Diagnostic yield of nasal scrape biopsies in primary ciliary dyskinesia: A multicenter experience

Examination of ciliary ultrastructure remains the cornerstone diagnostic test for primary ciliary dyskinesia (PCD), a disease of abnormal ciliary structure and/or function. Obtaining a biopsy with sufficient interpretable cilia and producing quality transmission electron micrographs (TEM) is challenging. Methods for processing tissues for optimal preservation of axonemal structures are not standardized. This study describes our experience using a standard operating procedure (SOP) for collecting nasal scrape biopsies and processing TEMs in a centralized laboratory. We enrolled patients with suspected PCD at research sites of the Genetic Disorders of Mucociliary Clearance Consortium. Biopsies were performed according to a SOP whereby curettes were used to scrape the inferior surface of the inferior turbinate, with samples placed in fixative. Specimens were shipped to a central laboratory where TEMs were prepared and blindly reviewed. Four hundred forty‐eight specimens were obtained from 107 young children (0–5 years), 189 older children (5–18 years), and 152 adults (> 18 years), and 88% were adequate for formal interpretation. The proportion of adequate specimens was higher in adults than in children. Fifty percent of the adequate TEMs showed normal ciliary ultrastructure, 39% showed hallmark ultrastructural changes of PCD, and 11% had indeterminate findings. Among specimens without clearly normal ultrastructure, 72% had defects of the outer and/or inner dynein arms (IDA), while 7% had central apparatus defects with or without IDA defects. In summary, nasal scrape biopsies can be performed in the outpatient setting and yield interpretable samples, when performed by individuals with adequate training and experience according to an SOP. Pediatr. Pulmonol. 2011; 46:483–488. © 2011 Wiley‐Liss, Inc.     

Mechanistic details of CRISPR-associated transposon recruitment and integration revealed by cryo-EM

CRISPR-associated transposons (CASTs) are Tn7-like elements that are capable of RNA-guided DNA integration. Although structural data are known for nearly all core transposition components, the transposase component, TnsB, remains uncharacterized. Using cryo-electron microscopy (cryo-EM) structure determination, we reveal the conformation of TnsB during transposon integration for the type V-K CAST system from Scytonema hofmanni (ShCAST). Our structure of TnsB is a tetramer, revealing strong mechanistic relationships with the overall architecture of RNaseH transposases/integrases in general, and in particular the MuA transposase from bacteriophage Mu. However, key structural differences in the C-terminal domains indicate that TnsB’s tetrameric architecture is stabilized by a different set of protein–protein interactions compared with MuA. We describe the base-specific interactions along the TnsB binding site, which explain how different CAST elements can function on cognate mobile elements independent of one another. We observe that melting of the 5′ nontransferred strand of the transposon end is a structural feature stabilized by TnsB and furthermore is crucial for donor–DNA integration. Although not observed in the TnsB strand-transfer complex, the C-terminal end of TnsB serves a crucial role in transposase recruitment to the target site. The C-terminal end of TnsB adopts a short, structured 15-residue “hook” that decorates TnsC filaments. Unlike full-length TnsB, C-terminal fragments do not appear to stimulate filament disassembly using two different assays, suggesting that additional interactions between TnsB and TnsC are required for redistributing TnsC to appropriate targets. The structural information presented here will help guide future work in modifying these important systems as programmable gene integration tools.

CRISPR-associated transposons (CASTs) have co-opted Cas genes for RNA-guided DNA integration and are promising candidates for novel genome-editing methods (1, 2). CAST elements are fascinating because of their ability to integrate DNA payloads contained within the element at a precise position, with a specific orientation, and in a programmable manner (3–6). CAST elements are evolutionarily related to Tn7 elements and are often referred to as “Tn7-like” (2). Accordingly, Tn7 and Tn7-like CAST elements contain multiple conserved genes that likely share common functions, leading to newfound appreciation for decades of biochemical, genetic, and structural work on Tn7 and related elements (7, 8).Despite remarkable diversity (1, 8, 9), all RNA-directed transposition systems characterized to date share multiple components: a CRISPR effector (Cas12k or Cascade), proteins dedicated to target capture (TniQ + TnsC), and a transposase called TnsB. By analogy to work from prototypic Tn7 (2), TnsB carries out transposon end recognition, pairing, and the chemical steps which result in integration of cognate element DNA. The V-K CAST system from Scytonema hofmanni (ShCAST) is especially appealing as a model system for mechanistic studies due to its simplicity (a single polypeptide chain encodes the effector) and robust in vitro activity (4). Currently, structural information on components Cas12k (10, 11), TniQ, and TnsC (11, 12) exists except for the TnsB transposase, and it remains mysterious how these indispensable components interact to precisely direct insertions into a guide RNA–directed target site. More generally, structural information is required for the TnsB transposase to obtain a mechanistic understanding of the Tn7 and Tn7-like elements given their broad distribution across diverse bacteria with many interesting targeting modalities, including all of the functionally described CAST elements.Despite their similarities, the transposase components of the aforementioned transposons do not behave identically, and components are not interchangeable. ShCAST, like bacteriophage Mu, likely uses a replicative transposition mechanism (13) involving host-primed DNA replication of the element to generate cointegrates between the donor and target DNAs in vivo (14, 15). In contrast, prototypic Tn7 uses a cut-and-paste mechanism that directly forms a simple insertion (16) based on the heteromeric TnsA+TnsB transposase (17). TnsA and TnsB form a protein complex for which the nuclease activities of both proteins (TnsA and TnsB) are required to generate simple insertions (17–20), but the regulatory details of this process remain unresolved with Tn7 and related elements. A structure of the TnsB transposase would set the foundation for understanding the similarities that link related Tn7 and CAST elements, as well as the key differences that would explain their distinct behavior.     

Capsid expansion mechanism of bacteriophage T7 revealed by multistate atomic models derived from cryo-EM reconstructions

Many dsDNA viruses first assemble a DNA-free procapsid, using a scaffolding protein-dependent process. The procapsid, then, undergoes dramatic conformational maturation while packaging DNA. For bacteriophage T7 we report the following four single-particle cryo-EM 3D reconstructions and the derived atomic models: procapsid (4.6-Å resolution), an early-stage DNA packaging intermediate (3.5 Å), a later-stage packaging intermediate (6.6 Å), and the final infectious phage (3.6 Å). In the procapsid, the N terminus of the major capsid protein, gp10, has a six-turn helix at the inner surface of the shell, where each skewed hexamer of gp10 interacts with two scaffolding proteins. With the exit of scaffolding proteins during maturation the gp10 N-terminal helix unfolds and swings through the capsid shell to the outer surface. The refolded N-terminal region has a hairpin that forms a novel noncovalent, joint-like, intercapsomeric interaction with a pocket formed during shell expansion. These large conformational changes also result in a new noncovalent, intracapsomeric topological linking. Both interactions further stabilize the capsids by interlocking all pentameric and hexameric capsomeres in both DNA packaging intermediate and phage. Although the final phage shell has nearly identical structure to the shell of the DNA-free intermediate, surprisingly we found that the icosahedral faces of the phage are slightly (∼4 Å) contracted relative to the faces of the intermediate, despite the internal pressure from the densely packaged DNA genome. These structures provide a basis for understanding the capsid maturation process during DNA packaging that is essential for large numbers of dsDNA viruses.Many dsDNA viruses, including tailed phages and herpes viruses, initially assemble a DNA-free procapsid with assistance of a network of scaffold proteins. Accompanying the exit of scaffolding proteins during subsequent ATP-driven DNA packaging, the icosahedral shell of the procapsid undergoes dramatic conformational changes and matures into a typically larger and more angular shell of the infectious phage (1–6). However, structural details, including those of capsid intermediates, are limited to the phage HK97 system (5, 7–9), for which recombinantly produced procapsid and nonphysiological conversion products were analyzed.The packaging of the 39.937-kbp DNA genome of the short-tail Escherichia coli bacteriophage, T7, is a model for understanding basic principles common to dsDNA tailed phages and herpes viruses. The T7 system is also of interest because it has been used for popular biotechnologies, such as recombinant protein expression (10) and protein display on the capsid surface (11). The T7 capsid contains 415 copies of the major shell protein gp10 (12) that form a T = 7L icosahedral lattice. From low-resolution cryo-EM 3D reconstructions the tertiary topology of gp10 can be divided into four regions: N-arm, E-loop, A-domain, and P-domain, which together place the gp10 protein in the HK97 fold category (2, 13, 14). The T7 procapsid, capsid I, contains 110–140 molecules of scaffolding protein, gp9 (4, 15, 16). After scaffolding protein expulsion the spherical T7 capsid I expands to more angular intermediates, which are collectively called capsid II (2, 4, 14, 16–18).Two DNA-free capsid IIs are purified in quantity sufficient for structural studies by cryo-EM (16). Both are produced during the normal process of wild-type T7 DNA packaging in vivo. One has an unusually low density during buoyant density centrifugation in a metrizamide density gradient (1.086 g/mL; metrizamide low density, or MLD, capsid II) and the other has a density as expected for hydrated proteins (1.28 g/mL; metrizamide high density, or MHD, capsid II) (16). The low density of MLD capsid II is caused by impermeability to metrizamide (789 Da) (16). The MLD capsid II particles are produced before MHD capsid II particles based on kinetic studies (16).The DNA packaging of T7 phage starts at capsid I state where the DNA is packaged by the ATPases (gp18 and gp19) to pass through the portal (gp8) apparatus (19). By analyzing kinetics of in vivo-produced capsids, MLD capsid II was found to be the first postcapsid I capsid. MLD capsid II appears with the kinetics of an intermediate (16) but is obviously no longer in the DNA packaging pathway because it has detached from the DNA molecule that it was packaging. MLD capsid II is not produced when a nonpermissive host is infected with a T7 amber mutant defective in DNA packaging (summarized in ref. 16). Thus, MLD capsid II is an intermediate that has been altered during either cellular lysis or subsequent purification. MHD capsid II also has the appearance kinetics of an intermediate of packaging, but one that occurs later (16). Whereas MLD capsid II has the internal core stack including proteins gp8, gp14, gp15, and gp16 (16), MHD capsid II does not have the internal core stack proteins, which were presumably lost when packaged DNA exited the capsid (16).The existence of these various capsids provides an opportunity to obtain a high-resolution (3–4 Å) analysis of structural dynamics that occur in vivo. Here we report cryo-EM structures of the shells of the following bacteriophage T7 capsids: capsid I (4.6 Å), MLD capsid II (3.5 Å), MHD capsid II (6.6 Å), and phage (3.6 Å). The two capsid II shells are the first postprocapsid, in vivo-generated shells (for any packaging system) to be subjected to high-resolution structural analysis, to our knowledge. The results reveal (i) an HK97-fold shell protein with an intracapsomere, noncovalent topological linking and another intercapsomere, joint interaction, neither interaction having been found for other dsDNA tailed phages; (ii) details of the interaction of gp9 scaffolding protein with the inner surface of the capsid I shell; (iii) a novel refolding and externalization of the N terminus of major capsid protein, gp10; and (iv) a subtle, surprising contraction of the gp10 shell in transit from MLD capsid II to phage. Based on these observations, we propose a general procapsid assembly and maturation pathway for dsDNA viruses.     

Impairment of nitric oxide output of conducting airways in primary ciliary dyskinesia

Nasal nitric oxide (NO) concentration is dramatically reduced in primary ciliary dyskinesia (PCD). The aims of this study were to apply a multiple-flow NO analysis to investigate whether NO output from the bronchial tree was affected in a similar way to nasal NO output, and to search for a relationship between flow-independent exchange parameters and airflow limitation. Multiple flow rate analysis of exhaled NO, allowing the calculation of maximum airway wall flux and alveolar NO concentration, was performed in 17 PCD patients (median age, 25-75th percentiles: 13.5, 12.1-17.6) with documented ultrastructural cilia abnormalities and 28 healthy subjects (16.0, 11.0-21.0). Median maximum airway wall flux and median alveolar NO concentration were significantly reduced in PCD patients compared to healthy subjects: 16.0, 7.5-29.5, vs. 25.0, 15.0-32.5 nl/min (P<0.05) and 2.5, 1.6-3.3, vs. 5.0, 3.6-6.5 ppb (P<0.01), respectively. Significant correlations between maximum airway wall flux and airflow limitation were found, i.e., resistance of respiratory system (rho=0.74, P<0.005), forced expiratory volume in one second (FEV(1))/VC (rho= -0.61, P<0.05), FEV(1) (rho=-0.52, P< 0.05), mid expiratory flow between 25 and 75% of forced vital capacity (MEF(25-75)) (rho=-0.54, P<0.05), and maximal instantaneous expiratory flow at 50% of the vital capacity (MEF(50)) (rho=-0.55, P<0.05). In conclusion, the impairment of NO output is less pronounced in the lower than in the upper (nasal) respiratory tract in PCD. A decrease in maximal NO output from conducting airways is associated with limited airflow impairment.     

Regulation of flagellar dynein activity by a central pair kinesin

The motility of cilia and flagella is powered by dynein ATPases associated with outer doublet microtubules. However, a flagellar kinesin-like protein that may function as a motor associates with the central pair complex. We determined that Chlamydomonas reinhardtii central pair kinesin Klp1 is a phosphoprotein and, like conventional kinesins, binds to microtubules in vitro in the presence of adenosine 5'-[beta,gamma-imido]triphosphate, but not ATP. To characterize the function of Klp1, we generated RNA interference expression constructs that reduce in vivo flagellar Klp1 levels. Klp1 knockdown cells have flagella that either beat very slowly or are paralyzed. EM image averages show disruption of two structures associated with the C2 central pair microtubule, C2b and C2c. Greatest density is lost from part of projection C2c, which is in a position to interact with doublet-associated radial spokes. Klp1 therefore retains properties of a motor protein and is essential for normal flagellar motility. We hypothesize that Klp1 acts as a conformational switch to signal spoke-dependent control of dynein activity.     

Uncoating of common cold virus is preceded by RNA switching as determined by X-ray and cryo-EM analyses of the subviral A-particle

During infection, viruses undergo conformational changes that lead to delivery of their genome into host cytosol. In human rhinovirus A2, this conversion is triggered by exposure to acid pH in the endosome. The first subviral intermediate, the A-particle, is expanded and has lost the internal viral protein 4 (VP4), but retains its RNA genome. The nucleic acid is subsequently released, presumably through one of the large pores that open at the icosahedral twofold axes, and is transferred along a conduit in the endosomal membrane; the remaining empty capsids, termed B-particles, are shuttled to lysosomes for degradation. Previous structural analyses revealed important differences between the native protein shell and the empty capsid. Nonetheless, little is known of A-particle architecture or conformation of the RNA core. Using 3D cryo-electron microscopy and X-ray crystallography, we found notable changes in RNA–protein contacts during conversion of native virus into the A-particle uncoating intermediate. In the native virion, we confirmed interaction of nucleotide(s) with Trp³⁸ of VP2 and identified additional contacts with the VP1 N terminus. Study of A-particle structure showed that the VP2 contact is maintained, that VP1 interactions are lost after exit of the VP1 N-terminal extension, and that the RNA also interacts with residues of the VP3 N terminus at the fivefold axis. These associations lead to formation of a well-ordered RNA layer beneath the protein shell, suggesting that these interactions guide ordered RNA egress.Human rhinoviruses (HRVs) cause the common cold. Although seldom severe, this disease is widespread and frequent in man; HRVs thus have considerable economic impact due to expenditure on medication and lost working days. More than 150 serotypes belong to the genus Enteroviruses (EVs) of the Picornaviridae family, which includes serious human and animal pathogens. In addition to phylogenetic classification into species A, -B, and -C, HRVs are divided into a minor receptor group (12 HRV-A) that bind low-density lipoprotein receptors (LDLRs), and a major receptor group (more than 89 HRV-A and -B serotypes) that use intercellular adhesion molecule 1 (ICAM-1) for cell entry (1). HRV-C binds an unknown receptor (2).The EV icosahedral shell is built from four viral proteins (VP1–4) that encase a single-stranded (+)–sense RNA genome. Sixty copies each of these four polypeptides assemble on a T = 1 (pseudo T = 3) lattice, ∼30 nm in diameter. VP1, VP2, and VP3 are surface-exposed; the small myristoylated VP4 is internal. In the mature virion, the N-terminal extensions of VP1, VP2, and VP3, together with the entire VP4, interact in an intricate network beneath the shell (Fig. S1) (3, 4).In the cytosol, the viral RNA is translated into a ∼250 kDa precursor polyprotein that is processed by viral proteinases. Assembly of the viral shell involves immature pentamers built from VP0, VP1, and VP3. VP2 and VP4 arise late in infection through VP0 cleavage, concomitant with RNA encapsidation. In addition to mature virions, native empty capsids (NECs) of HRV2, HRV14, and equine rhinovirus (5), and presumably of other EVs, are assembled in the infected cell. They might be direct precursors of native virions, a capsid protein reservoir (6), and/or the end-product of an abortive assembly process (7). NECs attach to the receptor and undergo conformational changes similar to those of the native virus, except that they retain VP4, as it remains connected to VP2 (3, 8).All EVs are thought to undergo a similar sequence of events leading to infection. After binding their respective receptors, they are endocytosed. In poliovirus (PV), receptor attachment catalyzes uncoating, but in some HRVs the acidic pH in endosomes is an additional trigger for the structural changes needed for RNA exit (9, 10). In minor group HRVs, low pH alone induces these changes (11). As shown for HRV2 (12), the acid-triggered beta-propeller switch of the LDLR assists rhinovirus infection. Once the virion is in the late endosomal compartment, it dissociates from its receptor and is simultaneously transformed into the A-particle, which has an expanded shell, lacks VP4, and is more hydrophobic than native virus and NEC due to surface exposure of the amphipathic VP1 N-terminal extension (13). The A-particle can bind directly to the endosomal membrane for RNA translocation (14), leaving behind the empty B-particle.Recent work on PV caught in the act of releasing its genome shows the RNA exiting through channels near the twofold axes (15). The X-ray structure of the HRV2 B-particle showed the details of the structural rearrangements that lead to the end-product of uncoating (16). A hinge movement around the hydrophobic pocket in VP1 induces a coordinated displacement of VP2 and VP3, resulting in capsid expansion and the opening of channels in the shell. Similar alterations were observed in EV-71 when the X-ray structures of native virus and expanded NECs were compared (17).By solving the 3D cryo–electron microscopy (3D cryo-EM) structures of native HRV2 and its A-particle (the intermediate between native virus and empty capsid) produced by incubation in acidic buffer that mimics the endosomal environment, we identified important changes in the interactions between RNA and the protein shell. We confirm these RNA–protein contacts in the A-particle in our 6.4 Å X-ray structure. The well-ordered RNA layer close to the inner capsid face is stabilized by numerous contacts; this framework might facilitate exit of the genome in a highly ordered, coordinated manner.     

Resection of the radial head and partial open synovectomy of the elbow in the young adult with haemophilia: long-term results

Resection of the radial head and partial open synovectomy of the elbow have been used in the treatment of repeated haemarthroses of the elbows in 15 patients with haemophilia. The indications for the index operation were: severe pain and bleeding in the elbow that occurred in spite of appropriate, episodic, replacement therapy over a period of more than 6 months, associated with hypertrophy of the radial head and a significant loss of pronation-supination. The long-term results were assessed retrospectively according to the classification recommended by the Orthopaedic Advisory Committee of the World Federation of Hemophilia. In the operated group, three patients had a good result, seven were fair, and five poor. It is concluded that this procedure appears to reduce the incidence of haemarthrosis but did not slow the rate of evolution of radiographic changes.     

经桡动脉与经股动脉两种途径冠心病介入治疗的对比研究

目的 :比较经桡动脉途径和股动脉途径冠心病介入的手术方法、结果和并发症。方法 :10 3 3例冠心病住院患者分别接受经桡动脉 （ A）组 （ n=419）或经股动脉 （ F）组 （ n=614 ）途径的冠心病介入 （ PCI）治疗 ,观察两组手术成功率和并发症发生率。结果 :两组患者 PCI成功率没有显著性差异。A组局部血肿、不适反应等发生率显著低于 F组 ,假性动脉瘤、动静脉瘘、局部感染及表皮坏死等并发症在 A组未出现 ,F组分别发生 11,4,6,7例 ,但两组间无显著性差异。两组均未出现远端肢体缺血、神经损伤等。结论 :实施 PCI术的两种途径都是安全、有效和可行的方法 ,与经股动脉比较 ,经桡动脉途径可减少并发症的发生     

血管钠肽对人桡动脉的舒张作用及其机制

目的 :研究血管钠肽 （VNP）对人桡动脉 （humanradialartery,HRA）的舒张作用及其机制。方法 :采用离体血管灌流的方法 ,观察VNP对内皮完整和去内皮HRA的舒张作用 ,以及 8 Br cGMP、NPR A、B选择性抑制剂HS 14 2 1、鸟苷酸环化酶选择性抑制剂美蓝 （methyleneblue ,MB）和Ca2 + 激活K+ 通道 （Ca2 + activatedK+ channel,KCa）的选择性抑制剂TEA对这一过程的影响。结果 :VNP（10 10 ～ 10 -6mol/L）可剂量依赖性地舒张HRA ,该作用无内皮依赖性。 8 Br cGMP（10 -7～ 10 -3 mol/L）可模拟VNP的血管舒张效应。HS 14 2 1（2× 10 -5mol/L）或MB（10 -5mol/L）完全阻断VNP舒张作用。TEA（10 -3 mol/L）减弱VNP的舒血管作用。结论 :VNP对HRA具有不依赖内皮的舒张作用 ,它主要是通过作用于NPR A、B ,即钠尿肽的鸟苷酸环化酶耦联受体 ,升高细胞内的cGMP水平。KCa可能是其舒张作用的重要效应分子。为应用VNP防治血管移植术后的痉挛提供了重要的理论依据。目的 :研究血管钠肽 （VNP）对人桡动脉 （humanradialartery,HRA）的舒张作用及其机制。方法 :采用离体血管灌流的方法 ,观察VNP对内皮完整和去内皮HRA的舒张作用 ,以及 8 Br cGMP、NPR A、B选择性抑制剂HS 14 2 1、鸟苷酸环化酶选择性抑制剂美蓝 （methyleneblue ,MB）和Ca2 +     

Randomized comparison of operator radiation exposure during coronary angiography and intervention by radial or femoral approach.

Controversial data have been published on the amount of radiation exposure during radial coronary procedures. We hypothesized that in the current era, high-volume operators with optimal technique would not be exposed to higher radiation doses during radial procedures. A total of 297 patients undergoing cardiac catheterization (195 elective diagnostic coronary angiograms and 102 elective coronary interventions) were prospectively assigned in a random fashion to the radial access (RA) or femoral access (FA). All procedures were performed by the same operator with vast experience in radial procedures and standard measures for radiation protection were used. Operator radiation exposure was measured with an electronic radiation dosimeter attached to the breast pocket of the operator on the outside of the lead apron and estimates of the ambient dose equivalent were derived. For coronary angiograms, fluoroscopy time (2.8 +/- 2.1 vs. 1.7 +/- 1.4 min; P < 0.001) and dose-area product (15.1 +/- 8.4 vs. 13.1 +/- 8.5 Gy x cm(2); P < 0.05) were increased by 18% and 15%, respectively, for RA vs. FA. Operator radiation exposure was 100% higher for the RA compared to the FA (64 +/- 55 vs. 32 +/- 39 microSv; P < 0.001). For coronary interventions, fluoroscopy time (11.4 +/- 8.4 vs. 10.4 +/- 6.8 min; P = NS) and dose-area product (46.3 +/- 28.7 vs. 51.0 +/- 29.4 Gy x cm(2); P = NS) for RA and FA were not statistically different. However, operator radiation exposure was increased by 51% for the RA compared to the FA (166 +/- 188 vs. 110 +/- 115 microSv; P < 0.05). This study demonstrates that the radial approach is burdened with a 100% increase in operator radiation exposure during diagnostic coronary catheterization procedures and a 50% increase during coronary interventions, provided that no special devices for radiation protection are used. Measurements of radiation dose, such as fluoroscopy time and dose-area product, substantially underestimate the disproportionate rise in radiation exposure. Special precautions are warranted to improve radiation protection during invasive coronary procedures via the radial approach.     

Feasibility of complex coronary and peripheral interventions by trans‐radial approach using large sheaths

Background : Trans‐radial approach (TRA) reduces vascular access‐site complications but has some technical limitations. Usually, TRA procedures are performed using 5 Fr or 6 Fr sheaths, whereas complex interventions requiring larger sheaths are approached by trans‐femoral access. Methods : During 4 years, at two Institutions with high TRA use, we have attempted to perform selected complex coronary or peripheral interventions by TRA using sheaths larger than 6 Fr. Clinical and procedural data were prospectively collected. Attempt to place a 7 Fr or 8 Fr sheath (according to the planned strategy of the procedure) was performed after 5–6 Fr sheath insertion, administration of intra‐arterial nitrates and radial artery angiography. Late (>3 months) patency of the radial artery was checked (by angiography in the case of repeated procedures or by palpation + reverse Allen test). Results : We collected 60 patients in which TRA large sheath insertion was attempted. The large sheath (87% 7 Fr, 13% 8 Fr) was successfully placed in all cases. Most of the procedures were complex coronary interventions (bifurcated or highly thrombotic or calcific chronic total occlusive lesions), whereas 8.3% were carotid interventions. Procedural success rate was 98.3% (1 failure to reopen a chronic total occlusion). No access‐site related complication occurred. In 57 (95%) patients, late radial artery patency was assessed and showed patency in 90% of the cases, the remaining patients having asymptomatic collateralized occlusion. Conclusions : In selected patients, complex percutaneous interventions requiring 7–8 Fr sheaths can be successfully performed by RA approach without access‐site clinical consequences. © 2011 Wiley Periodicals, Inc.