the established boundaries between scaffold and transport-channel Nups. We conclude that, similar to coated vesicles, several copies of the same structural building block--although compositionally identical--engage in different local sets of interactions and conformations.

Speckle tracking echocardiography in the indeterminate form of Chagas disease.

BACKGROUND: Chagas disease is one of the most common diseases in Latin-America, and cardiac involvement is a significant cause of death. Assessment of myocardial strain may detect early myocardial damage. OBJECTIVES: To determine differences in longitudinal strain using speckle tracking to assess regional and global left ventricular function in patients with the indeterminate form of Chagas disease, in comparison with a control group. METHODS: This is a retrospective matched case-control study, conducted in a single center. We evaluated 45 adult patients with Chagas disease, diagnosed with 2 serological methods, without evidence of cardiac involvement, who were compared with 45 healthy control subjects, who were sex- and age-matched. All patients underwent Doppler echocardiography and longitudinal strain with speckle tracking. RESULTS: Median age was 59 years, and 60% were female. Echocardiographic parameters were similar in patients with Chagas and control subjects. In patients with Chagas, global strain differed significantly from that of control subjects (-17 vs -20.3, P < .001). Segmental strain showed 7 abnormal segments in patients with Chagas (P < .05). CONCLUSIONS: In patients with the indeterminate form of Chagas disease, global and segmental longitudinal peak systolic strain is reduced compared with healthy subjects, thus suggesting that it could be a sensitive technique to detect early myocardial damage. These findings could provide useful information regarding the pathophysiology of cardiac involvement and understand whether they might have prognostic usefulness or help develop strategies to modify the course and prognosis of patients with Chagas disease. A longitudinal prospective study would be necessary to validate our findings.

Can the Femoro-Epiphyseal Acetabular Roof (FEAR) Index Be Used to Distinguish Dysplasia from Impingement?

BACKGROUND: Classifying hips with structural deformity on the spectrum from impingement to dysplasia is often subjective and frequently inexact. Currently used radiographic measures may inaccurately predict a hip's morphological stability in borderline hips. A recently described radiographic measure, the Femoro-Epiphyseal Acetabular Roof (FEAR) index, has demonstrated an ability to predict stability in the borderline hip. This measure is attractive to clinicians because procedures can be used on the basis of a hip's pathomechanics. This study was designed to further validate and characterize the FEAR index in a skeletally immature population, in hips with dysplasia/femoroacetabular impingement (FAI), and in asymptomatic hips. QUESTIONS/PURPOSES: (1) What are the characteristics of the FEAR index in children and how does the index change with skeletal maturation? (2) How does the FEAR index correlate with clinical diagnosis and surgical treatment in a large cohort of symptomatic hips and asymptomatic controls? (3) How does the FEAR index correlate with clinical diagnosis in the borderline hip (lateral center-edge angle [LCEA] 20°-25°) group? METHODS: A total of 220 participants with symptomatic investigational hips with a clinical diagnosis of dysplasia or FAI between January 2008 and January 2018 were retrospectively collected from the senior author's practice. Investigational hips were excluded if they had any femoral head abnormalities preventing LCEA measurement (for example, Perthes disease), Tönnis osteoarthritis grade greater than 1, prior hip surgery, or prior femoral osteotomy. In the 220 participants, 395 hips met inclusion criteria. Once exclusion criteria were applied, 15 hips were excluded due to prior hip surgery or prior femoral osteotomy, and 12 hips were excluded due to femoral head deformity. A single hip was then randomly selected from each participant, resulting in 206 investigational hips with a mean age of 13 ± 3 years. Between January 2017 and December 2017, 70 asymptomatic control participants were retrospectively collected from the senior author's institutional trauma database. Control hips were included if the AP pelvis film had the coccyx centered over the pubic symphysis and within 1 to 3 cm of the superior aspect of the symphysis. Control hips were excluded if there was any fracture to the pelvis or ipsilateral femur or the participant had prior hip/pelvis surgery. After exclusion criteria were applied, 16 hips were excluded due to fracture. One hip was then randomly selected from each participant, resulting in 65 control hips with a mean age of 16 ± 8 years. Standardized standing AP pelvis radiographs were used to measure the FEAR index, LCEA, and Tönnis angle in the investigational cohort. Standardized false-profile radiographs were used to measure the anterior center-edge angle (ACEA) in the investigational cohort. Two blinded investigators measured the FEAR index with an intraclass correlation coefficient of 0.92 [95% CI 0.84 to 0.96]. Question 1 was answered by comparing the above radiographic measures in age subgroups (childhood: younger than 10 years; adolescence: 10 to 14 years old; maturity: older than 14 years) of dysplastic, FAI, and control hips. Question 2 was answered by comparing the radiographic measures in all dysplastic, FAI, control hips, and a subgroup of operatively or nonoperatively managed dysplasia and FAI hips. Question 3 was answered by comparing the radiographic measures in borderline (LCEA 20°-25°) dysplastic, FAI, and control hips. RESULTS: The FEAR index was lower in older dysplastic of hips (younger than 10 years, 6° ± 9°; 10 to 14 years, 4° ± 10°; older than 14 years, 5° ± 9°; p < 0.001) and control hips (younger than 10 years, -6° ± 5°; 10 to 14 years, -15° ± 4°; older than 14 years, -16° ± 7°; p < 0.001). The diagnosis and age groups were independently correlated with the FEAR index (p < 0.001). The relationship between the FEAR index and diagnosis remained consistent in each age group (p = 0.11). The FEAR index was higher in all dysplastic hips (mean 5° ± 10°) than in asymptomatic controls (mean -13° ± 7°; p < 0.001) and FAI hips (mean -10° ± 11°; p < 0.001). Using -1.3° as a cutoff for FAI/control hips and dysplastic hips, 81% (112 of 139) of hips with values below this threshold were FAI/control, and 89% (117 of 132) of hips with values above -1.3° were dysplastic. The receiver operator characteristics area under the curve (ROC-AUC) was 0.91. Similarly, the FEAR index was higher in borderline dysplastic hips than in both asymptomatic borderline controls (p < 0.001) and borderline FAI hips (p < 0.001). Eighty-nine percent (33 of 37) of hips with values below this threshold were FAI/control, and 90% (37 of 41) of hips with values above -1.3° were dysplastic. The ROC-AUC for borderline hips was 0.86. CONCLUSION: The FEAR index was associated with the diagnosis of hip dysplasia and FAI in a patient cohort with a wide age range and with varying degrees of acetabular deformity. Specifically, a FEAR index greater than -1.3° is associated with a dysplastic hip and a FEAR index less than -1.3° is associated with a hip displaying FAI. Using this reliable, developmentally based radiographic measure may help hip preservation surgeons establish a correct diagnosis and more appropriately guide treatment. LEVEL OF EVIDENCE LEVEL: III, diagnostic study.