Gene-Wise Burden of Coding Variants Correlates to Noncoding Pharmacogenetic Risk Variants.

Genetic variability can modulate individual drug responses. A significant portion of pharmacogenetic variants reside in the noncoding genome yet it is unclear if the noncoding variants directly influence protein function and expression or are present on a haplotype including a functionally relevant genetic variation (synthetic association). Gene-wise variant burden (GVB) is a gene-level measure of deleteriousness, reflecting the cumulative effects of deleterious coding variants, predicted in silico. To test potential associations between noncoding and coding pharmacogenetic variants, we computed a drug-level GVB for 5099 drugs from DrugBank for 2504 genomes of the 1000 Genomes Project and evaluated the correlation between the long-known noncoding variant-drug associations in PharmGKB, with functionally relevant rare and common coding variants aggregated into GVBs. We obtained the area under the receiver operating characteristics curve (AUC) by comparing the drug-level GVB ranks against the corresponding pharmacogenetic variants-drug associations in PharmGKB. We obtained high overall AUCs (0.710 ± 0.022-0.734 ± 0.018) for six different methods (i.e., SIFT, MutationTaster, Polyphen-2 HVAR, Polyphen-2 HDIV, phyloP, and GERP++), and further improved the ethnicity-specific validations (0.759 ± 0.066-0.791 ± 0.078). These results suggest that a significant portion of the long-known noncoding variant-drug associations can be explained as synthetic associations with rare and common coding variants burden of the corresponding pharmacogenes.

Ideal Dimers of Gold Nanospheres for Precision Plasmonics: Synthesis and Characterization at the Single-Particle Level for Identification of Higher Order Modes.

Ideal dimers comprising gold nanoparticles with a smooth surface and high sphericity are synthesized by a substrate-based assembly strategy with efficient cetyltrimethylammonium bromide removal. An unprecedented structural and plasmonic uniformity at the single-particle level is observed since inhomogeneities resulting from variations in gap morphology are eliminated. Single ideal dimers are analyzed by polarization-resolved dark-field scattering spectroscopy. Contributions from transverse as well as quadrupolar and octupolar longitudinal plasmon coupling modes can be discriminated because of their orthogonal polarization behavior. The assignment of these higher order coupling modes is supported by computer simulations.

Gold and silver nanoparticle monomers are non-SERS-active: a negative experimental study with silica-encapsulated Raman-reporter-coated metal colloids.

Noble metal nanoparticles (NPs) are the most commonly employed plasmonic substrates in surface-enhanced Raman scattering (SERS) experiments. Computer simulations show that monomers of Ag and Au nanocrystals ("spherical" NPs) do not exhibit a notable plasmonic enhancement, i.e., they are essentially non-SERS-active. However, in experiments, SERS enhanced by spherical NP colloids has been frequently reported. This implies that the monomers do not have strong SERS activity, but detectable enhancement should more or less be there. Because of the gap between theory and practice, it is important to demonstrate experimentally how SERS-active the metal colloid actually is and, in case a SERS signal is observed, where it originates from. In particular the aggregation of the colloid, induced by high centrifugal forces in washing steps or due to a harsh ionic environment of the suspension medium, should be controlled since it is the very high SERS activity of NP clusters which dominates the overall SERS signal of the colloid. We report here the experimental evaluation of the SERS activity of 80 nm Au and Ag NP monomers. Instead of showing fancy nanostructures and super SERS enhancement, we present the method on how to obtain negative experimental data. In this approach, no SERS signal was obtained from the colloid with a Raman reporter on the metal surface when the NPs were encapsulated carefully within a thick silica shell. Without silica encapsulation, if a very low centrifugation speed is used for the washing steps, only a negligible SERS signal can be detected even at very high NP concentrations. In contrast, strong SERS signals can be detected when the NPs are suspended in acidic solutions. These results indicate that Au and Ag NP monomers essentially exhibit no SERS activity of practical relevance.

Probing interfacial interactions using core-satellite plasmon rulers.

Understanding molecular interactions at the interfaces of nanoparticles is fundamentally important because they determine the stability, affinity, functionality, and assembly of nanoparticles. However, probing the governing intermolecular forces at the interfaces, particularly for the nanoparticles dispersed in solution, remains challenging. Here, we demonstrate that the interfacial interactions between citrate-capped gold nanoparticles and various molecular functional groups can be probed using a plasmon ruler, based on a well-defined core-satellite nanoassembly structure. Different nature of the interactions causes a subtle change in the interparticle distance, and the change is sensitively measured as a shift in the plasmon coupling band of the core-satellite nanoassemblies. Molecular interactions including covalent bonding, hydrogen bonding, electrostatic interactions, and van der Waals interactions are explored.

Arthroscopically assisted reconstruction of triangular fibrocartilage complex foveal avulsion in the ulnar variance-positive patient.

PURPOSE: Our aim was to evaluate the clinical results of patients treated by arthroscopically assisted reconstruction of foveal avulsion injury of the triangular fibrocartilage complex (TFCC) using a suture anchor. METHODS: We retrospectively reviewed the results of 15 patients (11 men and 4 women; mean age, 30.5 years) who underwent surgical procedures for the treatment of TFCC foveal avulsion at our hospital. The patients were followed up for a mean of 29 months. The patients had TFCC foveal avulsion caused by sprains (n = 8), falls (n = 4), playing baseball (n = 2), and a motor vehicle accident (n = 1). All the patients underwent magnetic resonance imaging. Radiographs obtained to assess ulnar variance (UV), ulnar-dorsal subluxation, and function of the wrist based on grip power; Disabilities of the Arm, Shoulder and Hand score; and Mayo wrist score were examined for all patients both preoperatively and postoperatively. RESULTS: On preoperative magnetic resonance imaging, TFCC foveal avulsion was observed in 13 of 15 cases. The mean UV value based on preoperative simple radiographic findings was 1.7 ± 1.0 mm, and dorsal subluxation at the distal ulna improved from 2.9 ± 3.0 mm to 0.2 ± 0.9 mm (P = .017). In all cases the distal radioulnar joint instability disappeared postoperatively. Grip power (compared with the uninvolved limb) was 79.3% preoperatively and 82.9% postoperatively (P = .086). The Disabilities of the Arm, Shoulder and Hand scores were 28.4 points preoperatively and 16.6 points postoperatively (P = .061). The Mayo wrist scores were excellent in 10 cases, good in 2, and fair in 3, and the mean score improved significantly from 64 points preoperatively to 84 points postoperatively (P = .007). CONCLUSIONS: Arthroscopic-assisted suture anchor reattachment of the TFCC in patients with traumatic TFCC foveal avulsion can prevent or reduce distal radioulnar joint instability and reduce pain even in chronic cases with positive UV. LEVEL OF EVIDENCE: Level IV, therapeutic case series.

Photoisomerization of azobenzene derivatives confined in gold nanoparticle aggregates.

Photoisomerization is an important reaction that confers photoresponsive functionality on nanoparticles. Although photoisomerization of molecules forming self-assembled monolayers on two-dimensional surfaces or three-dimensional clusters has been studied, a detailed picture of interactions of molecules undergoing isomerization with nanoparticles is not available. In this paper, we report on the photoisomerization of azobenzene derivatives spatially confined in gold nanoparticle (AuNP) aggregates. AuNP aggregates allow us to simultaneously probe the structural changes of molecules via surface-enhanced Raman spectroscopy (SERS) and the accompanying changes in interparticle interactions via surface plasmon couplings. AuNP aggregates are formed by the adsorption of synthesized azobenzene-derivatized sulfides (Az) onto the surfaces of AuNPs. The photoisomerization of the adsorbed Az from trans to cis by excitation at 365 nm causes the AuNPs to move close to each other in the aggregates, leading to a redshift of the surface plasmon coupling band in the UV-vis spectra and a concomitant rise in SERS intensity. SERS spectra reveal that the vibrational modes containing the N=N stretching character redshift upon irradiation, suggesting that the N=N bond is significantly weakened when Az is in the cis form in the AuNP aggregates. The weakening of the N=N bond is attributed to the interaction of the N=N bond, which is more exposed to the outside in the cis conformation, with the nearby AuNPs that have come closer by the isomerization of adsorbed Az. We find that backisomerization from cis to trans occurs much faster in the AuNP aggregates (k = 1.9 × 10(-2) min(-1)) than in solution (k = 1.3 × 10(-3) min(-1)) because of the reduced N=N bond order of cis-Az in the aggregates.

Spatially controlled SERS patterning using photoinduced disassembly of gelated gold nanoparticle aggregates.

Controlling the assembly of the nanoparticles is important because the optical properties of noble metal nanoparticles, such as the surface plasmon resonance (SPR) and surface-enhanced Raman scattering (SERS), are critically dependent on interparticle distances. Among many approaches available, light-induced disassembly is particularly attractive because it enables spatial modification of the optical properties of nanoparticle assemblies. In this study, we prepare gold nanoparticle (AuNP) aggregates in a gel matrix. Irradiation of the gelated AuNP aggregates at 532 nm leads to the disassembly of the aggregates, changing the color (SPR) from dark blue to red and extinguishing the SERS signal along the irradiated pattern, which opens the possibility of facile fabrication of spatially controlled SERS-generating microstructures. The photoinduced disassembly of the AuNP aggregates in solution is also investigated using UV-vis spectroscopy and transmission electron microscopy.

Photooxidative coupling of thiophenol derivatives to disulfides.

Disulfide bonds play an important role in determining the structure and stability of proteins and nanoparticles. Despite extensive studies on the oxidation of thiols for the synthesis of disulfides, little is known about the photooxidation of thiols, which may be a clean, safe, and economical alternative to the use of harmful and expensive metal-containing oxidants and catalysts. In this paper, we report the photooxidative coupling of thiophenol derivatives to disulfides. Para-substituted thiophenol derivatives, p-SHC(6)H(4)X (X = NO(2), COOH, Cl, and OCH(3)), are irradiated, and disulfides, X(2)(C(6)H(4))(2)S(2), are identified as the major photoproducts using Raman, UV-vis, IR, and NMR spectroscopies. For p-nitrothiophenol (pNTP), 4,4'-dinitrodiphenyldisulfide (DNDPDS) is produced in 81% yield. The product yield changes with pH, being the highest at pH ≈ 5, suggesting that both neutral thiol and anionic thiolate forms of pNTP are required for the photoreaction to occur. Excitation at 455 nm, at which the thiolate form of pNTP absorbs strongly, leads to the largest yield of DNDPDS, whereas very little DNDPDS is formed by excitation of the thiol form of pNTP at 325 nm. Our observations suggest that the photooxidation occurs via collisions of the electronically excited thiolate form of pNTP with the surrounding neutral thiol forms of pNTP. The photooxidation reaction happens regardless of the electron-withdrawing or electron-donating properties of the substituents if the pH and excitation wavelengths are properly chosen. The versatility of light and generality of the photooxidative coupling reaction of thiophenol derivatives may open new possibilities for selective and site-specific photocontrol of disulfide bond formation in biology and nanomaterial science as well as in synthetic chemistry.

Time-dependent and symmetry-selective charge-transfer contribution to SERS in gold nanoparticle aggregates.

We report the time- and symmetry-dependent surface-enhanced Raman scattering (SERS) of gold nanoparticle (AuNP) aggregates. The addition of p-aminothiophenol (p-ATP) instantly induces the aggregation of AuNPs, confirmed by large absorption in the near-IR region. Dynamic light scattering measurements show that the addition of p-ATP immediately assembles the AuNPs (13 nm) to form aggregates with a mean diameter of approximately 200 nm, which then further grow to a size of approximately 300 nm. Raman spectra acquired via time lapse show that the a(1)-symmetry bands of p-ATP are enhanced simultaneously with the formation of the aggregates, indicating that the electromagnetic enhancement largely contributes to the SERS of the AuNP aggregates. In contrast, the enhancement of the b(2)-symmetry bands occurs approximately 10 h after the formation of the aggregates and slowly progresses. The enhancement of the b(2) mode is attributed to the charge transfer between AuNPs and adsorbates, rather than the reorientation of the adsorbates because thiophenol and p-methylthiophenol that have surface structures and intermolecular interactions similar to those of p-ATP do not exhibit a symmetry-specific Raman enhancement pattern. To elucidate the disparity in the timescale between the charge-transfer resonance and the formation of the aggregates, we propose two models. A further close approach of the AuNPs constituting the aggregates causes the additional adsorption of the initially adsorbed p-ATP onto neighboring AuNPs, tuning the charge transfer state to be in resonance with the Raman excitation laser. Density functional theory calculations confirm the resonance charge-transfer tunneling through the bridging p-ATP in the AuNP-p-ATP-AuNP structures. Alternatively, the gradual continuing adsorption of p-ATP increases the local Fermi level of AuNPs into the region of resonant charge transfer from the Fermi level to the LUMO of the adsorbates. This model is corroborated by the faster appearance of b(2)-mode enhancement for the AuNPs with initially higher zeta potentials.

Mobile Genome Express (MGE): A comprehensive automatic genetic analyses pipeline with a mobile device.

The development of next-generation sequencing (NGS) technology allows to sequence whole exomes or genome. However, data analysis is still the biggest bottleneck for its wide implementation. Most laboratories still depend on manual procedures for data handling and analyses, which translates into a delay and decreased efficiency in the delivery of NGS results to doctors and patients. Thus, there is high demand for developing an automatic and an easy-to-use NGS data analyses system. We developed comprehensive, automatic genetic analyses controller named Mobile Genome Express (MGE) that works in smartphones or other mobile devices. MGE can handle all the steps for genetic analyses, such as: sample information submission, sequencing run quality check from the sequencer, secured data transfer and results review. We sequenced an Actrometrix control DNA containing multiple proven human mutations using a targeted sequencing panel, and the whole analysis was managed by MGE, and its data reviewing program called ELECTRO. All steps were processed automatically except for the final sequencing review procedure with ELECTRO to confirm mutations. The data analysis process was completed within several hours. We confirmed the mutations that we have identified were consistent with our previous results obtained by using multi-step, manual pipelines.

Plasmon coupling between silver nanoparticles: Transition from the classical to the quantum regime.

We explore plasmon coupling between silver nanoparticles (AgNPs) as two AgNPs approach each other within a subnanometer distance. We prepare AgNP dimers with two 21-nm AgNPs separated by alkanedithiol linkers in high yield. Changing the length of the alkanedithiol linkers enables us to control the interparticle distance down to the subnanometer level on the molecular scale. We observe that the longitudinal plasmon coupling band, which is sensitive to the interaction between AgNPs, gradually redshifts as the interparticle distance decreases. This observation is fully consistent with the classical electromagnetic model. The redshift of the plasmon coupling, however, undergoes a drastic change when the interparticle distance reaches ∼1nm. The longitudinal plasmon coupling band vanishes and a new intense band appears at a shorter wavelength. This band redshifts as the nanogap further narrows, but crosses over to a blueshift at ∼0.7nm. A comparison of our observation with finite-difference time-domain simulations reveals that this band arises from quantum effects. Controlled assembly of AgNP dimers in combination with simulations allows us to observe the transition of the plasmon coupling from the classical to the quantum regime at the ensemble level.

GEE: An Informatics Tool for Gene Expression Data Explore.

OBJECTIVES: Major public high-throughput functional genomic data repositories, including the Gene Expression Omnibus (GEO) and ArrayExpress have rapidly expanded. As a result, a large number of diverse high-throughput functional genomic data retrieval systems have been developed. However, high-throughput functional genomic data retrieval remains challenging. METHODS: We developed Gene Expression data Explore (GEE), the first powerful, flexible web and mobile search application for searching whole-genome epigenetic data and microarray data in public databases, such as GEO and ArrayExpress. RESULTS: GEE provides an elaborate, convenient interface of query generation competences not available via various high-throughput functional genomic data retrieval systems, including GEO, ArrayExpress, and Atlas. In particular, GEE provides a suitable query generator using eVOC, the Experimental Factor Ontology (EFO), which is well represented with a variety of high-throughput functional genomic data experimental conditions. In addition, GEE provides an experimental design query constructor (EDQC), which provides elaborate retrieval filter conditions when the user designs real experiments. CONCLUSIONS: The web version of GEE is available at http://www.snubi.org/software/gee, and its app version is available from the Apple App Store.

Development of a robust DNA quality and quantity assessment qPCR assay for targeted next-generation sequencing library preparation.

Next-generation sequencing (NGS) is becoming a standard for genetic analyses of clinical samples. DNAs retrieved from formalin-fixed, paraffin-embedded (FFPE) tissue specimens are commonly degraded, and specimens such as core biopsies are sometimes too small to obtain enough DNA for NGS applications. Thus, it is important to measure both the DNA quantity and quality accurately from clinical samples. However, there is no standard method for DNA quantity and quality analyses for NGS library preparation. We tested four different methods (PicoGreen, Qubit® fluorometry, TaqMan and SYBR-Green-based qPCR assay) and compared each to RNase P TaqMan as a reference control. We found that SYBR-Green-based qPCR assay provides a consistent and accurate DNA quantification while keeping its cost relatively low and the throughput high. We designed a dual-probe SYBR-Green qPCR assay for DNA quantity and quality assessment for targeted NGS library preparation. This assay provides a Dscore (degradation score) of the interrogated DNA by analyzing two different sizes of amplicons. We show an example of a clinical sample with a very high Dscore (high degradation). With a regular DNA quantification, without considering the degradation status, no correct NGS libraries were obtained. However, after optimizing the library condition by considering its poor DNA quality, a reasonably good library and sequencing results were obtained. In summary, we developed and presented a new DNA quantity and quality analysis qPCR assay for the targeted NGS library preparation. This assay may be mostly efficient for the clinical samples with high degradation and poor DNA quality.

Comprehensive evaluation and validation of targeted next-generation sequencing performance in two clinical laboratories.

Next-generation sequencing (NGS) has led to breakthroughs for genetic and genomic analyses and personalized medicine approaches for many diseases. More and more clinical laboratories are using NGS as a genetic screening tool for providing mutation information that is used to select the best treatment regimens for cancer patients. However, several obstacles prevent the routine implementation of NGS technology into the clinical molecular diagnosis setting: the sophisticated sample preparation process, high cost, time-consuming data analyses, as well as the reproducibility and accuracy of interpretation. To systematically evaluate the performance and quality of targeted NGS cancer panel analyses in clinical laboratories, we performed three different tests: i) laboratory-to-laboratory accuracy test, ii) intra-laboratory precision validation, and iii) limit of detection test, using formalin-fixed, paraffin-embedded cancer tissue specimens, cell lines and mutation positive DNA. A laboratory-to-laboratory accuracy test performed using 51 samples showed 100% sensitivity and 99.97% specificity. For the intra-laboratory precision test, 100% reproducibility was observed. For the limit of detection test, KRAS mutations from samples diluted from 70 to 2% of mutant allele frequencies were detected correctly. We believe that the present study demonstrated the feasibility of clinical implementation of a targeted NGS cancer panel analysis for personalized medicine.

Whole exome and targeted deep sequencing identify genome-wide allelic loss and frequent SETDB1 mutations in malignant pleural mesotheliomas.

Malignant pleural mesothelioma (MPM), a rare malignancy with a poor prognosis, is mainly caused by exposure to asbestos or other organic fibers, but the underlying genetic mechanism is not fully understood. Genetic alterations and causes for multiple primary cancer development including MPM are unknown. We used whole exome sequencing to identify somatic mutations in a patient with MPM and two additional primary cancers who had no evidence of venous, arterial, lymphovascular, or perineural invasion indicating dissemination of a primary lung cancer to the pleura. We found that the MPM had R282W, a key TP53 mutation, and genome-wide allelic loss or loss of heterozygosity, a distinct genomic alteration not previously described in MPM. We identified frequent inactivating SETDB1 mutations in this patient and in 68 additional MPM patients (mutation frequency: 10%, 7/69) by targeted deep sequencing. Our observations suggest the possibility of a new genetic mechanism in the development of either MPM or multiple primary cancers. The frequent SETDB1 inactivating mutations suggest there could be new diagnostic or therapeutic options for MPM.

Probing quantum plasmon coupling using gold nanoparticle dimers with tunable interparticle distances down to the subnanometer range.

The assembly of noble metal nanoparticles is an appealing means to control the plasmonic properties of nanostructures. Dimers are particularly interesting because they are a model system that can provide fundamental insights into the interactions between nanoparticles in close proximity. Here, we report a highly efficient and facile assembly method for dimers and other forms of assemblies. Gold nanoparticles (AuNPs) adsorbed on aminosilanized glass surfaces protect the silanes underneath the nanoparticles from hydrolysis. This masked desilanization allows us to prepare AuNP homodimers on glass slides with remarkably high yield (∼90%). The interparticle distance and, accordingly, the surface plasmon coupling are readily tuned at the molecular level using self-assembled monolayers of alkanedithiols. As the interparticle distance is reduced, the resonance surface plasmon coupling progressively redshifts, following the classical electromagnetic model. When the interparticle distance enters the subnanometer regime, however, the resonance band begins to blueshift and significantly broadens. The comparison of our observations with theoretical studies reveals that quantum tunneling effects play a significant role in the plasmonic response of AuNP dimers in the subnanometer gap region. The assembly method based on the masked desilanization is extendable to the formation of various other forms of nanoassemblies and, thus, will further our understanding of plasmonic interactions in nanoassemblies.

Surface Plasmon Coupling of Compositionally Heterogeneous Core-Satellite Nanoassemblies.

Understanding plasmon coupling between compositionally heterogeneous nanoparticles in close proximity is intriguing and fundamentally important because of the energy mismatch between the localized surface plasmons of the associated nanoparticles and interactions beyond classical electrodynamics. In this Letter, we explore surface plasmon coupling between silver nanoparticles (AgNPs) and gold nanoparticles (AuNPs), assembled in the form of core-satellite structures. A recently developed assembly method allows us to prepare ultrapure core-satellite nanoassemblies in solution, where 50 nm AgNPs are surrounded by 13 nm AuNPs via alkanedithiol linkers. We observe changes in the plasmon coupling between the AgNP core and AuNP satellites as the core-to-satellite gap distance varies from 2.3 to 0.7 nm. Comparison with theoretical studies reveals that the traditional hybridized plasmon modes are abruptly replaced by charge-transfer plasmons at a ∼1 nm gap. Changes with the number of satellites are also discussed.

Controlled assembly and plasmonic properties of asymmetric core-satellite nanoassemblies.

The assembly of noble metal nanoparticles offers an appealing means to control and enhance the plasmonic properties of nanostructures. However, making nanoassemblies with easily modifiable gap distances with high efficiency has been challenging. Here, we report a novel strategy to assemble gold nanoparticles (AuNPs) into Janus-type asymmetric core-satellite nanostructures. Markedly different desorption efficiency between large and small AuNPs in ethanol allows us to prepare the asymmetric core-satellite nanoassemblies in a dispersed colloidal state with near 100% purity. The resulting nanoassemblies have well-defined structures in which a core AuNP (51 nm) is covered by an average of 13 ± 3 satellite AuNPs (13 nm) with part of the core surfaces left unoccupied. Strong surface plasmon coupling is observed from these nanoassemblies as a result of the close proximity between the core and the satellites, which appears significantly red-shifted from the surface plasmon resonance frequencies of the constituting nanoparticles. The dependence of the surface plasmon coupling on a gap distance of less than 3 nm is systematically investigated by varying the length of the alkanedithiol linkers. The asymmetric core-satellite nanoassemblies also serve as an excellent surface-enhanced Raman scattering substrate with an enhancement factor of ~10(6). Finally, we demonstrate that the presented assembly method is extendible to the preparation of compositionally heterogeneous core-satellite nanoassemblies.

Calcaneal reconstruction for the late complication of calcaneus fracture.

Calcaneal fracture is the most common fracture in the tarsal bones. Treatment is difficult because the patterns of fracture are various and complications occur frequently. The purpose of this study was to evaluate the clinical results of calcaneal reconstruction for chronic complications after calcaneal fracture. rom September 2001 to November 2004, calcaneal reconstruction was performed in 24 patients (25 feet). We reviewed 21 patients (22 feet) who could be followed up >2 years postoperatively. Patients who underwent subtalar arthrodesis and simple bone resection were excluded. Patients who underwent calcaneal sliding osteotomy were included. Nineteen men and 2 women ranged in age from 27 to 54 years (mean, 44.4 years). The mean interval between the first operation and reconstructive operation was 11.2 months (range, 3-31 months). The mean follow-up period after calcaneal reconstruction was 29.8 months (range, 24-38 months). Böhler angle, calcaneal pitch, and talocalcaneal height were checked pre- and postoperatively. The most common patient report was pain on the inferior aspect of the lateral malleolus (16 patients) and calcaneal tuberosity (3 patients). All the mean values of talocalcaneal height, calcaneal pitch, and Böhler angle improved, which was statistically significant. In the postoperative period, 10 patients were very satisfied, 9 were satisfied, and 3 were not satisfied. Although pain did not completely resolve, all patients were satisfied postoperatively.