PCR amplification from single DNA molecules on magnetic beads in emulsion: application for high-throughput screening of transcription factor targets

We have developed a novel method of genetic library construction on magnetic microbeads based on solid-phase single-molecule PCR in a fine and robust water-phase compartment formed in water-in-oil (w/o) emulsions. In this method, critically diluted DNA fragments were distributed over the emulsion as templates, where beads crosslinked with multiple primers and other PCR components were encapsulated to form multiple reaction compartments. The delivered DNA was then amplified and covalently immobilized on the beads in parallel, within individual compartments, to construct a genetic library on beads (GLOBE), which was readily applicable to a genomewide global scanning of genetic elements recognized by a defined DNA-binding protein. We constructed a GLOBE of Paracoccus denitrificans and selected gene beads that were bound to the His-tagged transcription factor PhaR by flow cytometry. As a result of flow cytometry screening with an anti-His fluorescent antibody, the PhaR target fragments were enriched 1200-fold from this library with this system. Therefore, this system is a powerful tool for analyzing the transcription network on a genomewide scale

Cellulitis caused by Streptococcus pyogenes at the Bacille Calmette-Guérin vaccination site: A case report

Recently, the number of reports on skin manifestations, including tuberculids, developing as adverse reactions to the Bacille Calmette-Guérin (BCG) vaccine has increased in Japan. We encountered a case in which an infant developed cellulitis at the vaccination site. The patient was a girl who received the BCG vaccine 5 months after birth. Two months after vaccination, she developed a fever of 39ºC, and redness and swelling spread to the entire left upper arm. After being treated with oral cefditoren pivoxil, she was referred to our hospital and received treatment with intravenous sulbactam/ampicillin. Streptococcus pyogenes was detected in a culture of the vaccination scars, and the reaction was very likely caused by pathogenic bacteria. There has been no previous report on a patient developing cellulitis following BCG vaccination caused by S. pyogenes at the vaccination site. Here, we describe such a case with a literature review

Children with unilateral aural atresia

The effects of FM system fitted into the normal hearing ear (NHE) or a cartilage conduction hearing aid (CCHA) fitted into the affected ear (AE) on the speech recognition ability in noise were examined in children with unilateral congenital aural atresia (UCAA). In children with bilateral normal hearing (BNH), speech recognition score (SRS) was significantly decreased in the noisy environment of -5 dB signal-to-noise ratio (SNR), compared with those in quiet. In children with UCAA, SRS was significantly decreased in noisy environments of 0 and -5 dB SNR, compared with those in quiet. In noisy environments of 0 and -5 dB SNR, SRS in children with UCAA was significantly decreased, compared those in children with BNH. In the noisy environment of -5 dB SNR, SRS in UCAA children aided by FM system fitted into NHE was significantly better than those in unaided children in the same group. In the noisy environment of 0 dB SNR, SRS in UCAA children aided by CCHA into AE tended to be higher than those in unaided children in the same group. FM system and CCHA can be recommended as an audiological management for the improvement of speech recognition in children with UCHL in classrooms

Cardiac magnetic resonance imaging-based myocardial strain study for evaluation of cardiotoxicity in breast cancer patients treated with trastuzumab: A pilot study to evaluate the feasibility of the method

Background: Trastuzumab, used to treat breast cancer overexpressing human epidermal growth factor receptor 2, may be cardiotoxic. Cardiac magnetic resonance (CMR) imaging with myocardial strain studies has been used to evaluate subclinical biventricular myocardial changes, however, its clinical utility during chemotherapy has not been evaluated. Methods: The clinical outcomes, CMR and cardiac biomarkers of 9 women aged 62.3 ± 12.6 years with early or locally advanced breast cancer were evaluated at baseline, and at 3, 6 and 12 months after the initiation of trastuzumab. Results: None of the patients developed heart failure or elevated serum cardiac biomarkers. Global left ventricular (LV) peak systolic longitudinal and circumferential strains were significantly decreased at 6 months (longitudinal strains, –21.1 ± 1.7% [baseline] vs. –19.5 ± 1.0% [6 months], p = 0.039, and circumferential strains, –23.4 ± 1.8% [baseline] vs. –21.6 ± 2.5% [6 months], p = 0.036). These changes were analogous to those observed in the LV ejection fraction. Right ventricular (RV) free wall peak systolic circumferential strains were decreased at 6 months (–20.9% ± 2.4% [baseline] vs. –19.1% ± 2.3% [6 months], p = 0.049), whereas RV longitudinal strains and ejection fraction remained unchanged. The LV longitudinal strain was the most reproducible of the 4 peak strain parameters. Conclusions: The LV longitudinal and circumferential strains measured by CMR decreased during trastuzumab therapy, although their predictive value for later heart failure or association with RV parameters was not determined. These techniques may be a useful means of diagnosing and monitoring trastuzumab-related cardiotoxicity

Successful Certolizumab Pegol Treatment of Chronic Anterior Uveitis Associated with Psoriasis Vulgaris

This report presents details on a 45-year-old male Japanese patient with chronic and refractory anterior uveitis associated with psoriasis vulgaris who was administered certolizumab pegol (CZP), which is an anti-tumor necrosis factor alpha (TNF-α) monoclonal antibody. Although CZP has only been formally approved for rheumatoid arthritis treatment in Japan, a clinical trial allowed us to assess CZP effectiveness in this patient. The grade 3+ anterior chamber inflammation (for both the cells and flare) observed at baseline improved to grade 0 at 3 months post-treatment. Dermatologically, the psoriasis area severity index (PASI) score was 25.4, while the body surface area (BSA) was 88% at baseline. At 3 months after treatment, the scores improved to 2.8 for PASI and less than 1% for BSA. After the treatment, remission has lasted for at least 9 months. No adverse events were seen during the CZP treatment. These findings suggest that CZP could be an effective therapeutic alternative in some refractory anterior uveitis patients with psoriasis vulgaris

ABCC6

Purpose. To report the spectrum of ABCC6 variants in Japanese patients with angioid streaks (AS). Patients and Methods. This was a single-center cohort study. The medical records of 20 patients with AS from 18 unrelated Japanese families were retrospectively reviewed. Screening of the ABCC6 gene (exons 1 to 31) was performed using PCR-based Sanger sequencing. Results. Eight ABCC6 variants were identified as candidate disease-causing variants. These eight variants included five known variants (p.Q378X, p.R419Q, p.V848CfsX83, p.R1114C, and p.R1357W), one previously reported variant (p.N428S) of unknown significance, and two novel variants (c.1939C>T [p.H647Y] and c.3374C>T [p.S1125F]); the three latter variants were determined to be variants of significance. The following four variants were frequently identified: p.V848CfsX83 (14/40 alleles, 35.0%), p.Q378X (7/40 alleles, 17.5%), p.R1357W (6/40 alleles, 15.0%), and p.R419Q (4/40 alleles, 10.0%). The ABCC6 variants were identified in compound heterozygous or homozygous states in 13 of 18 probands. Two families showed a pseudodominant inheritance pattern. Pseudoxanthoma elasticum was seen in 15 of 17 patients (88.2%) who underwent dermatological examination. Conclusions. We identified disease-causing ABCC6 variants that were in homozygous or compound heterozygous states in 13 of 18 families (72.2%). Our results indicated that ABCC6 variants play a significant role in patients with AS in the Japanese population

Cathepsins B and L in synovial fluids from patients with rheumatoid arthritis and the effect of cathepsin B on the activation of pro-urokinase

To clarify the pathophysiological role of cathepsins in rheumatoid arthritis (RA), we investigated whether cathepsin B or cathepsin L was increased in synovial fluid (SF) of RA joints, and whether the cathepsin isolated from SF of RA patients activated pro-urokinase or not. Thus, we estimated the content of cathepsins in SF of RA patients by measuring their activities by fluorospectrometry, using Z-Phe-Arg-MCA as the substrate. Cathepsin activity was approxymately 4-fold higher in the SF of RA patients than in those of patients with osteoarthritis. Cathepsin B and cathepsin L were separated by cation-exchange column chromatography. As a result, a large peak corresponding to cathepsin B and a very small peak correponding to cathepsin L were detected. Biochemical sequential fractionation of the cathepsin purified from the SF showed that the large peak was mainly composed of cathepsin B. This purified enzyme induced conversion of pro-urokinase to urokinase, and the Km for pro-urokinase was approximately 8.27μM. These findings indicated that an imbalance between cathepsin B and its inhibitors occurred due to increased concentrations of active cathepsin B in RA articular lesions, and that cathepsin B might be related to the degradation of cartilage in RA by activating the fibrinolytic cascade

The Effect of Cranial Change on Oropharyngeal Airway and Breathing During Sleep

Mandibular micrognathia is one of the characteristics of obstructive sleep apnea syndrome. The purpose of this study was to assess the effects of bimaxillary surgery without maxillary advancement on the upper airway using computational fluid dynamics (CFD) results of comparing pre- and post-operative finite element model. Seven female patients with jaw deformity, who underwent two-jaw surgery (Le Fort1 osteotomy and bilateral sagittal split ramus osteotomy; BSSRO) were enrolled. Maxillary was moved for correcting occlusal plane and mandibular was moved to advancement. Pharyngeal airway space and breathing during sleep were evaluated, comparing the periods of 2 days before and 6 months after the operation. The cross-sectional area of the level of the hard palate (HP) and the level of the tip of the uvula (TU), and airway volume of total, HP-TU, and TP- the level of the base of the epiglottis (BE) were increased. AI and AHI in 2 days before and 6 months after were decreased. As the result of nasal ventilation condition, velocity of HP and TU in 2 days before and 6 months after were decreased. We think that it was revealed that movement of the maxilla without advancement did not affect to the morphology and function of airway