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1.  Nano-Mole Scale Side-Chain Signal Assignment by 1H-Detected Protein Solid-State NMR by Ultra-Fast Magic-Angle Spinning and Stereo-Array Isotope Labeling 
PLoS ONE  2015;10(4):e0122714.
We present a general approach in 1H-detected 13C solid-state NMR (SSNMR) for side-chain signal assignments of 10-50 nmol quantities of proteins using a combination of a high magnetic field, ultra-fast magic-angle spinning (MAS) at ~80 kHz, and stereo-array-isotope-labeled (SAIL) proteins [Kainosho M. et al., Nature 440, 52–57, 2006]. First, we demonstrate that 1H indirect detection improves the sensitivity and resolution of 13C SSNMR of SAIL proteins for side-chain assignments in the ultra-fast MAS condition. 1H-detected SSNMR was performed for micro-crystalline ubiquitin (~55 nmol or ~0.5mg) that was SAIL-labeled at seven isoleucine (Ile) residues. Sensitivity was dramatically improved by 1H-detected 2D 1H/13C SSNMR by factors of 5.4-9.7 and 2.1-5.0, respectively, over 13C-detected 2D 1H/13C SSNMR and 1D 13C CPMAS, demonstrating that 2D 1H-detected SSNMR offers not only additional resolution but also sensitivity advantage over 1D 13C detection for the first time. High 1H resolution for the SAIL-labeled side-chain residues offered reasonable resolution even in the 2D data. A 1H-detected 3D 13C/13C/1H experiment on SAIL-ubiquitin provided nearly complete 1H and 13C assignments for seven Ile residues only within ~2.5 h. The results demonstrate the feasibility of side-chain signal assignment in this approach for as little as 10 nmol of a protein sample within ~3 days. The approach is likely applicable to a variety of proteins of biological interest without any requirements of highly efficient protein expression systems.
PMCID: PMC4391754  PMID: 25856081
2.  Solution NMR Structure of Proteorhodopsin 
PMCID: PMC4234116  PMID: 22034093
proteorhodopsin; membrane proteins; NMR spectroscopy; cell-free expression; structural biology
3.  Characteristics and significance of fever during 4 weeks after primary total knee arthroplasty 
Most previous studies on postoperative fever (POF; ≥38 °C) after total knee arthroplasty (TKA) have reported findings from only the immediate postoperative days (PODs). The hypothesis of the current study is that 4 weeks of follow-up may reveal differences in the characteristics of POF and fever-related factors between a normal inflammatory response and an early acute infection-related response.
A total of 400 consecutive TKAs (314 patients) were retrospectively investigated. Patients were stratified into those who developed an early acute periprosthetic infection that required subsequent surgical treatment (STG; n = 5 TKAs) and those who did not (non-STG; n = 395 TKAs).
Among the 400 knees, 149 (37 %) developed POF, with most reaching a maximum temperature (MT) on POD 0. In 13 TKA patients who had POF with a peak daily temperature ≥38 °C during postoperative weeks 2–4, the causes of POF were respiratory and urinary tract infections (n = 5 for each), superficial infection (n = 2), and periprosthetic infection (n = 1). The STG and non-STG differed significantly with regard to the rate of POF (p = 0.0205) and MT (p = 0.0003), including MTs less than 38 °C, during postoperative weeks 2–4. All five STG patients had elevated C-reactive protein levels and local symptomatic findings before the additional surgery.
The occurrence of POF and MT along with elevated C-reactive protein and local symptomatic findings at 2–4 weeks postoperatively may indicate the need for a positive fever workup to recognize early acute periprosthetic infection.
PMCID: PMC3990857  PMID: 24522863
Total knee arthroplasty; Postoperative fever; Maximum temperature; Periprosthetic infection
4.  Anteroposterior Translation Does Not Correlate With Knee Flexion After Total Knee Arthroplasty 
Stiffness after a TKA can cause patient dissatisfaction and diminished function, therefore it is important to characterize predictors of ROM after TKA. Studies of AP translation in conscious individuals disagree whether AP translation affects maximum knee flexion angle after implantation of a highly congruent sphere and trough geometry PCL-substituting prosthesis in a TKA.
We investigated whether AP translation correlated with maximum knee flexion angle (1) in patients who were awake, and (2) who were under anesthesia (to minimize the effects of voluntary muscle contraction) in a TKA with implantation of a PCL-substituting mobile-bearing prosthesis.
AP translation was examined under both conditions in 34 primary TKAs. Measurements under anesthesia were performed when the patients were having anesthesia for a contralateral TKA. Awake measurements were made within 4 days of that anesthetic session in patients who had no residual sedative effects. The average postoperative interval for the index TKA flexion measurements was 23 months (range, 6–114 months). AP translation was evaluated at 75° flexion using an arthrometer.
There was no correlation between postoperative maximum knee flexion and AP translation at 75° during consciousness. There was no correlation between postoperative maximum knee flexion and AP translation under anesthesia.
AP translation at 75° flexion did not correlate with postoperative maximum knee flexion in either awake or anesthetized patients during a TKA with implantation of a posterior cruciate-substituting prosthesis.
Level of Evidence
Level II, therapeutic study. See the Instructions for Authors for a complete description of levels of evidence.
PMCID: PMC3890185  PMID: 24005980
5.  Posterior Condylar Offset Does Not Correlate With Knee Flexion After TKA 
Studies of medial and lateral femoral posterior condylar offset have disagreed on whether posterior condylar offset affects maximum knee flexion angle after TKA.
We asked whether posterior condylar offset was correlated with knee flexion angle 1 year after surgery in (1) a PCL-retaining meniscal-bearing TKA implant, or in (2) a PCL-substituting mobile-bearing TKA implant.
Knee flexion angle was examined preoperatively and 12 months postoperatively in 170 patients who underwent primary TKAs to clarify the effect of PCL-retaining (85 knees) and PCL-substituting (85 knees) prostheses on knee flexion angle. A quasirandomized design was used; patients were assigned to receive one or the other implant using chart numbers. A quantitative three-dimensional technique with CT was used to examine individual changes in medial and lateral posterior condylar offsets.
In PCL-retaining meniscal-bearing knees, there were no significant correlations between posterior condylar offset and knee flexion at 1 year. In these knees, the mean (± SD) postoperative differences in medial and lateral posterior condylar offsets were 0.0 ± 3.6 mm and 3.8 ± 3.6 mm, respectively. The postoperative change in maximum knee flexion angle was −5° ± 15°. In PCL-substituting rotating-platform knees, similarly, there were no significant correlations between posterior condylar offset and knee flexion 1 year after surgery. In these knees, the mean postoperative differences in medial and lateral posterior condylar offsets were −0.5 ± 3.3 mm and 3.3 ± 4.2 mm, respectively. The postoperative change in maximum knee flexion angle was −2° ± 18°.
Differences in individual posterior condylar offset with current PCL-retaining or PCL-substituting prostheses did not correlate with changes in knee flexion 1 year after TKA. We should recognize that correctly identifying which condyle affects the results of the TKA may be difficult with conventional radiographic techniques.
Level of Evidence
Level II, therapeutic study. See Guidelines for Authors for a complete description of levels of evidence.
PMCID: PMC3734430  PMID: 23609812
6.  Safety and efficacy of a new tourniquet system 
BMC Surgery  2012;12:17.
In upper limb surgery, the pneumatic tourniquet is an essential tool to provide a clean, bloodless surgical field, improving visualization of anatomical structures and preventing iatrogenic failure. Optimal inflation pressure to accomplish these objects without injuring normal tissue and inducing complications is not yet established. Use of the minimum tourniquet pressure necessary to produce a bloodless surgical field is preferable in order to prevent injury to normal tissue. Various methods have been implemented in an effort to lower effective cuff pressure. The purpose of this study is to report clinical experience with a new tourniquet system in which pressure is synchronized with systolic blood pressure (SBP) using a vital information monitor.
We routinely used the tourniquet system in 120 consecutive upper limb surgeries performed under general anaesthesia in our operating room instead of our clinic. Cuff pressure was automatically regulated to additional 100 mmHg based on the SBP and was renewed every 2.5 minutes intervals.
An excellent bloodless field was obtained in 119 cases, with the exception of one case of a 44-year-old woman who underwent internal screw fixation of metacarpal fracture. No complications, such as compartment syndrome, deep vein disorder, skin disorder, paresis, or nerve damage, occurred during or after surgery.
This new tourniquet system, synchronized with SBP, can be varied to correspond with sharp rises or drops in SBP to supply adequate pressure. The system reduces labor needed to deflate and re-inflate to achieve different pressures. It also seemed to contribute to the safety in upper limb surgery, in spite of rare unexpected oozing mid-surgery, by reducing tissue pressure.
PMCID: PMC3465201  PMID: 22894765
Tourniquet; Upper limb surgery; Systolic blood pressure; Autoregulation
7.  Construction and performance of an NMR tube with a sample cavity formed within magnetic susceptibility-matched glass 
We describe the construction and performance of an NMR tube with a magnetic susceptibility matched sample cavity that confines the solution within the detection zone in the axial direction and in a quasi-rectangular region in the radial direction. The slot-like sample cavity provides both good sample volume efficiency and tolerance to sensitivity loss in the sample space. The signal-to-noise ratio per unit volume of the constructed tube was 2.2 times higher than that of a cylindrical tube of 5 mm outer diameter with a sample containing 300 mM NaCl at a static magnetic field of 14.1 T. Even the overall signal-to-noise ratio of the slot tube was 35% higher than that of the conventional 5 mm tube for a sample containing 300 mM NaCl. Similar improvements over existing sample tube geometries were obtained at 950 MHz. Moreover the temperature rise resulting from RF heating was found to be significantly lower for the slot tube even when compared to 3 and 4 mm outer diameter cylindrical tubes as measured in a 5 mm cryoprobe. A further advantage of this type of tube is that a sample cavity of any desired size and shape can be formed within a cylindrical tube for use in a single cryogenic probe.
PMCID: PMC3065960  PMID: 21316281
8.  Clinical use of a new tourniquet system for foot and ankle surgery 
International Orthopaedics  2009;34(3):355-359.
This study reports the results of the clinical use of a new tourniquet system for surgery of foot and ankle that can determine tourniquet pressure in synchrony with systolic blood pressure (SBP). We prospectively applied additional pressure of 100 mmHg based on the SBP recorded before the skin incision in 100 consecutive procedures. There were 34 open reduction internal fixation procedures, 26 lateral colateral ligament repair or reconstruction, 16 Achilles tendon repairs, nine arthroscopic procedures such as removal of loose body or accessory bone and synovectomy, seven corrective osteotomy and eight others such as removal of tumour, ankle fusion, and bone graft. The average initial tourniquet pressure was 211 mmHg. The average maximum SBP change during surgery was 28 mmHg. All cases maintained an excellent operative field without measurable bleeding and there were no postoperative complications. Fifty-five cases had a lower intra-operative SBP than the initial value. Since a tourniquet should be applied at the lowest pressure possible for maintaining a bloodless surgical field, the new system appears to be practical and reasonable, as compared to conventional tourniquets, which maintain the initial pressure.
PMCID: PMC2899285  PMID: 19455329
9.  Preoperative laxity in osteoarthritis patients undergoing total knee arthroplasty 
International Orthopaedics  2007;33(1):105-109.
A preoperative quantitative evaluation of soft tissues is helpful for planning total knee arthroplasty, in addition to the conventional clinical examinations involved in moving the knee manually. We evaluated preoperative coronal laxity with osteoarthritis in patients undergoing total knee arthroplasty by applying a force of 150 N with an arthrometer. We examined a consecutive series of 120 knees in 102 patients. The median laxity was 0° in abduction and 8° in adduction. The femorotibial angle on non-weight-bearing standard anteroposterior radiographs was 180° and correlated with both abduction (r = −0.244, p = 0.007) and adduction (r = 0.205, p = 0.025) laxity. The results of a regression analysis suggested that the femorotibial angle is helpful for estimating both laxities. Considering the many reports on how to obtain well-balanced soft tissues, stress radiographs might help to improve the preoperative planning for gaining the optimal laxity deemed appropriate by surgeons.
PMCID: PMC2899222  PMID: 17938923
10.  Structure of the Putative 32 kDa Myrosinase Binding Protein from Arabidopsis (At3g16450.1) Determined by SAIL-NMR 
The FEBS journal  2008;275(23):5873-5884.
The product of gene At3g16450.1 from Arabidopsis thaliana is a 32 kDa, 299-residue protein classified as resembling a myrosinase-binding protein (MyroBP). MyroBPs are found in plants as part of a complex with the glucosinolate-degrading enzyme, myrosinase, and are suspected to play a role in myrosinase-dependent defense against pathogens. Many MyroBPs and MyroBP-related proteins are composed of repeated homologous sequences with unknown structure. We report here the three-dimensional structure of the At3g16450.1 protein from Arabidopsis, which consists of two tandem repeats. Because the size of the protein is larger than that amenable to high-throughput analysis by uniformly 13C/15N labeling methods, we used our stereo-array isotope labeling (SAIL) technology to prepare an optimally 2H/13C/15N-labeled sample. NMR data sets collected with the SAIL-protein enabled us to assign 1H, 13C and 15N chemical shifts to 95.5% of all atoms, even at the low concentration (0.2 mM) of the protein product. We collected additional NOESY data and solved the three-dimensional structure with the CYANA software package. The structure, the first for a MyroBP family member, revealed that the At3g16450.1 protein consists of two independent, but similar, lectin-fold domains composed of three β-sheets.
PMCID: PMC2702212  PMID: 19021763
lectin; NMR structure; stereo-array isotope labeling; structural genomics

Results 1-10 (10)