Evidence of effectiveness of a fracture liaison service to reduce the re-fracture rate.

SUMMARY: We assessed the ability of a fracture liaison service (FLS) to directly reduce re-fracture risk. Having a FLS is associated with a ∼40% reduction in the 3-year risk of major bone and ∼30% of any bone re-fracture. The number needed to treat to prevent a re-fracture is 20. INTRODUCTION: FLS have been promoted as the most effective interventions for secondary fracture prevention, and while there is evidence of increased rate of investigation and treatment at institutions with a FLS, only a few studies have considered fracture outcomes directly. We therefore sought to evaluate the ability of our FLS to reduce re-fracture risk. METHODS: Historical cohort study of all patients ≥50 years presenting over a 6-month period with a minimal trauma fracture (MTF) to the emergency departments of a tertiary hospital with a FLS, and one without a FLS. Baseline characteristics, mortality and MTFs over a 3-year follow-up were recorded. RESULTS: Five hundred fifteen patients at the FLS hospital and 416 patients at the non-FLS hospital were studied. Over 3 years, 63/515 (12%) patients at the FLS hospital and 70/416 (17%) at the non-FLS hospital had a MTF. All patients were analysed in an intention-to-treat analysis regardless of whether they were seen in the FLS follow-up clinic. Statistical analysis using Cox proportional hazard models in the presence of a competing risk of death from any cause was used. After adjustment for baseline characteristics, there was a ∼30% reduction in rate of any re-fracture at the FLS hospital (hazard ratio (HR) 0.67, confidence interval (CI) 0.47-0.95, p value 0.025) and a ∼40% reduction in major re-fractures (hip, spine, femur, pelvis or humerus) (HR 0.59, CI 0.39-0.90, p value 0.013). CONCLUSIONS: We found a ∼30% reduction in any re-fractures and a ∼40% reduction in major re-fractures at the FLS hospital compared with a similar non-FLS hospital. The number of patients needed to treat to prevent one new fracture over 3 years is 20.

A DFT and multi-configurational perturbation theory study on O2 binding to a model heme compound via the spin-change barrier.

Dioxygen binding to a model heme compound via intersystem crossing (ISC) was investigated with a multi-state multi-configurational self-consistent field method with second-order perturbation theory (MS-CASPT2) and density functional theory (DFT) calculations. In elongated Fe-O distances, the energy levels of the S0 and T1 states are separated, which decreases the probability of intersystem crossing in these structures. At the DFT(B97D) level of calculation, the Fe-O distances of the S0 and T1 states were 1.91 and 2.92 Å, respectively. The minimum energy intersystem crossing point (MEISCP) was located as a transition state at a Fe-O distance of 2.17 Å with an energy barrier of 1.0 kcal mol(-1) from the T1 minimum. The result was verified with MS-CASPT2 calculations including the spin-orbit interaction which also showed the intersystem crossing point at a Fe-O distance of 2.05 Å. An energy decomposition analysis on the reaction coordinate showed the important contribution of the ring-shrinking mode of the porphyrin ring, indicating that the reaction coordinates which control the relative energy level of the spin-states play a key role in intersystem crossing.

Early optimization of antimicrobial therapy improves clinical outcomes of patients administered agents targeting methicillin-resistant Staphylococcus aureus.

WHAT IS KNOWN AND OBJECTIVE: Antimicrobial stewardship is required to ensure the appropriate use of antimicrobials. However, no reports have been published on clinical outcomes of implementation of antimicrobial stewardship in patients receiving pathogen-specific antibiotics. METHOD: To evaluate the clinical outcomes of patients who received drugs, we conducted a single-centre, retrospective study of the effects of an antimicrobial stewardship programme targeting methicillin-resistant Staphylococcus aureus (MRSA). RESULTS: The time to administer effective antimicrobials was significantly (median number of days, 3 before vs. 0 after, P < 0·001) shortened, and the rate of de-escalation was significantly elevated (47·1% vs. 96·2%, P < 0·001) after implementation of daily review. The 60-day clinical failure associated with Gram-positive bacterial infection was significantly reduced (33·3% vs. 17·6%, P = 0·007) after intervention. WHAT IS NEW AND CONCLUSIONS: Daily review of administration of antimicrobials targeting MRSA was highly effective in improving clinical outcomes by optimizing early antimicrobial therapy.

Anti-platelet effects of chalcones from Angelica keiskei Koidzumi (Ashitaba) in vivo.

Angelica keiskei Koidzumi (Ashitaba) is a traditional folk medicine that is also regarded in Japan as a health food with potential antithrombotic properties. The ability of the major chalcones, xanthoangelol (XA) and 4-hydroxyderricin (4-HD) extracted from Ashitaba roots to inhibit platelet aggregation activity in vitro was recently determined. However, the anti-platelet activities of Ashitaba chalcones in vivo have remained unclear. The present study examines the anti-platelet effects of Ashitaba exudate and its constituent chalcones using mouse tail-bleeding models that reflect platelet aggregation in vivo. Ashitaba exudate and the major chalcone subtype XA, suppressed the lipopolysaccharide (LPS)-induced shortening of mouse tail bleeding. However, trace amounts of other Ashitaba chalcone subtypes including xanthoangelols B (XB), D (XD), E (XE) and F (XF) did not affect tail bleeding. These results suggest that the major chalcone subtype in Ashitaba, XA, has anti-platelet-activities in vivo.

Thyroid signaling in immune organs and cells of the teleost fish rainbow trout (Oncorhynchus mykiss).

Thyroid hormones are involved in modulating the immune system in mammals. In contrast, there is no information on the role played by these hormones in the immune system of teleost fish. Here we provide initial evidence for the presence of active thyroid signaling in immune organs and cells of teleosts. We demonstrate that immune organs (head kidney and spleen) and isolated leukocytes (from head kidney and peripheral blood) of the rainbow trout (Oncorhynchus mykiss) express both thyroid receptor α (THRA) and ß (THRB). Absolute mRNA levels of THRA were significantly higher than those of THRB. THRA showed higher expression in immune organs and isolated immune cells compared to the reference organ, liver, while THRB showed the opposite. In vivo exposure of trout to triiodothryronine (T3) or the anti-thyroid agent propylthiouracil (PTU) altered THR expression in immune organs and cells. Effect of T3 and PTU over the relative expression of selected marker genes of immune cell subpopulations was also studied. Treatments changed the relative expression of markers of cytotoxic, helper and total T cells (cd4, cd8a, trb), B lymphocytes (mIgM) and macrophages (csf1r). These findings suggest that the immune system of rainbow trout is responsive to thyroid hormones.

Suitability of polymers as screw post materials in primary teeth: an in vitro study.

AIM: Primary teeth undergo physiological root resorption during the transition to permanent dentition. The aim of this study was to assess the potential use of screw posts in core build-up for primary teeth while adequately retaining the crown restoration and allowing smooth physiological root resorption. METHODS: To determine whether biodegradable polymers such as polyglycolic acid (PGA) and poly-L-lactic acid (PLLA) were appropriate as post materials, bending strength test and bending elastic modulus test were performed according to ISO standards. The prepared screw posts were immersed in 0.01 mol/L phosphate-buffered saline at 37 degrees Celsius, and changes due to hydrolysis were observed. Results In the bending strength test and bending elastic modulus test, PGA and PLLA showed similar values to composite resins used for core build-up. Although both showed adequate hydrolysis, the hydrolysis rate of PGA was higher than that of PLLA. CONCLUSION: PGA and PLLA may be suitable as biodegradable screw posts for primary teeth because they have appropriate strength and hydrolysis ability.

A detailed analysis of the spin-crossover reaction of H2S binding to heme and the six-coordinated FeP(Im)-HS- porphyrin complex.

The potential energy surfaces of the H2S binding to iron-porphyrin (FeP) with the imidazole (Im) ligand via intersystem crossings are investigated by using density functional theory. The minimum energy intersystem crossing point (MEISCP) between the quintet and triplet states (MEISCPTQ) for the Fe(II)P(Im)-H2S complex is located at a Fe-S distance of 3.39 Šwith only 1.1 kcal/mol above the quintet state minimum. The second spin-crossover point, where a change from the triplet to the singlet state occurs, comes at a much shorter Fe-S distance of 2.79 Å, and the MEISCPST is located at 3.7 kcal/mol above the triplet state minimum. The nature of the chemical bonding along the Fe-S reaction coordinate from the ground state singlet to the quintet state along the path to the separated species is analyzed. An inspection of the vibrational modes reveals that the largest contribution to the triplet-quintet transition around the quintet and triplet state minimum comes from the symmetric shrinking of the pyrrole units of the porphyrin ring, indicating that the related reaction coordinate plays a main role in the intersystem crossing. The fully optimized structures of the Fe(II)P(Im)-HS- complex corresponding to three different spin multiplicities (M = 1, 3, 5) are characterized by a bent Fe-H-S conformation. The binding of the hydrosulfide anion to Fe(II)P(Im) in the quintet state induces a 0.2 Šdisplacement of the Fe atom out of the nitrogen porphyrin (Npyr) plane. The fully optimized structure of the ground state of Fe(II)P(Im)-HS- agrees well with experimental data for the corresponding heme models.

Correction: Synergistic anti-AML effects of the LSD1 inhibitor T-3775440 and the NEDD8-activating enzyme inhibitor pevonedistat via transdifferentiation and DNA rereplication.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Identification of ABCG2 as an Exporter of Uremic Toxin Indoxyl Sulfate in Mice and as a Crucial Factor Influencing CKD Progression.

Chronic kidney disease (CKD) patients accumulate uremic toxins in the body, potentially require dialysis, and can eventually develop cardiovascular disease. CKD incidence has increased worldwide, and preventing CKD progression is one of the most important goals in clinical treatment. In this study, we conducted a series of in vitro and in vivo experiments and employed a metabolomics approach to investigate CKD. Our results demonstrated that ATP-binding cassette transporter subfamily G member 2 (ABCG2) is a major transporter of the uremic toxin indoxyl sulfate. ABCG2 regulates the pathophysiological excretion of indoxyl sulfate and strongly affects CKD survival rates. Our study is the first to report ABCG2 as a physiological exporter of indoxyl sulfate and identify ABCG2 as a crucial factor influencing CKD progression, consistent with the observed association between ABCG2 function and age of dialysis onset in humans. The above findings provided valuable knowledge on the complex regulatory mechanisms that regulate the transport of uremic toxins in our body and serve as a basis for preventive and individualized treatment of CKD.

Identification of sequence elements that confer cell-type-specific control of MF alpha 1 expression in Saccharomyces cerevisiae.

The MF alpha 1 gene of Saccharomyces cerevisiae, a major structural gene for mating pheromone alpha factor, is an alpha-specific gene whose expression is regulated by the mating-type locus. To study the role of sequences upstream of MF alpha 1 in its expression and regulation, we generated two sets of promoter deletions: upstream deletions and internal deletions. By analyzing these deletions, we have identified a TATA box and two closely related, tandemly arranged upstream activation sites as necessary elements for MF alpha 1 expression. Two upstream activation sites were located ca. 300 and 250 base pairs upstream of the MF alpha 1 transcription start points, which were also determined in this study. Each site contained a homologous 22-base-pair sequence, and both sites were required for maximum transcription level. The distance between the upstream activation sites and the transcription start points could be altered without causing loss of transcription efficiency, and the sites were active in either orientation with respect to the coding region. These elements conferred cell type-specific expression on a heterologous promoter. Analysis with host mating-type locus mutants indicates that these sequences are the sites through which the MAT alpha 1 product exerts its action to activate the MF alpha 1 gene. Homologous sequences with these elements were found in other alpha-specific genes, MF alpha 2 and STE3, and may mediate activation of this set of genes by MAT alpha 1.

Synergistic anti-AML effects of the LSD1 inhibitor T-3775440 and the NEDD8-activating enzyme inhibitor pevonedistat via transdifferentiation and DNA rereplication.

Lysine-specific demethylase 1A (LSD1, KDM1A) specifically demethylates di- and monomethylated histones H3K4 and K9, resulting in context-dependent transcriptional repression or activation. We previously identified an irreversible LSD1 inhibitor T-3775440, which exerts antileukemic activities in a subset of acute myeloid leukemia (AML) cell lines by inducing cell transdifferentiation. The NEDD8-activating enzyme inhibitor pevonedistat (MLN4924, TAK-924) is an investigational drug with antiproliferative activities in AML, and is also reported to induce cell differentiation. We therefore tested the combination of these two agents in AML models. The combination treatment resulted in synergistic growth inhibition of AML cells, accompanied by enhanced transdifferentiation of an erythroid leukemia lineage into granulomonocytic-like lineage cells. In addition, pevonedistat-induced rereplication stress during the S phase was greatly augmented by concomitant treatment with T-3775440, as reflected by the increased induction of apoptosis. We further demonstrated that the combination treatment was markedly effective in subcutaneous tumor xenograft models as well as in a disseminated model of AML, leading to tumor eradication or prolonged survival in T-3775440/pevonedistat cotreated mice. Our findings indicate the therapeutic potential of the combination of LSD1 inhibitors and pevonedistat for the treatment of AML.

Signal transduction and cellular functions of the TEL/ARG oncoprotein.

The TEL/ARG oncogene is formed by t(1;12)(q25;p13) reciprocal translocation and is associated with human leukemia. We have previously demonstrated that the expression of TEL/ARG in Ba/F3 cells results in prolonged viability and hyper-responsiveness to IL-3. To determine the molecular mechanisms, a series of mutants of TEL/ARG were generated, and each cDNA was expressed in Ba/F3 or CHO cells. The PNT domain in TEL and K317 in ARG were essential for both signaling and biological effects. The SH3 domain in ARG was required for hyper-responsiveness to IL-3, but not for prolonged viability. The opposite was true for the SH2 domain in ARG. Mutation of Y314 in TEL, a putative GRB2-binding site, led to reduced viability, and loss of hyper-responsiveness to IL-3. All biological functions were profoundly impaired with deletion of the C-terminus in ARG, despite maintaining high levels of its kinase activity. When expressed in CHO cells, wild-type TEL/ARG induced the formation of fillopodia, in a fashion dependent on the C-terminal portion and intact kinase activity. Thus, these results suggest several critical domains within TEL/ARG necessary for function, and indicate that the signaling pathways necessary for viability, growth factor hyper-responsiveness and cytoskeletal reorganization are likely to be separate.

Tissue-specific carcinogenesis in transgenic mice expressing the RET proto-oncogene with a multiple endocrine neoplasia type 2A mutation.

Germ line mutations of the RET proto-oncogene are responsible for the development of multiple endocrine neoplasia type 2A (MEN 2A), an inherited cancer syndrome characterized by medullary thyroid carcinoma, pheochromocytoma, and parathyroid hyperplasia. To study the mechanism of tissue-specific tumor development by RET with a MEN2A (cysteine 634-->arginine) mutation, we generated transgenic mice by introducing the RET-MEN2A gene fused to Moloney murine leukemia virus long terminal repeat. Expression of the transgene and its product was detected at variable levels in a variety of tissues including thyroid, heart, liver, colon, parotid gland, and brain. All of 29 mice analyzed developed thyroid C-cell hyperplasia or medullary carcinoma, accompanying high levels of serum calcitonin. In addition, development of mammary or parotid gland adenocarcinoma was observed in one-half of the transgenic mice. RET dimerization and its complex formation with Shc and Grb2 adaptor proteins were detected in tumor tissues. Unexpectedly, no tumor formation was found in other tissues despite RET-MEN2A expression where RET dimerization was undetectable. Because these tissues but not tumors expressed glial cell line-derived neurotrophic factor family receptor alpha (GFR alpha) at high levels, this suggested that GFR alpha expression may interfere in the dimerization of the RET-MEN2A mutant proteins, leading to tissue-specific tumor development in vivo.

Endothelin-1 Immunoreactivity and its Association with Intramedullary Hemorrhage and Myelomalacia in Naturally Occurring Disk Extrusion in Dogs.

BACKGROUND: The pathophysiology of ascending/descending myelomalacia (ADMM) after canine intervertebral disk (IVD) extrusion remains poorly understood. Vasoactive molecules might contribute. HYPOTHESIS/OBJECTIVES: To investigate the immunoreactivity of endothelin-1 (ET-1) in the uninjured and injured spinal cord of dogs and its potential association with intramedullary hemorrhage and extension of myelomalacia. ANIMALS: Eleven normal control and 34 dogs with thoracolumbar IVD extrusion. METHODS: Spinal cord tissue of dogs retrospectively selected from our histopathologic database was examined histologically at the level of the extrusion (center) and in segments remote from the center. Endothelin-1 immunoreactivity was examined immunohistochemically and by in situ hybridization. Associations between the immunoreactivity for ET-1 and the severity of intramedullary hemorrhage or the extension of myelomalacia were examined. RESULTS: Endothelin-1 was expressed by astrocytes, macrophages, and neurons and only rarely by endothelial cells in all dogs. At the center, ET-1 immunoreactivity was significantly higher in astrocytes (median score 4.02) and lower in neurons (3.21) than in control dogs (3.0 and 4.54) (P < .001; P = .004) irrespective of the grade of hemorrhage or myelomalacia. In both astrocytes and neurons, there was a higher ET-1 immunoreactivity in spinal cord regions remote from the center (4.58 and 4.15) than in the center itself (P = .013; P = .001). ET-1 mRNA was present in nearly all neurons with variable intensity, but not in astrocytes. CONCLUSION AND CLINICAL IMPORTANCE: Enhanced ET-1 immunoreactivity over multiple spinal cord segments after IVD extrusion might play a role in the pathogenesis of ADMM. More effective quantitative techniques are required.

Establishment and characterization of a malignant melanocytic tumor cell line expressing the ret oncogene.

We established a cell line (designated Mel-ret) from a melanocytic tumor developed in a metallothionein/ret transgenic mouse. Unlike primary melanocytic tumors, which did not show malignant features, when the Mel-ret cells were transplanted into nude mice they invaded into surrounding tissues and had metastatic ability. Although the Ret proteins were expressed at similar levels in the cell line and the primary tumors, the level of tyrosine phosphorylation in the Mel-ret cells was much higher than that in the primary tumors. In particular, an 85-kDa tyrosine-phosphorylated band was specifically detected in the Mel-ret cells. These results suggest that the increase in tyrosine phosphorylation may be responsible for malignant transformation of the Mel-ret cells. Immunofluorescence and cell fractionation studies showed that the Ret proteins and most of tyrosine-phosphorylated proteins in the Mel-ret cells localized in the membrane fraction. No activation of phosphatidyl-inositol-3 kinase (PI-3 kinase), a target protein for several tyrosine kinases, was detected in the Mel-ret cells.

Characterization of two promoters that regulate alternative transcripts in the microtubule-associated protein (MAP) 1A gene.

We cloned and characterized the mouse gene for microtubule-associated protein (MAP) 1A, an important protein for neuronal morphology and mitotic spindle formation. We also investigated the 5' untranslated region of the gene to characterize the promoter units. Two alternative transcripts different in the 5' region were identified by 5' RACE. Both transcripts were principally observed in the brain. Genomic cloning revealed that exons 1, 2, and 4 generate the 5' part of a long transcript, whereas exons 3 and 4 generate a short transcript. Putative 5' and intronic promoters flanking exons 1 and 3, respectively, are GC-rich and lack a canonical TATA box. DNase I footprinting from mouse cells revealed that several potential cis-elements were occupied by nuclear proteins. A reporter assay system in conjunction with a number of deletion and mutation constructs was used to test the two putative promoters. Both putative promoters showed transactivity and their function was dependent upon Sp1 sites. In addition, an NF-1 site, an HNF3B site, and an AP-1/ATF site were necessary for basal promoter activity of the intronic promoter. Our data provide insight into the regulatory mechanisms that govern the expression of the MAP1A gene.

Prevalence of the coexistence of left ventricular false tendons and premature ventricular complexes in apparently healthy subjects: a prospective study in the general population.

The prevalence of left ventricular false tendons, premature ventricular complexes and their coexistence was evaluated prospectively in 187 healthy company workers aged 21 to 50 (mean 36) years. False tendons were demonstrated echocardiographically in 133 (71%). Eight subjects were withdrawn from the study because of silent mitral valve prolapse. In these 179 healthy subjects, false tendons were detected in 127 (71%) and premature ventricular complexes in 48 (27%). Their coexistence was observed in 40, which showed a significant correlation (p less than 0.05) of false tendons and premature ventricular complexes. In seven of the eight subjects without false tendons, premature ventricular complexes were uniform and infrequent (mean 3 beats/24 h). In the 40 subjects with false tendons, premature ventricular complexes were uniform in 29, multiform in 6 and repetitive in 5, and the mean frequency was 96 beats/24 h. Correlation of premature ventricular complexes with the type of false tendons showed that premature ventricular complexes were significantly associated with thick (greater than or equal to 2 mm) and longitudinal tendons (p less than 0.005). Although it is not certain that left ventricular false tendons are arrhythmogenic, the prevalence of the coexistence of left ventricular false tendons and premature ventricular complexes in the general population, and the special relation between the frequency and the form of premature ventricular complexes and the type of false tendons, suggests that false tendons may play an etiologic role in the genesis of premature ventricular complexes in apparently healthy subjects.

Mutations in microphthalmia, the mouse homolog of the human deafness gene MITF, affect neuroepithelial and neural crest-derived melanocytes differently.

The mouse microphthalmia (Mitf) gene encodes a basic-helix-loop-helix-zipper transcription factor whose mutations are associated with abnormalities in neuroepithelial and neural crest-derived melanocytes. In wild type embryos, Mitf expression in neuropithelium and neural crest precedes that of the melanoblast marker Dct, is then co-expressed with Dct, and gradually fades away except in cells in hair follicles. In embryos with severe Mitf mutations, neural crest-derived Mitf-expressing cells are rare, lack Dct expression, and soon become undetectable. In contrast, the neuroepithelial-derived Mitf-expressing cells of the retinal pigment layer are retained, express Dct, but not the melanogenic enzyme genes tyrosinase and Tyrp1, and remain unpigmented. The results show that melanocyte development critically depends on functional Mitf and that Mitf mutations affect the neural crest and the neuroepithelium in different ways.

Kit(+) melanocytes seem to contribute to melanocyte proliferation after UV exposure as precursor cells.

Melanogenesis caused by UV exposure is characterized by proliferation and differentiation of functioning melanocytes in epidermis. Recently, the existence of precursor melanocytes in normal skin was proposed immunohistochemically. Thus, this precursor melanocyte seems to proliferate and differentiate into mature pigmented melanocytes after UV exposure. To elucidate how these processes occur, we examined C57BL mouse epidermal sheets after UV exposure immunohistochemically. In normal epidermis, TRP-1(+) cells and Kit(+) cells were easily identified and the cellularities were 41.0 and 31.7 cells per mm(2), respectively. Only a few Mitf(+) cells and no TRP-2(+) cells were observed. On the first day after UV exposure, Mitf(+) cells and TRP-2(+) cells appeared, whereas the numbers of TRP-1(+) cells and Kit(+) cells decreased. Some Kit+ cells also expressed Mitf, but no TRP-1 and Mitf double positive cells were seen. In this period, some Mitf(+) cells were labeled with BrdU. The next day, Mitf(+) cells and TRP-2(+) cells increased to the maximum cellularity, and they decreased thereafter and disappeared on the seventh day. An increase of TRP-1(+) cells was first identified on the fifth day after UV exposure, and reached a cellularity four times as great as that of the normal control on the fourteenth day. The subepidermal injection of Kit antibody attenuated the increase of Mitf+ cells and TRP-1(+) cells. These findings suggested that precursor cells that reside in the skin, first differentiated into Mitf(+) and TRP-2(+) melanocytes by the activation of the Kit receptor, then become mature TRP-1(+) melanocytes after UV exposure.