Altered expression of microRNA during fracture healing in diabetic rats.

OBJECTIVES: Diabetes mellitus (DM) is known to impair fracture healing. Increasing evidence suggests that some microRNA (miRNA) is involved in the pathophysiology of diabetes and its complications. We hypothesized that the functions of miRNA and changes to their patterns of expression may be implicated in the pathogenesis of impaired fracture healing in DM. METHODS: Closed transverse fractures were created in the femurs of 116 rats, with half assigned to the DM group and half assigned to the control group. Rats with DM were induced by a single intraperitoneal injection of streptozotocin. At post-fracture days five, seven, 11, 14, 21, and 28, miRNA was extracted from the newly generated tissue at the fracture site. Microarray analysis was performed with miRNA samples from each group on post-fracture days five and 11. For further analysis, real-time polymerase chain reaction (PCR) analysis was performed at each timepoint. RESULTS: Microarray analysis showed that there were 14 miRNAs at day five and 17 miRNAs at day 11, with a greater than twofold change in the DM group compared with the control group. Among these types of miRNA, five were selected based on a comparative and extended literature review. Real-time PCR analysis revealed that five types of miRNA (miR-140-3p, miR-140-5p, miR-181a-1-3p, miR-210-3p, and miR-222-3p) were differentially expressed with changing patterns of expression during fracture healing in diabetic rats compared with controls. CONCLUSIONS: Our findings provide information to further understand the pathology of impaired fracture healing in a diabetic rat model. These results may allow the potential development of molecular therapy using miRNA for the treatment of impaired fracture healing in patients with DM.Cite this article: S. Takahara, S. Y. Lee, T. Iwakura, K. Oe, T. Fukui, E. Okumachi, T. Waki, M. Arakura, Y. Sakai, K. Nishida, R. Kuroda, T. Niikura. Altered expression of microRNA during fracture healing in diabetic rats. Bone Joint Res 2018;7:139-147. DOI: 10.1302/2046-3758.72.BJR-2017-0082.R1.

Profiling microRNA expression in fracture nonunions: Potential role of microRNAs in nonunion formation studied in a rat model.

MicroRNAs (miRNAs ) are small non-coding RNAs that regulate gene expression. We hypothesised that the functions of certain miRNAs and changes to their patterns of expression may be crucial in the pathogenesis of nonunion. Healing fractures and atrophic nonunions produced by periosteal cauterisation were created in the femora of 94 rats, with 1:1 group allocation. At post-fracture days three, seven, ten, 14, 21 and 28, miRNAs were extracted from the newly generated tissue at the fracture site. Microarray and real-time polymerase chain reaction (PCR) analyses of day 14 samples revealed that five miRNAs, miR-31a-3p, miR-31a-5p, miR-146a-5p, miR-146b-5p and miR-223-3p, were highly upregulated in nonunion. Real-time PCR analysis further revealed that, in nonunion, the expression levels of all five of these miRNAs peaked on day 14 and declined thereafter. Our results suggest that miR-31a-3p, miR-31a-5p, miR-146a-5p, miR-146b-5p and miR-223-3p may play an important role in the development of nonunion. These findings add to the understanding of the molecular mechanism for nonunion formation and may lead to the development of novel therapeutic strategies for its treatment.

Complete traumatic backout of the blade of proximal femoral nail antirotation: a case report.

This article presents a rare case of traumatic complete expulsion of the helical blade after successful treatment of an intertrochanteric fracture with proximal femoral nail antirotation (PFNA). A 94-year-old woman sustained an intertrochanteric fracture of the left femur. Fracture fixation was performed by using PFNA-II. At six months FU, the patient presented with pain at the proximal lateral left thigh after she had fallen. A protrusion was noted. Radiographs showed a complete expulsion of the helical blade with a healed intertrochanteric fracture. The PFNA-II was removed and a cemented bipolar hemiarthroplasty was performed. At 5 months after surgery, the patient was able to walk with a walker without pain. Traumatic complete expulsion of the blade should be considered as a possible complication of PFNA/PFNA-II.

Efficient cell-seeding into scaffolds improves bone formation.

Bone marrow stromal cells (BMSCs)/beta-tricalcium phosphate (beta-TCP) composites have attracted a great deal of attention in bone tissue engineering. If more effective bone regeneration is to be achieved, efficient cell-seeding systems need to be clarified. In this study, we investigated the number of cells contained in composites, and the in vitro/vivo osteogenic differentiation capacity of composites using 4 conventional systems of seeding rat BMSCs into beta-TCP: soak, low-pressure, pipette, and syringe systems. The highest number of cells was contained in the composites from the syringe group. Moreover, after two-week osteogenic induction in vitro, the composites in the syringe group exhibited the highest osteogenic potential, which continued at 8 weeks after subcutaneous implantation in vivo. Our results indicated that efficient and appropriate cell-seeding could improve in vitro/vivo bone formation in composites and thus make a potential clinical contribution to successful bone tissue engineering.

[Acute pulmonary thromboembolism in polysplenia syndrome].

We present a case of azygos continuation of the inferior vena cava, combined with polysplenia syndrome in a 72-year-old woman who presented with circulatory collapse due to acute pulmonary thromboembolism. Patients with polysplenia have multiple spleens, and their splenic function is usually normal, but this case was not. In this case, defective splenic function was associated with a high risk of fulminant bacterial infections, especially with encapsulated bacteria. The clinical features and prognosis of this entity are discussed.

Pathologic pain distorts visuospatial perception.

Spatial perception is achieved by integrating multisensory information. Using visual subjective body midline (vSM) judgments in patients with unilateral limb pain (complex regional pain syndrome [CRPS]), we found that their vSM deviated toward the affected side; however, deafferentation of the affected limb caused a transient pain decrease and a transient shift of the vSM deviation toward the unaffected side. Our results indicate that the persistent pain state in CRPS distorts visuospatial perception.

[Drug-eluting stents restenosis 2 months after the procedure and necessitating semi-emergency off-pump coronary artery bypass].

Drug-eluting stents (sirolimus-eluting stent: Cypher stent) have showed a reducing the frequency of in-stent restenosis and a good safety profile. One case involving edge dissection with Cypher stent restenosis for 2 lesions was diagnosed in 2 months after the procedure. This case occurred in a hypertensive 63-year-old woman with complex coronary lesions. The coronary angiography showed uncovered proximal dissection with 90% restenosis in left anterior descending coronary artery to left main trunk and 75% restenosis in right coronary artery. We were able to perform semi-emergency off-pump coronary artery bypass (OPCAB). There is the relationship between coronary dissection and restenosis. Cypher stent delayed dissected arterial healing and promote some inflammation at the lesion. Patients implanted Cypher stent with uncovered coronary dissection need more frequent follow-up.

A novel heterozygous missense mutation in the vasopressin moiety is identified in a Japanese person with neurohypophyseal diabetes insipidus.

The autosomal dominant familial neurohypophyseal diabetes insipidus (adFNDI) is caused by diverse mutations in one allele of the gene that encodes the arginine vasopressin (AVP) precursor protein, AVP-neurophysin II (AVP-NP II). Most of the mutations identified so far are located in either the signal peptide or NP II moiety. Two recently published mutations in the AVP gene identified in kindreds with adFNDI predict a substitution of histidine for tyrosine at position 2 and a deletion of phenylalanine at position 3 in AVP. They are unique among adFNDI mutations in that they are the only adFNDI mutations that affect amino acid residues in the AVP moiety of the pro-hormone. Here, we report a novel heterozygous missense mutation in the AVP moiety of the AVP-NP II gene in a Japanese person with neurohypophyseal diabetes insipidus (DI). This mutation occurs at position 2 in AVP and predicts a substitution of serine for tyrosine (Y21S). It is expected to interfere with normal binding of AVP with NP II, and thus result in misfolding of the precursor proteins. The data of this study support the notion that mutations affecting the AVP moiety can result in the initiation of the pathological processes.

[Clinical outcome of aortic valve replacement with 19-mm prosthetic valve in elderly patients].

From 1979 to June 2005, 90 patients aged 65 or older underwent aortic valve replacement with 19-mm prosthetic valve. They were 84 women and 6 men, with a mean age of 74. The mean body surface area was 1.35 m2. Bioprosthetic valves were implanted in 77 patients (85.6%). In-hospital mortality was 2.2% (2 of 90). There were 13 late deaths. New York Heart Association (NYHA) functional class improved to class I in most of survivors. Survival rates for 5 and 10 years were 84.9 and 71.2%, respectively. The outcome of aortic valve replacement with 19-mm prosthetic valve in elderly patients was excellent.

[Cholesterol crystal embolization exacerbated after the off-pump coronary artery bypass; report of a case].

A 66-year-old man with left main trunk disease was treated under a diagnosis of acute myocardial infarction based on coronary angiography by off-pump coronary artery bypass (OPCAB). About 1 month after operation, his renal function deteriorated and purpura appeared on both feet, especially at the toe tips. In this case, steroid therapy was performed and the patient survived. Cholesterol embolism rarely occurs after angiographic procedure or cardiovascular surgery. In general, it is associated with high morbidity and mortality, but no optimal treatment has yet been developed. This underlines the importance of careful observation and skin biopsy for early diagnosis.

Safety and efficacy of water-in-oil-in-water emulsion vaccines containing Newcastle disease virus haemagglutinin-neuraminidase glycoprotein.

Subunit vaccines containing haemagglutinin-neuraminidase (HN) glycoprotein of Newcastle disease virus (NDV), formulated as water-in-oil-in-water (W/O/W) emulsions, were prepared. First, the suitable constituents of a W/O/W emulsion adjuvant were investigated with polyvalent vaccines using NDV, infectious bronchitis virus and Haemophilus paragallinarum. The W/O/W emulsion adjuvant, composed of the antigen in phosphate-buffered saline (PBS), liquid paraffin, squalene, diglyceryl monooleate, polysorbate 80 and PBS in a 30:25:10:5:2:28 ratio, induced a good antibody response with less adverse local reactions. HN protein of NDV was expressed by an improved baculovirus expression vector, a hybrid nucleopolyhedrovirus (HyNPV) between Autographa californica NPV and Bombyx mori NPV,and was prepared from silkworm pupae infected with the recombinant baculovirus, HyNPV-HN. Then, the W/O/W emulsion vaccine containing HN protein was prepared using the aforementioned constituents. Chickens showed 100, 100 and 80% protection against challenge exposure to virulent NDV at 4 weeks after vaccination with W/O/W emulsion vaccines containing 30, 6 and 3% of HyHPV-HN-infected pupae, respectively. The vaccines containing HN protein did not induce adverse local reactions at the site of injection. The subunit vaccine for NDV containing HN protein expressed in the recombinant baculovirus-infected pupae, formulated as a W/O/W emulsion vaccine composed of the antigen in PBS, liquid paraffin, squalene, diglyceryl monooleate, polysorbate 80 and PBS in a 30:25:10:5:2:28 ratio, was therefore found to be safe and effective.

Activating transcription factor-2 is a positive regulator in CaM kinase IV-induced human insulin gene expression.

Insulin plays a crucial role in the regulation of glucose-homeostasis, and its synthesis is regulated by several stimuli. The transcription of the human insulin gene, enhanced by an elevated intracellular concentration of calcium ions, was completely blocked by Ca2+/calmodulin-dependent protein kinase inhibitor. The activity of the transcription factor activating transcription factor-2 (ATF-2), which binds to the cAMP responsive elements of the human insulin gene, was enhanced by Ca2+/calmodulin-dependent protein kinase IV (CaMKIV). Mutagenesis studies showed that Thr69, Thr71, and Thr73 of ATF-2 are all required for activation by CaMKIV. CaMKIV-induced ATF-2 transcriptional activity was not altered by activation of cJun NH2-terminal protein kinase (JNK) or p38 mitogen-activated protein (MAP) kinase. Furthermore, when transfected into rat primary cultured islets, ATF-2 enhanced glucose-induced insulin promoter activity, whereas cAMP response element-binding protein (CREB) repressed it. These results suggest a mechanism in which ATF-2 regulates insulin gene expression in pancreatic beta-cells, with the transcriptional activity of ATF-2 being increased by an elevated concentration of calcium ions.

Sustained enhancement of Ca(2+) influx by glibenclamide induces apoptosis in RINm5F cells.

Cytosolic Ca(2+) elevations are known to be involved in triggering apoptosis in many tissues, but the effect of sustained enhancement of Ca(2+) influx on apoptosis in beta cells remains unknown. We have found that the viability of RINm5F cells is decreased dose-dependently by continuous exposure to glibenclamide at concentrations from 10(-7) to 10(-4) M, and that this effect is partially ameliorated by pretreatment with cycloheximide. Electrophoresis of the cells exposed to glibenclamide revealed ladder-like fragmentation characteristic of apoptosis, and which also is suppressed by cycloheximide pretreatment. By using terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining, we detected increased DNA fragmentation in the nuclei of the cells exposed to glibenclamide, and staining with Hoechst 33342 and propidium iodide showed a dose-dependent increase in the number of cells with the chromatin condensation and fragmentation in their nuclei that is characteristic of apoptosis. The effects of glibenclamide on cell viability and apoptotic cell death were partially inhibited by treatment with Ca(2+) channel blocker, and by reducing the extracellular Ca(2+) concentration during glibenclamide exposure, suggesting that they may be derived from increased Ca(2+) influx. Furthermore, only the percentage of apoptotic cells, and not that of necrotic cells, increased with the increasing intracellular Ca(2+) concentration during glibenclamide exposure. In conclusion, we have demonstrated that the sustained enhancement of Ca(2+) influx caused by glibenclamide exposure can induce apoptotic cell death in a pure beta cell line.

Antioxidant alpha-tocopherol ameliorates glycemic control of GK rats, a model of type 2 diabetes.

We have shown recently that oxidative stress by chronic hyperglycemia damages the pancreatic beta-cells of GK rats, a model of non-obese type 2 diabetes, which may worsen diabetic condition and suggested the administration of antioxidants as a supportive therapy. To determine if natural antioxidant alpha-tocopherol (vitamin E) has beneficial effects on the glycemic control of type 2 diabetes, GK rats were fed a diet containing 0, 20 or 500 mg/kg diet alpha-tocopherol. Intraperitoneal glucose tolerance test revealed a significant increment of insulin secretion at 30 min and a significant decrement of blood glucose levels at 30 and 120 min after glucose loading in the GK rats fed with high alpha-tocopherol diet. The levels of glycated hemoglobin A1c, an indicator of glycemic control, were also reduced. Vitamin E supplementation clearly ameliorated diabetic control of GK rats, suggesting the importance of not only dietary supplementation of natural antioxidants but also other antioxidative intervention as a supportive therapy of type 2 diabetic patients.

The MH1 domains of smad2 and smad3 are involved in the regulation of the ALK7 signals.

The biological responses of the transforming growth factor beta (TGF-beta) superfamily are induced by activation of a receptor complex and Smad proteins. We surveyed the TGF-beta superfamily receptors using the degenerate PCR strategy, and found activin receptor-like kinase 7 (ALK7) to be abundantly expressed in fetal rat pancreatic islets. ALK7 is also expressed in adult rat islets and pancreatic beta-cell-derived MIN6 cells. The constitutively active form of ALK7, ALK7(T194D), activated Smad3 and a chimeric Smad protein, Smad3-2, containing the MH1 domain of Smad3 and the MH2 domain of Smad2, and translocated them to nuclei and then induced activation of the human PAI-1 promoter. However, neither Smad2 nor Smad2-3 protein, containing the MH1 domain of Smad2 and the MH2 domain of Smad3 were activated. These results indicate that the ALK7 signal regulates nuclear localization and activation of Smad2 and Smad3, and the MH1 domain of Smad2 has inhibitory effects on the nuclear localization.

Incorporation and distribution of tritium in rats exposed to tritiated rice or tritiated soybean.

The incorporation and distribution of tritium were examined in rats exposed to tritiated rice or tritiated soybean by single ingestion or continuous feeding. Results were compared with those for tritiated wheat and tritiated water in a previous study done under the same experimental conditions. All the tritiated crops examined were more efficiently incorporated into rat tissues than was tritiated water, the extent of incorporation depending on the kind of crop. The differences in incorporation were clear in organically bound tritium determined as tritium in dried tissue. The respective concentrations of organically bound tritium after a single ingestion of tritiated rice, tritiated wheat or tritiated soybean were about 10-20, 20-30 and 25-60 times higher than after the ingestion of tritiated water. After continuous feeding for 22 days with tritiated rice, tritiated wheat or tritiated soybean, the respective concentrations of organically bound tritium were 5-8, 6-11 and 10-25 times the values after continuous ingestion of tritiated water. At the end of continuous ingestion, the radiation dose rates to almost of the tissues from all three tritiated crops were estimated to be 2-3 times that for tritiated water.

Tritium concentration in Japanese rice.

Polished rice samples harvested in 1985 were collected from 25 prefectures throughout Japan. Concentrations of both tissue free water tritium (TFWT) and organically bound tritium (OBT) were determined. Nearly uniform distribution of the TFWT and OBT concentrations was observed in Japan, taking into account a relatively large counting error. The average values for all Japanese polished rice were 2.69 +/- 0.74 Bq/L for the TFWT concentration, 2.01 +/- 0.78 Bq/L for the OBT concentration and 0.83 +/- 0.32 for the specific activity ratio. The TFWT concentration was within the range of tritium concentration of the land water reported by other researchers. The specific activity ratio below unity also confirmed by analyses of the 4 Chinese polished rice samples suggests that OBT in the rice is not equilibrated with TFWT or HTO in the environment because of isotope discrimination.