ChIP-Seq Assays from Mammalian Cartilage and Chondrocytes.

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a powerful tool to identify binding profiles of transcriptional regulators and chromatin regulators as well as histone modification patterns in a genome-wide manner. ChIP-seq consists of five major steps: (1) preparation of cells and chromatin, (2) ChIP, (3) ChIP-seq library construction, (4) sequencing of ChIP DNA with a next-generation sequencer (NGS), and (5) computational analysis of sequence data. Recent ChIP-seq studies in skeletal tissues enable us to understand the modes of action of key skeletal regulators, functional interaction among the enhancers bound by the regulators, the complex nature of regulatory inputs, and thereby the gene regulatory landscape in skeletal development. Here we describe a ChIP-seq protocol that we have employed in our studies, with particular focus on chromatin preparation and subsequent ChIP in skeletal cells, including chondrocytes.

[Effectiveness of Prothrombin Complex Concentrate for Warfarin-related Intracranial Hemorrhage].

BACKGROUND AND PURPOSE: Warfarin-associated intracranial hemorrhage(w-ICH)usually increases and results in unfavorable outcomes. Administration of prothrombin complex concentrate(PCC)can reverse anticoagulation and correct prothrombin time-international normalized ratio(PT-INR)immediately; it is recommended by some guidelines for cases of w-ICH. We assessed the effect of PCC on blood coagulation. METHODS: We administered PCC and vitamin K to 11 patients with w-ICH who were admitted to our hospital between October 2016 and November 2017. We measured the PT-INR at baseline and immediately, 1 hour, 6 hours, and on the day after PCC administration. RESULTS: Patients' mean(range)PT-INR normalized from 1.92(1.64-3.26)to 1.08(1.03-1.29)immediately after receiving PCC. Patients' PT-INR was 1.17(1.08-1.29)1 hour after receiving PCC, 1.22(1.16-1.52)6 hours after receiving PCC, and 1.17(1.05-1.29)on the day after receiving PCC. In all the cases, no side effects emerged. Five patients had a safe operation. All the patients' modified Rankin Scale scores at discharge were stable or within a permissive limit in comparison with the symptoms on admission. CONCLUSION: In our cases, administration of PCC corrected the PT-INR immediately and contributed to a better outcome of w-ICH.

[Tentorial Dural Arteriovenous Fistula Successfully Treated with Transvenous Embolization Using a Double Catheterization Technique through Venous Drainage:A Case Report].

BACKGROUND: Tentorial dural arteriovenous fistulas(dAVFs)are a rare clinical entity accounting for less than 10% of all intracranial dAVFs. Because these lesions are characterized by high hemorrhagic risk, aggressive treatment should be considered. Although the number of reported cases treated with endovascular transarterial embolization(TAE)using glue has been increasing, little is known about the transvenous approach. Here, we report the case of a patient with a tentorial dAVF who was successfully treated with transvenous embolization(TVE)through venous drainage using a double catheterization technique. CASE PRESENTATION: A 68-year-old male patient who had a history of left putaminal hemorrhage treated with a craniotomy was diagnosed with a tentorial dAVF on a magnetic resonance angiogram. Because the patient refused another craniotomy for surgical interruption of the dAVF, an endovascular approach was considered. We first attempted to perform TAE with glue, but catheterization into the tortuous meningohypophyseal trunk failed. We then performed a TVE of the venous drainage near the shunt with detachable coils and achieved complete obliteration of the fistula. During coil embolization of the venous drainage, insertion of small coils near the shunt was supported by another anchor coil that was delivered using a double catheterization technique. CONCLUSIONS: The method of TVE through venous drainage using a double catheterization technique, which involved placing coils in the fragile drainage vein, was safe and effective in a case of tentorial dAVF. This technique should be considered as another option for the management of complex tentorial dAVFs.

Nanostructure and physical properties of cellulose nanofiber-carbon nanotube composite films.

We studied the nanostructure and physical properties of cellulose nanofiber-multi-walled carbon nanotube (CNF-MWNT) composite films prepared via MWNT aqueous dispersion using 4-O-methyl-α-d-glucuronoxylan as a MWNT dispersion aid. The composite film had high electrical conductivity (1.05S/cm), good mechanical properties (Young's modulus: 10.1GPa, tensile strength: 173.4MPa) and a low coefficient of thermal expansion (7ppm/K). FE-SEM imaging showed that the carbon nanotubes dispersed homogeneously and made reinforcing networks in the matrix of cellulose nanofibers. Improvement in the physical properties of cellulose nanofiber film by adding MWNTs is due to this composite structure.

Reliability of MR Quantification of Rotator Cuff Muscle Fatty Degeneration Using a 2-point Dixon Technique in Comparison with the Goutallier Classification: Validation Study by Multiple Readers.

RATIONALE AND OBJECTIVES: Presurgical assessment of fatty degeneration is important in the management of patients with rotator cuff tears. The Goutallier classification is widely accepted as a qualitative scoring system, although it is highly observer-dependent and has poor reproducibility. The objective of this study was to quantify fatty degeneration of the supraspinatus muscle using a 2-point Dixon technique in patients with rotator cuff tears by multiple readers, and to evaluate the reproducibility compared to Goutallier classification. MATERIALS AND METHODS: Two hundred patients with rotator cuff tears who underwent magnetic resonance imaging (MRI), including 2-point Dixon sequence at 3.0-T, were selected retrospectively. Qualitative and quantitative analyses of fatty degeneration were performed by two radiologists and three orthopedic surgeons independently. The fat quantification was performed by measuring signal intensity values of in phase (S(In)) and fat image (S(Fat)), and calculating fat fraction as S(Fat)/S(In). The reproducibility of MR quantification was analyzed by the intra- and interclass correlation coefficients and Bland-Altman plots. RESULTS: The interobserver agreement of the Goutallier classification among five readers was moderate (k = 0.51), whereas the interclass correlation coefficient regarding fat fraction value quantified in 2-point Dixon sequence was excellent (0.893). The mean differences in fat fraction values from the individual segmentation results were from -0.072 to 0.081. Proposed fat fraction grading and Goutallier grading showed similar frequency and distribution in severity of rotator cuff tears. CONCLUSIONS: Fat quantification in the rotator cuff muscles using a 2-point Dixon technique at 3.0-T MRI is highly reproducible and clinically feasible in comparison to the qualitative evaluation using Goutallier classification.

The H2 blocker famotidine suppresses progression of ossification of the posterior longitudinal ligament in a mouse model.

BACKGROUND: Ossification of the posterior longitudinal ligament (OPLL) of the spine is a common human myelopathy that leads to spinal cord compression. No disease-modifying drug for OPLL has been identified, whereas surgery and conservative management have been established. OBJECTIVES: To evaluate the therapeutic potential of the H2 blocker famotidine for ectopic ossification in the cervical spine in an OPLL mouse model. METHODS: The H2 blocker famotidine was orally administered to Enpp1 (ttw/ttw) mice, a model of OPLL, at either 4 or 15 weeks of age. Radiological and survival rate analyses were performed to assess the effects of famotidine on OPLL-like lesions and mortality in Enpp1 (ttw/ttw) mice. RESULTS: Oral administration of famotidine suppressed the progression of OPLL-like ectopic ossification and reduced mortality in Enpp1 (ttw/ttw) mice when administration began at 4 weeks of age, early in the development of ossification. CONCLUSIONS: This study points to the use of famotidine as a disease-modifying drug for ectopic ossification of spinal soft tissue, including OPLL.

Combined Arthroscopic Bankart Repair and Coracoid Process Transfer to Anterior Glenoid for Shoulder Dislocation in Rugby Players: Evaluation Based on Ability to Perform Sport-Specific Movements Effectively.

PURPOSE: To evaluate the outcomes of a combination of an arthroscopic Bankart repair and an open Bristow procedure in relation to the subjective quality of performance in movements that are typical in rugby. METHODS: Forty shoulders in 38 players who underwent surgery for traumatic anterior instability of the shoulder were reviewed. In all cases, arthroscopic Bankart repair was followed by a Bristow procedure, with preservation of the repaired capsular ligaments, during the same operation. The mean age at the time of surgery was 21 years. Patients were asked to describe common rugby maneuvers (tackle, hand-off, jackal, and saving) preoperatively and postoperatively as "no problem," "insufficient," or "impossible." RESULTS: There were no recurrent dislocations at a mean follow-up of 30.5 months. The mean Rowe score improved significantly from 65.0 (range, 55 to 75) to 97.5 (range, 95 to 100) (P < .001) after surgery. Preoperatively, regarding the tackling motion, none of the patients reported having no problem, whereas the ability was described as insufficient for 23 shoulders and impossible for 17 shoulders. Postoperatively, no problem with tackling was reported for 36 shoulders, whereas insufficiency was reported for 4. The results for the hand-off, jackal, and saving maneuvers were similar (P < .001). No patient rated any of the motions as impossible postoperatively. CONCLUSIONS: This combined surgical procedure clearly is effective in preventing recurrent dislocation in rugby players; however, some players complained of insufficiency in the quality of their play when they were tackling or performing other rugby-specific movements. LEVEL OF EVIDENCE: Level IV, case series.

Quantitative and qualitative analyses of subacromial impingement by kinematic open MRI.

PURPOSE: Quantitative and qualitative kinematic analyses of subacromial impingement by 1.2T open MRI were performed to determine the location of impingement and the involvement of the acromioclavicular joint. METHODS: In 20 healthy shoulders, 10 sequential images in the scapular plane were taken in a 10-s pause at equal intervals from 30° to maximum abduction in neutral and internal rotation. The distances between the rotator cuff (RC) and the acromion and the acromioclavicular joint were measured. To comprehend the positional relationships, cadaveric specimens were also observed. RESULTS: Although asymptomatic, the RC came into contact with the acromion and the acromioclavicular joint in six and five cases, respectively. The superior RC acted as a depressor for the humeral head against the acromion as the shoulder elevated. The mean elevation angle and distance at the closest position between the RC and the acromion in neutral rotation were 93.5° and 1.6 mm, respectively, while those between the RC and the acromioclavicular joint were 86.7° and 2.0 mm. When comparing this distance and angle, there was no significant difference between the RC to the acromion and to the acromioclavicular joint. The minimum distance between the RC and the acromion was significantly shorter than that between the greater tuberosity and the acromion. The location of RC closest to the acromion and the acromioclavicular joint differed significantly. CONCLUSION: Although asymptomatic, contact was found between the RC and the acromion and the acromioclavicular joint. The important role of the RC to prevent impingement was observed, and hence, dysfunction of the RC could lead to impingement that could result in a RC lesion. The RC lesions may differ when they are caused by impingement from either the acromion or the acromioclavicular joint.

A functional role of the glycosylated N-terminal domain of chondromodulin-I.

Chondromodulin-I (ChM-I) is a 25-kDa glycoprotein that specifically localizes in the extracellular matrix of cartilage and negatively regulates angiogenesis. ChM-I comprises two domains: an N-terminal hydrophilic domain (domain 1) containing an N-linked glycosylation site and a C-terminal hydrophobic domain (domain 2) with all four disulfide bonds that are present in this protein. We generated a nonglycosylated recombinant human ChM-I (NG-hChM-I) and compared its bioactivity with that of the glycosylated form of human ChM-I (G-hChM-I) expressed in Chinese hamster ovary cells in vitro. NG-hChM-I exhibited the growth factor/inhibitor activity in the cultures of chondrocytes and vascular endothelial cells but required markedly higher doses. Although domain 1 is predicted to be hydrophilic per se on the basis of its amino acid sequence, NG-hChM-I remains insoluble in aqueous solution as much as ΔN-hChM-I that lacks the N-terminal 37 amino acids containing an N-glycosylation site. Circular dichroism measurements revealed that the content of α-helix was calculated to be 34% in G-hChM-I, whereas the content of the characteristic secondary structures in NG-hChM-I was distinctly lower than those in G-hChM-I. These results indicate that glycosylation in domain 1 is critical for the structural integrity for biological functions of ChM-I in vitro.

Crystallization and preliminary X-ray analysis of two inhibitor complexes of the catalytic domain of death-associated protein kinase.

The catalytic domain of death-associated protein kinase (DAPK) has been overexpressed, purified and crystallized using the sitting-drop vapour-diffusion method with PEG 8000 and magnesium acetate as precipitants. Complexes with the inhibitor staurosporine and its analogue BDB402 were also crystallized in the presence of PEG 400 and PEG 8000, respectively. Diffraction data were collected to 2.4 A for the native catalytic domain, to 2.9 A for the staurosporine complex and to 2.7 A for the BDB402 complex. All three crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 77.992, b = 109.909, c = 50.063 A for the catalytic domain, a = 78.911, b = 113.162, c = 50.658 A for the staurosporine complex and a = 77.337, b = 108.869, c = 50.186 A for the BDB402 complex. In both complexes the inhibitor molecule was clearly assigned in the difference Fourier map calculated on the basis of the phases obtained from the structure of the catalytic domain.