Gene expression of type II collagen is regulated by direct interaction with Kruppel-like factor 4 and AT-rich interactive domain 5B.

We have previously found and characterized two pairs of enhancer elements, E1 and E2, in the type II collagen alpha 1 chain (COL2A1) gene. Subsequent studies have suggested that these enhancers function differently in the regulation of gene expression. For example, histone deacetylase 10 modifies only the E2 enhancer region to affect gene expression. Therefore, in this study, we aimed to clarify the transcriptional complex formed at each enhancer region by identifying transcription factors that specifically bind to each enhancer element. To this end, we used chondrocytic cell lines established using our unique silent reporter system and overexpressed candidate transcription factors in these cells. We found two transcription factors, other than the SOX trio, that directly bound to COL2A1 and regulated its expression. The first was Kruppel-like factor-4 (KLF4), which bound to the promoter proximal region, and the second was AT-rich interactive domain 5B (ARID5B) which bound to the E1 enhancer element. Further studies are needed to identify factors that specifically bind to the E2 enhancer element. In any case, our findings provide an important insight into the molecular mechanisms underlying the regulation of COL2A1. In this paper, we reevaluated the previous analysis of transcription factors involved in the regulation of COL2A1 expression.

Levansucrase from Bacillus amyloliquefaciens KK9 and Its Y237S Variant Producing the High Bioactive Levan-Type Fructooligosaccharides.

Levan-typed fructooligosaccharide (LFOS), a ß-2,6 linked oligofructose, displays the potential application as a prebiotic and therapeutic dietary supplement. In the present study, LFOS was synthesized using levansucrase from Bacillus amyloliquefaciens KK9 (LsKK9). The wild-type LsKK9 was cloned and expressed in E. coli, and purified by cation exchanger chromatography. Additionally, Y237S variant of LsKK9 was constructed based on sequence alignment and structural analysis to enhance the LFOS production. High-performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) analysis indicated that Y237S variant efficiently produced a higher amount of short-chain LFOS than wild type. Also, the concentration of enzyme and sucrose in the reactions was optimized. Finally, prebiotic activity assay demonstrated that LFOS produced by Y237S variant had higher prebiotic activity than that of the wild-type enzyme, making the variant enzyme attractive for food biotechnology.

Galactomannan Pentasaccharide Produced from Copra Meal Enhances Tight Junction Integration of Epithelial Tissue through Activation of AMPK.

Mannan oligosaccharide (MOS) is well-known as an effective fed supplement for livestock to increase their nutrients absorption and health status. Pentasaccharide of mannan (MOS5) was reported as a molecule that possesses the ability to increase tight junction of epithelial tissue, but the structure and mechanism of action remains undetermined. In this study, the mechanism of action and structure of MOS5 were investigated. T84 cells were cultured and treated with MOS5 compared with vehicle and compound C, a 5'-adenosine monophosphate-activated protein kinase (AMPK) inhibitor. The results demonstrated that the ability of MOS5 to increase tight junction integration was inhibited in the presence of dorsomorphine (compound C). Phosphorylation level of AMPK was elevated in MOS5 treated group as determined by Western blot analysis. Determination of MOS5 structure was performed using enzymatic mapping together with 1H, 13C NMR, and 2D-NMR analysis. The results demonstrated that the structure of MOS5 is a ß-(1,4)-mannotetraose with α-(1,6)-galactose attached at the second mannose unit from non-reducing end.

Production and purification of mannan oligosaccharide with epithelial tight junction enhancing activity.

BACKGROUND: Mannanan oligosaccharide (MOS) is well-known as effective supplement food for livestock to increase their nutrients absorption and health status, but the structure and identification of bioactive MOS remain unclear. In this study, MOS production was accomplished, using enzymatic hydrolysis of pretreated coconut meal substrate with recombinant mannanase. METHODS: The mannanase gene was cloned from Bacillus subtilis cAE24, then expressed in BL21. Purified Mannanase exhibit stability over a wide range of pH and temperature from pH 6-8 and 4 °C to 70 °C, respectively. SEM analysis revealed that sonication could change the surface characteristic of copra meal, which gave better MOS yield, compared to untreated substrates. The separation and purification of each MOS were achieved using Biogel-P2 column chromatography. Determination of biological active MOS species was also investigated. T84 cells were cultured and treated with each of the purified MOS species to determine their tight junction enhancing activity. RESULTS: Scanning electron microscope imaging showed that pretreatment using sonication could disrupt the surface of copra meal better than grinding alone, which can improve the production of MOS. Pentamer of MOS (M5) significantly increased tight junction integration of T84 cells measured with TEER (p < 0.0001).

Nilotinib vs. imatinib in Japanese patients with newly diagnosed chronic myeloid leukemia in chronic phase: long-term follow-up of the Japanese subgroup of the randomized ENESTnd trial.

In the ongoing, international, phase 3 study Evaluating Nilotinib Efficacy and Safety in Clinical Trials-Newly Diagnosed Patients (ENESTnd), nilotinib 300 and nilotinib 400 mg, both twice daily, are compared with imatinib 400 mg once daily for the treatment of newly diagnosed chronic myeloid leukemia in the chronic phase (CML-CP). Results for the overall population in ENESTnd (n = 846) showed that nilotinib resulted in higher response rates vs. imatinib and was well tolerated. Outcomes among Japanese patients in ENESTnd were specifically analyzed after 1 year of follow-up, and showed similar trends to the overall population; we present updated analysis of the Japanese subgroup based on 5 years of follow-up. Among Japanese patients in the nilotinib 300-mg (n = 29), nilotinib 400-mg (n = 23), and imatinib (n = 25) arms, 86.2, 78.3, and 60.0%, respectively, achieved major molecular response [BCR-ABL1 ≤ 0.1% on the International Scale (BCR-ABL1 IS)] by 5 years, and 65.5, 69.6, and 40.0%, respectively, achieved MR4.5 (BCR-ABL1 IS ≤ 0.0032%). Safety results were consistent with prior reports. In this subgroup, one death occurred during treatment in the nilotinib 400-mg twice-daily arm (unknown cause), and one patient in each arm progressed to accelerated phase/blast crisis by the data cutoff.

Ruxolitinib is effective and safe in Japanese patients with hydroxyurea-resistant or hydroxyurea-intolerant polycythemia vera with splenomegaly.

Ruxolitinib, a potent JAK1/JAK2 inhibitor, was found to be superior to the best available therapy (BAT) in controlling hematocrit, reducing splenomegaly, and improving symptoms in the phase 3 RESPONSE study of patients with polycythemia vera with splenomegaly who experienced an inadequate response to or adverse effects from hydroxyurea. We report findings from a subgroup analysis of Japanese patients in RESPONSE (n = 18). The composite response rate (hematocrit control and spleen response) was higher in patients receiving ruxolitinib (50.0%) than in those receiving BAT (8.3%). A total of 50.0% of patients randomized to ruxolitinib achieved a spleen response vs 8.3% of those receiving BAT; 100 and 33.3% of patients in the respective groups achieved hematocrit control, with mean hematocrit in ruxolitinib-treated patients remaining stable at < 45% throughout the study. Similarly, a higher proportion of ruxolitinib-treated patients achieved complete hematologic remission (33.3 vs 16.7%). Ruxolitinib also led to rapid improvements in pruritus. All responses with ruxolitinib were durable to week 80, and its safety profile was consistent with that in the overall study. These findings suggest that ruxolitinib is an effective and well-tolerated treatment option for Japanese patients with polycythemia vera with an inadequate response to or adverse effects from hydroxyurea.

Mouse embryonic dorsal root ganglia contain pluripotent stem cells that show features similar to embryonic stem cells and induced pluripotent stem cells.

In the present study, we showed that the dorsal root ganglion (DRG) in the mouse embryo contains pluripotent stem cells (PSCs) that have developmental capacities equivalent to those of embryonic stem (ES) cells and induced pluripotent stem cells. Mouse embryonic DRG cells expressed pluripotency-related transcription factors [octamer-binding transcription factor 4, SRY (sex determining region Y)-box containing gene (Sox) 2, and Nanog] that play essential roles in maintaining the pluripotency of ES cells. Furthermore, the DRG cells differentiated into ectoderm-, mesoderm- and endoderm-derived cells. In addition, these cells produced primordial germ cell-like cells and embryoid body-like spheres. We also showed that the combination of leukemia inhibitor factor/bone morphogenetic protein 2/fibroblast growth factor 2 effectively promoted maintenance of the pluripotency of the PSCs present in DRGs, as well as that of neural crest-derived stem cells (NCSCs) in DRGs, which were previously shown to be present there. Furthermore, the expression of pluripotency-related transcription factors in the DRG cells was regulated by chromodomain helicase DNA-binding protein 7 and Sox10, which are indispensable for the formation of NCSCs, and vice versa. These findings support the possibility that PSCs in mouse embryonic DRGs are NCSCs.

Posteromedial papillary muscle rupture due to squeezing of the left anterior descending coronary artery.

An unusual case of posteromedial papillary muscle (PPM) rupture due to isolated left anterior descending (LAD) artery ischaemia, associated with severe myocardial bridge contraction, is presented. The unusual blood supply to the PPM was associated with its apical origin and apex-forming LAD.

CHD7, Oct3/4, Sox2, and Nanog control FoxD3 expression during mouse neural crest-derived stem cell formation.

Neural crest-derived stem cells (NCSCs) are tissue-specific stem cells derived from multipotent neural crest cells. NCSCs are present in some adult tissues such as dorsal root ganglia, sciatic nerve, and bone marrow. However, little is known about the formation mechanisms of these cells. We have shown that BMP2/Wnt3a signaling and a chromatin remodeler, CHD7, in mice help to maintain the multipotency of neural crest cells and lead to the formation of NCSCs. In the present study, we analyzed a regulatory gene cascade in the formation of mouse NCSCs. The inhibition of FoxD3 expression significantly suppressed the expression of Sox10, which is an indispensable transcription factor for mouse NCSC formation, in the presence of BMP2/Wnt3a. CHD7, Oct3/4, Sox2, and Nanog occupied multiple conserved regions of mouse FoxD3, mE1, mE2, and mE3, in a BMP2/Wnt3a-dependent manner. Furthermore, siRNA of CHD7, Oct3/4, Sox2, and Nanog significantly suppressed FoxD3 expression. The inhibition of histone H3K4 mono- or trimethylation also repressed FoxD3 expression. The present data suggest that CHD7, Oct3/4, Sox2, and Nanog directly induce FoxD3 expression when stimulated by BMP2/Wnt3a signaling, that FoxD3 promotes Sox10 expression, and that histone H3K4 methylation plays important roles in this process of mouse NCSC formation.

Development and application of a new Silent reporter system to quantitate the activity of enhancer elements in the type II Collagen Gene.

Type II collagen is a major component of cartilage, which provide structural stiffness to the tissue. As a sufficient amount of type II collagen is critical for maintaining the biomechanical properties of cartilage, its expression is tightly regulated in chondrocytes. Therefore, it is essential to elucidate in detail the transcriptional mechanism that controls expression of type II collagen, in particular by two enhancer elements we recently discovered. To systematically analyze and compare enhancer activities, we developed a novel reporter assay system that exploits site-specific integration of promoter and enhancer elements to activate a transcriptionally silent reporter gene. Using this system, we found that the enhancer elements have distinct characteristics, with one exhibiting additive effects and the other exhibiting synergistic effects when repeated in tandem.

Hydrodynamic evaluation of a new dispersive aortic cannula (Stealthflow).

The aim of this study was to evaluate flow from a new dispersive aortic cannula (Stealthflow) in the aortic arch using flow visualization methods. Particle image velocimetry was used to analyze flow dynamics in the mock aortic model. Flow patterns, velocity distribution, and streamlines with different shape cannulas were evaluated in a glass aortic arch model. We compared flow parameters in two different dispersive type cannulas: the Stealthflow and the Soft-flow cannula. A large vortex and regurgitant flow were observed in the aortic arch with both cannulas. With the Stealthflow cannula, a high-velocity area with a maximum velocity of 0.68 m/s appeared on the ostium of the cannula in the longitudinal plane. With the Soft-flow cannula, 'multiple jet streams, each with a velocity less than 0.60 m/s, were observed at the cannula outlet. Regurgitant flow from the cannula to the brachiocephalic artery and to the ascending aorta on the greater curvature was specific to the Soft-flow cannula. The degree of regurgitation on the same site was lower with the Stealthflow cannula than with the Soft-flow cannula. The Stealthflow cannula has similar flow properties to those of the Soft-flow cannula according to glass aortic model analysis. It generates gentle flow in the aortic arch and slow flow around the ostia of the aortic arch vessels. The Stealthflow cannula is as effective as the Soft-flow cannula. Care must be taken when the patient has thick atheromatous plaque or frail atheroma on the lesser curvature of the aortic arch.

A multinational, open-label, phase 2 study of ruxolitinib in Asian patients with myelofibrosis: Japanese subset analysis.

Ruxolitinib is a potent Janus kinase (JAK) 1/JAK2 inhibitor that has demonstrated rapid and durable improvements in splenomegaly and symptoms and a survival benefit in 2 phase 3 trials in patients with myelofibrosis. Ruxolitinib was well tolerated and effectively reduced splenomegaly and symptom burden in Asian patients with myelofibrosis in the Asian multinational, phase 2 Study A2202. We present a subset analysis of Japanese patients (n = 30) in Study A2202. At data cutoff, 22 patients were ongoing; 8 discontinued, mainly due to adverse events (n = 4). At week 24, 33 % of patients achieved ≥35 % reduction from baseline in spleen volume; 56.0 % achieved ≥50 % reduction from baseline in total symptom score, as measured by the 7-day Myelofibrosis Symptom Assessment Form v2.0. The most common adverse events were anemia (63 %), thrombocytopenia (40 %), nasopharyngitis (37 %), decreased platelet counts (30 %), and diarrhea (30 %). Dose reductions or interruptions due to hemoglobin decreases were more frequent in Japanese patients; no loss of efficacy and no discontinuations due to hematologic abnormalities were observed. Ruxolitinib was well tolerated in Japanese patients and provided substantial reductions in splenomegaly and myelofibrosis-related symptoms similar to those observed in the overall Asian population and phase 3 COMFORT studies.

Cannulation needle-induced anterior wall tenting of internal jugular vein causing posterior wall penetration.

Unintentional posterior venous wall penetration during internal jugular vein (IJV) cannulation may cause critical arterial injuries in spite of ultrasound guidance. We aimed to evaluate whether small venous diameter and anterior venous wall tenting by a needle would be associated with posterior venous wall penetration, and to seek factors related to the venous wall tenting. We conducted a retrospective review in patients who underwent IJV cannulation. Using an ultrasound view obtained when puncturing, venous diameter, venous wall thickness, anterior venous wall tenting length, and needle angle were measured, and posterior venous wall penetration was determined. Eleven cannulations in 56 patients were assigned to posterior venous wall penetration. Small venous diameter (p = 0.004), and long anterior venous wall tenting (p = 0.007) were associated with posterior venous wall penetration. The longer anterior venous tenting would be expected with reducing needle angle (p = 0.004) or increasing anterior venous wall thickness (p = 0.006). In conclusion, small IJV and anterior venous wall tenting lead to posterior venous wall penetration. Anterior venous wall tenting is longer with reducing needle angle, or increasing the anterior venous wall thickness.

Efficacy and safety of ruxolitinib in Asian patients with myelofibrosis.

Myelofibrosis is characterized by progressive cytopenias, bone marrow fibrosis, splenomegaly and severe constitutional symptoms. In the phase 3 Controlled Myelofibrosis Study with Oral JAK Inhibitor Treatment (COMFORT) studies, ruxolitinib, a potent Janus kinase 1 (JAK1)/JAK2 inhibitor, provided substantial improvements in splenomegaly, symptoms, quality-of-life measures and overall survival compared with placebo or best available therapy. No assessments of the efficacy and safety of ruxolitinib have been conducted in Asian patients. Here, we describe results from an open-label, single-arm, phase 2 trial evaluating ruxolitinib in Asian patients with myelofibrosis (n = 120). The primary endpoint was met, with 31.7% of patients achieving a ≥ 35% reduction from baseline spleen volume at week 24. As measured by the 7-day Myelofibrosis Symptom Assessment Form v2.0, 49% of patients achieved a ≥ 50% reduction from baseline in total symptom score. Adverse events were consistent with those seen in the COMFORT studies. Ruxolitinib was well tolerated in Asian patients with myelofibrosis and provided substantial reductions in splenomegaly and improvements in symptoms.

Roles of chromatin remodelers in maintenance mechanisms of multipotency of mouse trunk neural crest cells in the formation of neural crest-derived stem cells.

We analyzed roles of two chromatin remodelers, Chromodomain Helicase DNA-binding protein 7 (CHD7) and SWItch/Sucrose NonFermentable-B (SWI/SNF-B), and Bone Morphogenetic Protein (BMP)/Wnt signaling in the maintenance of the multipotency of mouse trunk neural crest cells, leading to the formation of mouse neural crest-derived stem cells (mouse NCSCs). CHD7 was expressed in the undifferentiated neural crest cells and in the dorsal root ganglia (DRG) and sciatic nerve, typical tissues containing NCSCs. BMP/Wnt signaling stimulated the expression of CHD7 and participated in maintaining the multipotency of neural crest cells. Furthermore, the promotion of CHD7 expression maintained the multipotency of these cells. The inhibition of CHD7 and SWI/SNF-B expression significantly suppressed the maintenance of the multipotency of these cells. In addition, BMP/Wnt treatment promoted CHD7 expression and caused the increase of the percentage of multipotent cells in DRG. Thus, the present data suggest that the chromatin remodelers as well as BMP/Wnt signaling play essential roles in the maintenance of the multipotency of mouse trunk neural crest cells and in the formation of mouse NCSCs.

A candidate enhancer element responsible for high-level expression of the aggrecan gene in chondrocytes.

Aggrecan is the most abundant proteoglycan in cartilage. It contains a lot of negatively charged glycosaminoglycan chains along the core protein, providing a large osmotic swelling pressure within the cartilage. Therefore, the biomechanical properties of cartilage, such as its compressive load-bearing capacity, are highly dependent on the presence of abundant aggrecan in the cartilage matrix. To elucidate the transcriptional mechanism that leads to abundant expression of aggrecan by chondrocytes, we screened for enhancer elements in 130 kb of the aggrecan gene, Agc1, using a reporter assay system that we previously developed. The system is based on co-transfection of candidate enhancer elements and reporter constructs into Swarm rat chondrosarcoma chondrocytes that retain a high level of aggrecan expression. We found an element that might be involved in high-level expression of Agc1 gene in chondrocytes in vivo. The element is located 30 kb upstream of the Agc1 transcription start site.

Contamination of poultry products with Listeria monocytogenes at poultry processing plants.

This study aimed to confirm that poultry products packed at poultry processing plants have already been contaminated with Listeria monocytogenes and that poultry products contaminated with L. monocytogenes are derived from broiler flocks infected with L. monocytogenes. L. monocytogenes was isolated from 16.8% (58/345) of chicken breast products and 2.3% (8/345) of chicken liver products. In contrast, L. monocytogenes was isolated from the pooled cecal content sample from only 1 (4%) of 25 flocks and was never isolated from any pooled dropping samples collected from 25 farms. The results of our study indicate that cecal content does not seem to be an important source of L. monocytogenes in poultry products.

Prevalence and antimicrobial susceptibility of foodborne bacteria in wild boars (Sus scrofa) and wild deer (Cervus nippon) in Japan.

This study aimed to evaluate the role of wild boars and deer as reservoirs of foodborne bacteria. We investigated the prevalence and antimicrobial susceptibility of Campylobacter spp., Salmonella spp., Shiga toxin-producing Escherichia coli (STEC) O157 and O26, and Listeria monocytogenes isolated from wild boars and deer in Japan, from July through December 2010. Campylobacter spp. and Salmonella spp. were isolated from 43.8% (95% confidence interval [CI]: 35.0-52.6) and 7.4% (95% CI: 2.8-12.1) of rectal content samples of wild boars, respectively, but not from wild deer. The most common Campylobacter species was C. lanienae and C. hyointestinalis. The nine Salmonella serovars isolated were S. enterica subsp. enterica serovar Agona (three isolates), S. Narashino (two), S. Enteritidis (one), S. Havana (one), S. Infantis (one), and S. Thompson (one). Five (16%) and 6 (29%) isolates of C. lanienae and C. hyointestinalis, respectively, were resistant to enrofloxacin. STEC O157 and O26 and L. monocytogenes were isolated from 2.3% (95% CI: 0-5.0), 0.8% (95% CI: 0-2.3), and 6.1% (95% CI: 1.7-10.5) of the rectal content samples of wild deer, respectively, but not from wild boars. This first nationwide survey of the prevalence of foodborne bacteria in wild boars and wild deer in Japan suggests that consumption of meat from these animals is associated with the risk of causing infection with these bacteria in humans. Moreover, these animals are potential vehicles for distribution of antimicrobial-resistant bacteria into their habitat. The prevalence and antimicrobial susceptibility of such foodborne bacteria in these wild animals should be monitored periodically.

Interactive effect of galanin-like peptide (GALP) and spontaneous exercise on energy metabolism.

Galanin-like peptide (GALP) is a neuropeptide involved in energy metabolism. The interactive effect of GALP and exercise on energy metabolism has not been investigated. The aim of this study was to determine if energy metabolism in spontaneously exercising mice could be promoted by intracerebroventricular (ICV) GALP administration. Changes in respiratory exchange ratio in response to GALP ICV administration indicated that lipids were primarily consumed followed by a continuous consumption of glucose throughout the dark period in non-exercising mice. In mice permitted to spontaneously exercise on a running-wheel, GALP ICV administration increased the consumed oxygen volume and heat production level from 5 to 11h after administration. These effects occurred independently from the total running distance. The interaction between GALP ICV administration and spontaneous exercise decreased body weight within 24h (F(1,16)=5.772, p<0.05), with no significant interaction observed regarding food and water intake or total distance. Energy metabolism-related enzymes were assessed in liver and skeletal muscle samples, with a significant interaction on mRNA expression between GALP ICV administration and spontaneous exercise observed in phosphoenolpyruvate carboxykinase (F(1,16)=18.602, p<0.001) that regulates gluconeogenesis and glucose transporter-4 (F(1,16)=21.092, p<0.001). GALP significantly decreased the mRNA expression of sterol regulatory element-binding protein-1c (p<0.05) that regulates fatty acid synthesis regardless of spontaneous exercise with no changes to acetyl-CoA carboxylase a and fatty acid synthetase. These results indicate the GALP ICV administration can further promote energy metabolism when administered to spontaneously exercising mice.

A report from Fukushima: an assessment of bone health in an area affected by the Fukushima nuclear plant incident.

Bone health was assessed for inhabitants of an area affected by the Fukushima nuclear plant incident. Osteoporotic patients, who had been treated with active vitamin D3 and/or bisphosphonate at Soma Central Hospital before the Fukushima incident, were enrolled. Changes in bone turnover markers and bone mineral density were retrospectively analyzed. Serum levels of a bone resorption marker, serum type I collagen cross-linked N-telopeptide were decreased in all the treated groups, whereas those of a bone formation marker, bone-specific alkaline phosphatase, were increased. Accordingly, bone mineral density, estimated by dual-energy X-ray absorptiometry, was increased in the lumbar spine of all groups, but bone mass increase in the proximal femur was detected only in the group treated with the two agents in combination. From the degree of these parameter changes, the antiosteoporotic treatments looked effective and were equivalent to the expected potency of past observations. At this stage, the present study implies that the Fukushima nuclear incident did not bring an acute risk to bone health in the affected areas.