Normal Research Articles

Effect of Advanced Glycation End-Products (AGE) Lowering Drug ALT-711 on Biochemical, Vascular, and Bone Parameters in a Rat Model of CKD-MBD.

Chronic kidney disease-mineral bone disorder (CKD-MBD) is a systemic disorder that affects blood measures of bone and mineral homeostasis, vascular calcification, and bone. We hypothesized that the accumulation of advanced glycation end-products (AGEs) in CKD may be responsible for the vascular and bone pathologies via alteration of collagen. We treated a naturally occurring model of CKD-MBD, the Cy/+ rat, with a normal and high dose of the AGE crosslink breaker alagebrium (ALT-711), or with calcium in the drinking water to mimic calcium phosphate binders for 10 weeks. These animals were compared to normal (NL) untreated animals. The results showed that CKD animals, compared to normal animals, had elevated blood urea nitrogen (BUN), PTH, FGF23 and phosphorus. Treatment with ALT-711 had no effect on kidney function or PTH, but 3 mg/kg lowered FGF23 whereas calcium lowered PTH. Vascular calcification of the aorta assessed biochemically was increased in CKD animals compared to NL, and decreased by the normal, but not high dose of ALT-711, with parallel decreases in left ventricular hypertrophy. ALT-711 (3 mg/kg) did not alter aorta AGE content, but reduced aorta expression of receptor for advanced glycation end products (RAGE) and NADPH oxidase 2 (NOX2), suggesting effects related to decreased oxidative stress at the cellular level. The elevated total bone AGE was decreased by 3 mg/kg ALT-711 and both bone AGE and cortical porosity were decreased by calcium treatment, but only calcium improved bone properties. In summary, treatment of CKD-MBD with an AGE breaker ALT-711, decreased FGF23, reduced aorta calcification, and reduced total bone AGE without improvement of bone mechanics. These results suggest little effect of ALT-711 on collagen, but potential cellular effects. The data also highlights the need to better measure specific types of AGE proteins at the tissue level in order to fully elucidate the impact of AGEs on CKD-MBD. © 2019 American Society for Bone and Mineral Research.

Trisomy 21 Driven Pro-Inflammatory Signalling in Fetal Bone Marrow May Play a Role in Perturbed B-Lymphopoiesis and Acute Lymphoblastic Leukemia of Down Syndrome

Introduction: Children with Down syndrome (DS) have a markedly increased risk of acute lymphoblastic leukemia (ALL), suggesting that trisomy 21 (T21) has specific effects on hematopoietic stem and progenitor cell (HSPC) biology in early life. Data from human fetal liver (FL) indicates that T21 alters fetal hematopoiesis, causing multiple defects in lympho-myelopoiesis. The impact of T21 on fetal B lymphopoiesis and how this may underpin the increase in ALL is not well known. We have recently found that fetal bone marrow (FBM) rather than FL is the main site of B lymphopoiesis; with a marked enrichment of fetal-specific progenitors (early lymphoid progenitors, ELP and PreProB progenitors) that lie upstream of adult type ProB progenitors (O'Byrne et al, Blood, in press). Previous preliminary data suggested that B progenitors were also reduced in T21 FBM (Roy et al, Blood. 124, 4331). Aim: To dissect putative molecular mechanisms responsible for the defects in T21 FBM B-lymphopoiesis and its association with childhood DS ALL. Methods: Second trimester human FBM and paediatric ALL samples were obtained from the Human Developmental Biology Resource and UK Childhood Leukaemia Cell Bank respectively. Multiparameter flow cytometry/sorting, transcriptome analysis by RNA-sequencing and microarray, and stromal co-culture assays were used to characterize HSPC and mesenchymal stromal cells (MSC) from normal (NM) disomic (n=21-35) and T21 (n=7-12) human FBM; RNASeq was performed on cytogenetically matched non-DS (n=13) and DS ALL (n=7). Results: In contrast to NM FBM, fetal specific progenitors were virtually absent (CD34+CD10-CD19-CD127+ ELP 2.8±0.4% vs. 0.8±0.4% of CD34+ cells) or very severely reduced (CD34+CD10-CD19+ PreProB 12.8±1 vs 2.6±0.7%) in T21 FBM. This was despite a >4-fold increase in the frequency of immunophenotypic HSC (4.2±1.2% vs 0.9±0.2% of CD34+ cells) and similar frequencies of MPP and LMPP in T21 FBM. As in adult BM, the vast majority of B progenitors in T21 FBM were CD34+CD10+CD19+ ProB progenitors with a frequency (28.8±8.3%) similar to NM FBM (30.3±2.3% of CD34+ cells). Thus, T21 causes a severe block in B-progenitor commitment at the LMPP stage, in tandem with a compensatory expansion of ProB progenitors. Consistent with this, T21 FBM HSC, MPP and LMPP had reduced B cell potential in vitro compared to NM FBM in MS5 co-cultures. RNAseq of NM (n=3) and T21 (n=3) FBM HSPC demonstrated global transcriptomic disruption by T21, with increased gene expression in HSC, MPP, LMPP and ProB progenitors. Cell cycle genes were enriched in T21 ProB progenitors. Despite these functional and global gene expression differences, expression of key B-lineage commitment genes was maintained suggesting the defect in B-lymphopoiesis may be secondary to lineage skewing of multipotent progenitors towards a non-B lymphoid fate and/or mediated by extrinsic factors. GSEA pointed to a role for multiple inflammatory pathways in T21 hematopoiesis with dysregulation of IFNα, IL6 and TGFβ signalling pathways in T21 HSC/LMPP. To investigate the role of the T21 microenvironment, we co-cultured NM HSC, MPP and LMPP with T21 or NM primary FBM MSC. T21 FBM MSC (n=3) had reduced capacity to support B cell differentiation in vitro consistent with perturbation of MSC function by T21. Similar to T21 FBM HSPC, transcriptomic analysis of T21 FBM MSC by microarray showed enrichment for IFNα signalling compared to NM; and T21 HSPC and MSC both showed increased gene expression for IFNα receptors IFNAR1 and IFNAR2, which are encoded on chromosome 21. Since IFNα was undetectable by ELISA of conditioned media from NM and T21 MSC, differences in secreted IFNα from MSC are unlikely to fully explain the increased IFN signalling in T21 HSPC and MSC. This suggests that T21 may drive autocrine rather than paracrine IFN signalling in FBM cells. Finally, RNASeq showed perturbed inflammatory signalling in DS ALL compared to non-DS ALL, suggesting a role for T21-driven inflammatory pathways in the biology of DS ALL. Conclusions: These data show that T21 severely impairs B lymphopoiesis in FBM and is associated with expression of proinflammatory gene expression programs in T21 FBM HSPC and MSC and DS ALL. The compensatory expansion of T21 FBM ProB progenitors, through self-renewal or via an alternative differentiation pathway; with concomitant T21-driven proinflammatory signalling may underpin the increased risk of B progenitor ALL in childhood. Disclosures No relevant conflicts of interest to declare.

HDAC8 Promotes MLL-Rearranged Acute Myeloid Leukemia By Inhibiting p53 Acetylation

Chromosomal rearrangements involving the mixed lineage leukemia (MLL) gene at the 11q23 locus are found in a subset of acute myeloid leukemia (AML) generally associated with intermediate to poor prognosis. MLL-AF9 resulting from t(9;11)(p22;q23) is the most common in MLL-rearranged (MLL-r) AML. Chemotherapy is the current standard of care for AML and the overall 5-year survival rate is only 25%. We previously reported that HDAC8 functions to control p53 activity in hematopoietic stem cells via deacetylation of p53 protein (Hua et al, Blood 2017). Here, we investigated the contribution of HDAC8 in MLL-r AML pathogenesis and maintenance. We have previously generated a conditional Hdac8 floxed (f) allele and combined with Mx1-Cre (Mx1-Cre/Hdac8f/f(y)) to generate HDAC8△/△ mice upon induction with poly (I:C). To examine the role of HDAC8 in MLL-AF9-AML, we transduced Hdac8△/△ or control LSK (Lin-Sca1+cKit+) with a MSCV-ires-GFP (MIG)-MLL-AF9 (MA9) vector and sorted GFP+ cells for CFC assay. Hdac8△/△/MA9 cells generated significantly less CFC colonies compared to control, suggesting that HDAC8 promotes MA9 clonogenic activity. To assess their leukemogenic potential in vivo, we transplanted GFP+ control MA9 or Hdac8△/△/MA9 cells (5x103-104) into sublethally irradiated (6 Gy) wild-type (WT) recipients. All MA9 recipients (n=4) developed lethal AML with a median survival of 69 days while none of the Hdac8△/△/MA9 recipients (n=4) developed AML up to 4 months. To examine the contribution of HDAC8 in established AML, we transduced LSK from uninduced Hdac8f/f(y)/Mx1-Cre mice with MIG-MA9 and transplanted into WT recipients. Upon development of moribund leukemia, AML cells (5x 103) were transplanted into a cohort of recipients (n=16), which were treated with poly (I:C) (n=8; 14 mg/kg/dose) or PBS every other day for 2 weeks starting at day 10 (5-10% GFP+ in blood). Mice induced with poly (I:C) to delete Hdac8 showed dramatically lower white blood count [9.0 (103 /ul) vs. 109.7 (103 /ul); n=8, P<0.05] and lower AML burden (GFP+) in peripheral blood (9.3% vs 42.3%; n=8, P<0.05) at 3 weeks. Prolonged survival was observed in poly (I:C) treated mice compared to control (median survival 36 days vs 27 days, n=8, P<0.01). These results indicate that HDAC8 deletion impairs AML progression. Thus, we examined the effects of HDAC8-selective inhibitor (HDAC8i) on MA9 AML cell proliferation and survival. Treatment of MA9 AML cells with HDAC8i (22d) led to dose-dependent inhibition of cell survival (IC50 = 4.724 - 8.453 mM) and increased apoptosis (10 mM, 48 h) compared to vehicle treated control (40% vs. 7%). HDAC8i treatment significantly enhanced p53 acetylation, and upregulated p53 target genes, Gadd45d (fold change = 3.2; p< 0.01), Puma (fold change = 5.7; p< 0.01), Mdm2 (fold change = 3.0; p< 0.01) and Cdkn1a (fold change = 3.6; p< 0.01). To assess the effects on AML growth and leukemia-initiating capacity in vivo, we transplanted MA9 AML cells treated with HDAC8i (1 x 105, 2.5x105) or vehicle (2.5x105) into sublethally irradiated WT recipients (n=8-11). We observed significantly (p<0.0001) reduced GFP+ AML burden in recipients of HDAC8i-treated cells (both 1x105 & 2 x105) and significantly (p<0.0001) improved survival. Secondary transplantation of AML cells from HDAC8i- or vehicle-treated (2 x105) recipients revealed significantly improved survival in HDAC8i-treated group (22d 105 days n=4 vs. vehicle 33 days n=5; p< 0.01). These results indicate that HDAC8 inhibition enhanced elimination of MA9 AML cells and leukemia-initiating cells. We further evaluated the effect of HDAC8i in human AML cell lines and primary MLL-r AML patient cells. HDAC8i treatment increased p53 acetylation and enhanced p53-dependent apoptosis in MOLM-13 AML cells (22d: 50.8% vs. vehicle: 11.89%). Knock-down of p53 rescued the apoptosis induced by HDAC8i, indicating that HDAC8i induced apoptosis is mediated by p53. Similarly, HDAC8i (22d) treatment significantly increased apoptosis accompanied by elevated levels of p53 acetylation and p53 target gene expression in MLL-r AML CD34+ cells compared to normal (NL) CD34+ cells. Thus, HDAC8 inhibition targets human MLL-r AML cells and AML CD34+ cells by restoring p53 acetylation and inducing apoptosis. In conclusion, this study reveals a critical role of HDAC8 in promoting initiation and maintenance of MLL-AF9 AML and highlights HDAC8 inhibition as a promising approach to target MLL-r AML. Disclosures No relevant conflicts of interest to declare.

Evaluation of sweet cherry fruit quality after short-term storage in relation to the rootstock

Fruits of the sweet cherry cultivar ‘Regina’ collected from trees growing on seven rootstocks were stored in a cold room at 2 °C with a normal (NA) and controlled atmosphere (15% and 20% CO2 and 5% O2—CA1 and CA2) for 2 weeks. The rootstocks on which the trees grew and the storage conditions significantly affected all fruit parameters tested during both years of the experiment. Fruit from Damil rootstock exhibited higher mean firmness than fruit from Colt rootstock. The effect of rootstocks on the value of soluble solids content (SSC) varied, wherein the fruits from Tabel Edabriz and Damil were characterized by high SSC mean content. The organic acids content (TA) was significantly lower after storage than during harvest time. Fruits from Tabel Edabriz trees were characterized by faster ripening, as was evident by the higher SSC to TA ratio. The amount of mass lost depended significantly and only on the storage conditions—sweet cherries from both CA combinations had the lowest mass losses. The percentage of fruits showing disease symptoms was largely dependent on the weather conditions in the orchard the year before the fruit harvest, as well as atmosphere composition and RH during fruit storage. Cold storage conditions with a high (20%) CO2 content are recommended for the short-term storage of sweet cherry fruits because they preserve fruit quality parameters: a low decrease in firmness, maintenance of a high SSC/TA ratio, a low percent of fungal infections, and good preservation of green color in the peduncle.

Impact of CYP2C19 genotype on sertraline exposure in 1200 Scandinavian patients.

Sertraline is an (SSRI-)antidepressant metabolized by the polymorphic CYP2C19 enzyme. The aim of this study was to investigate the impact of CYP2C19 genotype on the serum concentrations of sertraline in a large patient population. Second, the proportions of patients in the various CYP2C19 genotype-defined subgroups obtaining serum concentrations outside the therapeutic range of sertraline were assessed. A total of 2190 sertraline serum concentration measurements from 1202 patients were included retrospectively from the drug monitoring database at Diakonhjemmet Hospital in Oslo. The patients were divided into CYP2C19 genotype-predicted phenotype subgroups, i.e. normal (NMs), ultra rapid (UMs), intermediate (IMs), and poor metabolisers (PMs). The differences in dose-harmonized serum concentrations of sertraline and N-desmethylsertraline-to-sertraline metabolic ratio were compared between the subgroups, with CYP2C19 NMs set as reference. The patient proportions outside the therapeutic concentration range were also compared between the subgroups with NMs defined as reference. Compared with the CYP2C19 NMs, the sertraline serum concentration was increased 1.38-fold (95% CI 1.26-1.50) and 2.68-fold (95% CI 2.16-3.31) in CYP2C19 IMs and PMs, respectively (p < 0.001), while only a marginally lower serum concentration (-10%) was observed in CYP2C19 UMs (p = 0.012). The odds ratio for having a sertraline concentration above the therapeutic reference range was 1.97 (95% CI 1.21-3.21, p = 0.064) and 8.69 (95% CI 3.88-19.19, p < 0.001) higher for IMs and PMs vs. NMs, respectively. CYP2C19 IMs and PMs obtain significantly higher serum concentrations of sertraline than NMs. Based on the relative differences in serum concentrations compared to NMs, dose reductions of 60% and 25% should be considered in PMs and IMs, respectively, to reduce the risk of sertraline overexposure in these patients.

296P - Patient-tailored tamoxifen dosing based on an increased quantitative understanding of its complex pharmacokinetics: A novel integrative modelling approach

BackgroundAbout 20% of breast cancer (BC) patients do not attain tamoxifen’s (TAM) active metabolite endoxifen (ENDX) target concentrations when receiving 20mg TAM once daily (q.d.). Thus, individualised dosing of up to 120mg TAM q.d. for ENDX target attainment (TA) has been proposed. Combining concentrations and antiestrogenic activities of ENDX and its 3 precursors, the antiestrogenic activity score (AAS) has been developed as alternative TA metric. We aimed to integrate experimental and clinical data from diverse sources in an innovative modelling approach to identify patient groups at risk of treatment failure and to assess whether ENDX- or AAS-guided dosing would be more favourable for TA applying in silico simulation. MethodsIn vitro data from enzyme kinetic experiments, pharmacokinetic (PK) parameters from previous clinical studies and in vivo data from three clinical trials were synthesised into a minimal nonlinear mixed-effects physiologically-based pharmacokinetic (NLME-PBPK) model. Using simulation, lowest doses needed for TA, applying either the ENDX (≥5.97ng/mL) or the AAS (≥1798) threshold, were investigated in a representative virtual BC population with various CYP2D6 activity scores (AS) and age. ResultsThe developed NLME-PBPK model captured individual TAM and 3 metabolite concentration profiles from 406 BC patients well. Bioactivation to ENDX was 4.5-fold higher in CYP2D6 normal (NM) than in poor metabolisers (PM). Patients with low CYP2D6 activity and young age showed highest risks for ENDX non-TA. Among all patients, 76% received the same dose irrespective of the TA metric used. For the remaining 23% with different dose selections, applying the AAS instead of the ENDX target, TA increased in NM and intermediate metabolisers (IM) (+21.7% and +6.9%, respectively), while TA decreased in PM (-11.7%). ConclusionsOur modelling approach combined pharmacogenetic factors, physiological changes and variability on PK parameters in a quantitative manner and allowed to translate PK information on TAM and its three major metabolites into individualised dosing. While ENDX-guided dosing was preferable for PM in our simulation, AAS-guided dosing was superior for NM and IM. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureL. Klopp-Schulze: Full / Part-time employment: Merck Healthcare KGaA. S.L. Koolen: Speaker Bureau / Expert testimony, Research grant / Funding (institution): Novartis; Speaker Bureau / Expert testimony: Pfizer; Speaker Bureau / Expert testimony: Roche; Research grant / Funding (institution): Astellas; Research grant / Funding (institution): Cristal Therapeutics; Travel / Accommodation / Expenses: Ipsen. R.H.J. Mathijssen: Research grant / Funding (institution), Travel / Accommodation / Expenses: Astellas; Research grant / Funding (institution): Bayer; Research grant / Funding (institution): Boehringer; Research grant / Funding (institution): Cristal Therapeutics; Advisory / Consultancy, Research grant / Funding (institution): Novartis; Research grant / Funding (institution): Pamgene; Research grant / Funding (institution): Roche; Research grant / Funding (institution): Sanofi; Research grant / Funding (institution), Travel / Accommodation / Expenses: Pfizer; Advisory / Consultancy: Servier. C. Kloft: Research grant / Funding (institution), Grants received outside the submitted work: DDMoRe; Research grant / Funding (institution), Grants received outside the submitted work: Industry consortium (AbbVie Deutschland GmbH & Co. KG, Boehringer Ingelheim Pharma GmbH & Co. KG, Gruenenthal GmbH, F. Hoffmann-La Roche Ltd., Merck KGaA and SANOFI); Research grant / Funding (institution), Grants received outside the submitted work: Federal Ministry of Education and Research; Research grant / Funding (institution), Grants received outside the submitted work: Diurnal Ltd. All other authors have declared no conflicts of interest.

Dysregulation of H11 Kinase in the Failing Left Ventricular Myocardium

Background: Increased expression of H11 kinase (H11K) has been observed in Left Ventricular (LV) myocardium of failed human hearts. Its potential role as a contributor to the progression of Heart Failure (HF) remains uncertain. In the present study we examined the expression of H11K in cytosolic and mitochondrial fractions of failing human and dog LV myocardium and assessed its interaction with Akt (cell-survival enzyme) and p38MAPK (programmed cell death enzyme). Methods: Total RNA and Sodium-Dodecyl Sulfate (SDS) extracts were prepared from homogenate of LV specimens of 6 dogs with intracoronary microembolization-induced HF, 6 normal (NL) dogs, 7 explanted failed human hearts due to Idiopathic Dilated Cardiomyopathy (IDC), 7 failed human hearts due to Ischemic Cardiomyopathy (ICM) and 7 non-failing human donor hearts (DNR). SDS extracts were also prepared from cytosolic and mitochondrial fractions isolated from LV specimens. Results: H11K mRNA and protein levels normalized to GAPDH increased significantly in LV tissue from ICM and IDC hearts compared to DNR hearts and in HF dogs compared with NL dogs. H11K protein levels increased in cytosolic fractions but decreased in mitochondrial fractions of both failed human and dog hearts compared to DNR hearts and NL dog hearts. Immunoprecipitation studies in specimens from HF dogs showed that H11K cytosolic fractions interacted predominantly with p38MAPK and least with Akt when compared with NL dogs. Conclusions: Enhanced interaction of H11K with p38MAPK in HF can promote cell death thus contributing to progressive LV dysfunction. Therapeutic modalities that restore interaction of H11K with Akt and augment H11K translocation to mitochondria can potentially and partially reverse the progression of LV dysfunction by promoting cell survival.

A Fully Automatic Framework for Parkinson's Disease Diagnosis by Multi-Modality Images.

BackgroundParkinson’s disease (PD) is a prevalent long-term neurodegenerative disease. Though the criteria of PD diagnosis are relatively well defined, current diagnostic procedures using medical images are labor-intensive and expertise-demanding. Hence, highly integrated automatic diagnostic algorithms are desirable.MethodsIn this work, we propose an end-to-end multi-modality diagnostic framework, including segmentation, registration, feature extraction and machine learning, to analyze the features of striatum for PD diagnosis. Multi-modality images, including T1-weighted MRI and 11C-CFT PET, are integrated into the proposed framework. The reliability of this method is validated on a dataset with the paired images from 49 PD subjects and 18 Normal (NL) subjects.ResultsWe obtained a promising diagnostic accuracy in the PD/NL classification task. Meanwhile, several comparative experiments were conducted to validate the performance of the proposed framework.ConclusionWe demonstrated that (1) the automatic segmentation provides accurate results for the diagnostic framework, (2) the method combining multi-modality images generates a better prediction accuracy than the method with single-modality PET images, and (3) the volume of the striatum is proved to be irrelevant to PD diagnosis.

Direct and indirect effects of environmental factors on daily CO2 exchange in a rainfed maize cropland—A SEM analysis with 10 year observations

The carbon budget of agricultural ecosystems is of great importance to the global carbon balance. In this study, the structural equation modeling (SEM) method was employed to quantify the direct and indirect effects of environmental factors on daily net ecosystem CO2 exchange (NEE) in a rainfed maize cropland, northeast China. The results showed that net radiation (Rn) was the most important factor controlling daily NEE, followed by the leaf area index (LAI), air temperature (Ta), vapor pressure deficit (VPD), and clearness index (Kt). The strong effect of Rn was mainly attributed to its direct effect, while Ta and VPD showed comparable or even higher indirect effects than direct effects, indicating that Ta and VPD influenced NEE mainly through regulating canopy development on a daily scale. Moreover, the responses of NEE to individual environmental factors differed greatly among multiyear climatic conditions. Ta and VPD were shown to be more important in warm (WM) years and warm and dry (WD) years than in normal (NM) years and cold and wet (CW) years. LAI was the primary controlling factor of daily NEE in WD years, resulting in large indirect effects of Ta and VPD on NEE. Kt had a large effect on daily NEE only in CW years. SWC had significant effects on daily NEE in WM and WD years but in the opposite direction, i.e., daily NEE increased with SWC in WD years but decreased with SWC in CW years. The partitioning of direct and indirect effects of environmental factors with SEM can greatly enhance the understanding of the controlling mechanism of NEE and remind researchers to consider the distinct effects of environmental factors on NEE among multiyear climatic conditions in future models.

Abstract 278: Reduced HtrA2 Protein Levels in Mitochondria but Elevated Levels in Cytosol of Left Ventricular Myocardium of Dogs with Chronic Heart Failure

Background: The high-temperature requirement serine peptidase 2 (HtrA2) is a pro-apoptotic mitochondrial serine protease involved in caspase-dependent as well as caspase-independent programmed cell death. When HtrA2 protein is tightly associated with mitochondria (MITO), its protease activity is masked. In contrast, once the protein is released from MITO into the cytosol (CS), its protease activity is exposed. Once active HtrA2 activates caspase-3, a pro-apoptotic enzyme, by degrading the caspase-3 inhibitor XIAP (x-linked inhibitor of apoptosis protein. Recent studies have shown that translocation of HtrA2 protein from the MITO to the CS occurs in mice hearts during experimental myocardial ischemic and reperfusion. Hypothesis: In this study we examined if translocation of HtrA2 from MITO to CS occurs in LV myocardium of dogs with chronic stable heart failure (HF). Methods: LV tissue from 7 dogs with chronic HF produced by multiple sequential intracoronary micro-embolizations (LV ejection fraction &lt;35%) and 7 normal (NL) dogs was used to prepare crude homogenate (HG) and to isolate MITO and CS fractions. Using primary antibodies, HtrA2 protein was determined in HG, MITO and CS fractions by Western blotting coupled with chemiluminescence. GAPDH was used as an internal loading control for both HG and CS fractions. Porin was used as an internal loading control for MITO fractions. HtrA2 band intensity was normalized to GAPDH for HG and CS and to porin for MITO. Results: GAPDH and porin protein levels were unchanged between NL and HF dogs. Compared to NL, HtrA2 protein levels in dogs with HF were unchanged in HG (0.76 ± 0.04 vs. 0.73 ± 0.08), significantly reduced in MITO (0.17 ± 0.02 vs. 0.40 ± 0.05, p&lt;0.05) and significantly increased in CS (0.90 ± 0.07 vs. 0.36 ± 0.05, p&lt;0.05). Conclusions: Compared to NL dogs, HtrA2 protein levels are significantly reduced in MITO but increased in cytosol fractions from LV myocardium of dogs with chronic stable HF; a maladaptations that promotes cardiomyocyte apoptosis and progressive LV dysfunction. Inhibiting the translocation of HtrA2 from MITO to CS may have therapeutic potential for the preventing progressive dysfunction of the failing heart.

The absence of lipoxygenase and 7S globulin of soybeans and heating temperatures on the properties of soymilks and soy yogurts

The aim of this study was to produce soy yogurt and investigate the physicochemical, textural, and volatile flavor compounds, as well as sensory characteristics using normal (NOR), lipoxygenase-lacking (LOX-lack), and 7S-globulin lacking (7S-lack) soybeans at different temperatures. The soy yogurts were produced by Streptococcus thermophilus and Lactobacillus delbruekii subsp. bulgaricus fermentation of the soymilk. SDS-PAGE results showed that the globulin, subunit, and polypeptide contents of soymilk were affected significantly by the absence of LOX and 7S. Heat treatment accelerated the protein aggregation of soymilk but the content of large non-disulfide aggregates was lower for LOX-lack than for NOR and 7S-lack. As a consequence, the gel firmness and storage modulus of LOX-lack soy yogurt were the highest among others during the heat treatment (80–115 °C), and its network structure was the most compact under CSLM observation. The LOX-lack soy yogurt had the lowest off-flavor compounds and the best sensory acceptability, which offered the best potential for producing soy yogurt with improved textural and volatile profiles.

Skeletal levels of bisphosphonate in the setting of chronic kidney disease are independent of remodeling rate and lower with fractionated dosing.

Chronic kidney disease (CKD) results in a dramatic increase in skeletal fracture risk. Bisphosphates (BP) are an effective treatment for reducing fracture risk but they are not recommended in advanced CKD. We have recently shown higher acute skeletal accumulation of fluorescently-tagged zoledronate (ZOL) in the setting of CKD but how this accumulation is retained/lost over time is unclear. Furthermore, it is unknown if alternative dosing approaches can modulate accumulation in the setting of CKD. To address these two questions normal (NL) and Cy/+ (CKD) rats were divided into control groups (no dosing), a single dose of a fluorescent-tagged ZOL (FAM-ZOL), a single dose of non-labelled zoledronate (ZOL) or ten weekly doses of FAM-ZOL each at 1/10th the dose of the single dose group. Half of the CKD animals in each group were provided water with 3% calcium in drinking water (CKD + Ca) to suppress PTH and remodeling. At 30 or 35 weeks of age, serum, tibia, ulna, radius, vertebra, femora, and mandible were collected and subjected to assessment methods including biochemistry, dynamic histomorphometry and multi-spectral fluorescence levels (using IVIS SpectrumCT). FAM-ZOL did not significantly reduce bone remodeling in either NL or CKD animals while Ca supplementation in CKD produced remodeling levels comparable to NL. At five- and ten-weeks post-dosing, both CKD and CKD + Ca groups had higher levels of FAM-ZOL in most, but not all, skeletal sites compared to NL with no difference between the two CKD groups suggesting that the rate of remodeling did not affect skeletal retention of FAM-ZOL. Fractionating the FAM-ZOL into ten weekly doses led to 20-32% less (p < 0.05) accumulation/retention of compound in the vertebra, radius, and ulna compared to administration as a single dose. The rate of bone turnover does not have significant effects on levels of FAM-ZOL accumulation/retention in animals with CKD. A lower dose/more frequent administration paradigm results in lower levels of accumulation/retention over time. These data provide information that could better inform the use of bisphosphonates in the setting of CKD in order to combat the dramatic increase in fracture risk.

Is There a Relationship Between Vitamin D Deficiency Status and PCOS in Infertile Women?

Introduction It is still unclear in the literature whether low vitamin D levels play a role in the pathogenesis of polycystic ovary syndrome (PCOS), especially with respect to the regulation of anti-Müllerian hormone (AMH). Therefore, we aimed to investigate whether there could be a relationship between vitamin D deficiency status and PCOS. Materials and Methods A total of 146 infertile women were divided into two groups according to their ovarian reserve patterns: (i) normal (NOR), and (ii) high (PCOS). The participants were also categorized into two groups according to vitamin D concentrations: (i) Group A < 10 ng/mL, and (ii) Group B 10 – 20 ng/mL. Samples were obtained and analyzed for estradiol (E2), follicle-stimulating hormone (FSH), luteinizing hormone (LH), total testosterone (TT), 17-hydroxyprogesterone (17-OHP), dehydroepiandrosterone sulfate (DHEA-S) and AMH. Results In the NOR group, there were significant differences between Group A and Group B in terms of anthropometric characteristics (p < 0.05, for all). The women in both Group A and Group B had similar AMH concentrations (p > 0.005). Only the NOR group showed a significant though moderate negative correlation between 25(OH)D levels and anthropometric parameters. AMH levels were not correlated with 25(OH)D levels in the NOR or the PCOS group (r = − 0.112, p = 0.008; r = 0.027, p = 0.836). Multivariate regression analysis showed no impact of 25(OH)D on other study parameters. Only AMH measurements were significant enough (p < 0.001) to differentiate between PCOS and NOR patterns. Conclusion We found no difference in serum 25(OH)D and AMH levels between women with and women without PCOS. No correlation could be demonstrated between 25(OH)D and AMH in the PCOS group or controls.

The Influence of Fin Thread Implant Design on Stress Distribution Comparative FEA Study

Background: one of the requirements of long term dental implant success lies within the implant structure and bone- implant interface ability to cope with functional stress. Dental implant ability to withstand different occlusal loads optimizes implant-supported prosthesis function, minimize stress over the surrounding bone, improve osseointegration and reduce the required peri od for implant loading. One of the areas of research on implant geometry is the thread design. Threads increase initial bone contact, enhance implant stability, maximize implant surface area with subsequent improvement of osseointegration. Recently, Fin Thread (FT) and Modified Fin Thread designs (IBS®) have been introduced. These designs on TiG5 dental implant did not seem to receive adequate attention in the current literature. Methods: four dental implant model s (Modified Fin Thread, Fin Thread, V-shaped and Buttress designs) of TiG5 were tested using FEA for stress distributi on using normal and extreme static occlusal loads on 0, 15 and 25ο angles. Results: Modified Fin Thread (MFT) desig n and Fin Thread (FT) design showed almost uniform stress distribution compared to other models under normal occlu sal load. However, MFT design showed better stress distribution with no cortical bone involvement in overloaded occlusal stress (200 and 400N) respectively. Conclusion: Modified Fin Thread Design of Ti G5 seems to be the more suitable dental implant model in terms of stress distribution in norm al and over occlusal load conditions. MFT designs need clinical studies to support this study findings on patients with long fol low up periods.

Classifying heart conditions based on class probability output networks

This paper presents a novel method of classifying heart conditions from an electrocardiography (ECG) signal. For this purpose, the R-R intervals of ECG signal are analyzed by Gamma distribution parameters and classified into normal (NR) or abnormal (AN) ECG waves. For the normal ECG waves, the heart condition is further investigated by analyzing the dynamic behavior of heart activity based on the correlation between successive R-R intervals and long-term analysis. The classification of heart conditions is made by estimating the conditional class probabilities using class probability output networks (CPONs). The simulation for classifying heart conditions using the MIT-BIH data sets reveals that the proposed approach is effective for classifying heart conditions and allows more accurate classification than the existing classifiers such as the k-NN and SVM.

A New Classification of Benign, Premalignant, and Malignant Endometrial Tissues Using Machine Learning Applied to 1413 Candidate Variables.

Benign normal (NL), premalignant (endometrial intraepithelial neoplasia, EIN) and malignant (cancer, EMCA) endometria must be precisely distinguished for optimal management. EIN was objectively defined previously as a regression model incorporating manually traced histologic variables to predict clonal growth and cancer outcomes. Results from this early computational study were used to revise subjective endometrial precancer diagnostic criteria currently in use. We here use automated feature segmentation and updated machine learning algorithms to develop a new classification algorithm. Endometrial tissue from 148 patients was randomly separated into 72-patient training and 76-patient validation cohorts encompassing all 3 diagnostic classes. We applied image analysis software to keratin stained endometrial tissues to automatically segment whole-slide digital images into epithelium, cells, and nuclei and extract corresponding variables. A total of 1413 variables were culled to 75 based on random forest classification performance in a 3-group (NL, EIN, EMCA) model. This algorithm correctly classifies cases with 3-class error rates of 0.04 (training set) and 0.058 (validation set); and 2-class (NL vs. EIN+EMCA) error rate of 0.016 (training set) and 0 (validation set). The 4 most heavily weighted variables are surrogates of those previously identified in manual-segmentation machine learning studies (stromal and epithelial area percentages, and normalized epithelial surface lengths). Lesser weighted predictors include gland and lumen axis lengths and ratios, and individual cell measures. Automated image analysis and random forest classification algorithms can classify normal, premalignant, and malignant endometrial tissues. Highest predictive variables overlap with those discovered independently in early models based on manual segmentation.

Multiparameter probability distributions for at-site frequency analysis of annual maximum wind speed with L-Moments for parameter estimation

Estimation of quantiles of Annual Maximum Wind Speed (AMWS) is needed in different environmental fields, engineering risk analysis, design of structures, renewable energy sources, agricultural operations, and climatology. Therefore, wind speed frequency analysis (WSFA) was carried out at nine stations from Pakistan. Multiparameter Probability Distributions (PDs), such as Generalized logistic (GLO), Generalized Extreme Value (GEV), Generalized Normal (GNO), Generalized Pareto (GPA), Weibull (WEI), Pearson type 3 (P3), Log Pearson type 3 (LP3); and two parameter PDs, such as Logistic (LOG), Normal (NOR), Gumbel (GUM), Exponential (EXP), and Uniform (UNI) were used to determine the most suitable distributions for the nine stations. The method of L-moments was used for estimating parameters of the distributions. The Kolmogorov-Smirnov (KS) test, Anderson-Darling (AD) test, Minimum L-Kurtosis (ML-K) Difference Criterion, and L-moment ratio diagram (L-ratio diagram) showed that four distributions, namely GEV, GNO, GPA, and GLO were the most suitable distributions for different stations and were superior to the two-parameter distributions. The quantile estimates (design estimates) from multiparameter PDs provide information on how fast the maximum wind will pass through a certain place and hence are important for policy makers and planners in the design and construction of different structures. The Multivariate Diebold–Mariano (DM) test was applied to check the accuracy of design estimates from the best fitted PDs and results indicated that they were significantly different.

Automatic detection of retinal regions using fully convolutional networks for diagnosis of abnormal maculae in optical coherence tomography images.

.In conventional retinal region detection methods for optical coherence tomography (OCT) images, many parameters need to be set manually, which is often detrimental to their generalizability. We present a scheme to detect retinal regions based on fully convolutional networks (FCN) for automatic diagnosis of abnormal maculae in OCT images. The FCN model is trained on 900 labeled age-related macular degeneration (AMD), diabetic macular edema (DME) and normal (NOR) OCT images. Its segmentation accuracy is validated and its effectiveness in recognizing abnormal maculae in OCT images is tested and compared with traditional methods, by using the spatial pyramid matching based on sparse coding (ScSPM) classifier and Inception V3 classifier on two datasets: Duke dataset and our clinic dataset. In our clinic dataset, we randomly selected half of the B-scans of each class (300 AMD, 300 DME, and 300 NOR) for training classifier and the rest (300 AMD, 300 DME, and 300 NOR) for testing with 10 repetitions. Average accuracy, sensitivity, and specificity of 98.69%, 98.03%, and 99.01% are obtained by using ScSPM classifier, and those of 99.69%, 99.53%, and 99.77% are obtained by using Inception V3 classifier. These two classification algorithms achieve 100% classification accuracy when directly applied to Duke dataset, where all the 45 OCT volumes are used as test set. Finally, FCN model with or without flattening and cropping and its influence on classification performance are discussed.

Masticatory function in complete denture wearers varying degree of mandibular bone resorption and occlusion concept: canine-guided occlusion versus bilateral balanced occlusion in a cross-over trial

PurposeThe present cross-over study evaluated the masticatory function and maximum occlusal force of edentulous patients with complete dentures with bilateral balanced occlusion (BBO) and canine guidance (CG), with normal (NR) and resorbed mandibular ridges (RR). MethodsParticipants received new complete dentures and both occlusal concepts (BBO and CG) were applied for a period of 30 days each. The masticatory performance was evaluated with the sieving method; masticatory ability with a visual analog scale (VAS) and maximum occlusal force was assessed by means of a gnatodynamometer. Data were analyzed using repeated-measure ANOVA or Generalized Estimating Equations (GEEs), α = 0.05. ResultsThirty women completed the trial. The height of mandibular ridge was significant for the masticatory performance (ANOVA, p < 0.001, NR = 46.35 ± 12.18%, RR = 30.39 ± 9.94%), regardless of the occlusion guidance. A significant effect of the occlusion guidance was observed for the maximum occlusal force (ANOVA, p = 0.021, CG = 31.99 ± 12.74 N, BBO = 28.37 ± 9.83 N). The occlusion guidance was not significant for the masticatory performance (ANOVA, p = 0.156) and the height of the mandibular ridge did not influence on the occlusal force (ANOVA, p = 0.060). The interaction of the factors (guidance × ridge) was not significant for masticatory performance (ANOVA, p = 0.184) and occlusal force (ANOVA, p = 0.236). The VAS scores showed a significant effect of the ridge on chewing of lettuce, resulting in greater ease (GEEs, p = 0.016) and chewing quality (GEE, p = 0.028) of this food for participants with resorbed ridges. Participants with CG reported greater ease of chewing fresh bread and beef, and expressed higher quality of chew raw carrot. Participants with CG and normal ridges exhibited the highest overall chewing ability, in comparison to BBO (Bonferroni, p < 0.05, CG = 91.9, BBO = 72.7). ConclusionsCG represents a viable alternative to the BBO in complete dentures with satisfactory clinical results related to the self-perception of chewing and occlusal force.

SUN-553 Atypical Spindle Cells on a Thyroid Aspirate: A Rare Case of a Thyroid Schwannoma Presenting as a Progressively Expanding Thyroid Nodule

Background: Mesenchymal tumors represent around 1% of all thyroid neoplasms. Schwannoma is one such tumor that arises from the peripheral nerve sheath within the thyroid gland. Case: A 40-year-old lady was referred to our center for evaluation of a thyroid mass that was initially noticed 2 years prior and had been rapidly enlarging over the past several months. The patient experienced occasional dysphagia, but no shortness of breath nor change in voice. There was no family history of thyroid cancer or exposure to head and neck radiation. On physical exam, a firm, non-tender, 3 cm nodule was palpated in the right thyroid lobe. There was no cervical lymphadenopathy. Pemberton sign was negative. There was no evidence of café-au-lait spots or cutaneous neurofibromas on skin exam. The thyroid function tests were normal (NL), with serum TSH of 2.06 µIU/mL (NL: 0.27 - 4.2) and free thyroxine of 1.2 ng/dL (NL: 0.9 - 1.7), anti-TPO antibody was elevated (483 IU/mL; NL: 0 - 34.9), serum calcitonin was undetectable (<5 pg/ml, NL <= 7.6 pg/ml), and serum PTH was NL (29.8 pg/ml; NL: 15 - 65). Thyroid ultrasound (US) revealed a solid, heterogeneous, hypoechoic, vascularized nodule, with regular borders and no microcalcifications, measuring 3.2cm x 1.7cm x 3.9cm - consistent with intermediate suspicion for malignancy per the American Thyroid Association risk stratification. There was no cervical lymphadenopathy. A CT scan of the neck revealed the mass causing mild tracheal deviation. A fine needle aspiration biopsy of the nodule revealed atypical spindle cells with hyperchromatic elongated nuclei and rare colloid, representing a spindle cell neoplasm. The patient underwent total thyroidectomy, right central neck lymph node dissection and a right cervical thymectomy. Histopathology was consistent with thyroid schwannoma characterized by a low Ki67 proliferation index, along with background Hashimoto’s thyroiditis. Immunohistochemical staining showed that the tumor cells were focally positive for TTF-1, negative for thyroglobulin and calcitonin and diffusely positive for S-100 and vimentin, further supporting the diagnosis of a schwannoma. Next generation sequencing of the DNA and RNA derived from tumor tissue using Oncomine Comprehensive Assay v3 detected NF2 p.Leu505fs frameshift mutation. The post-operative course was uneventful, and the patient was discharged on replacement levothyroxine. Upon 8 months follow-up, the patient continued to do well without any symptoms. Conclusion: Thyroid schwanommas are exceptionally rare, with around 20 cases reported to this day. Schwannomas may progressively enlarge causing compressive symptoms and mimicking more aggressive spindle cell tumors such as spindle cell papillary, medullary or anaplastic thyroid cancer and thyroid sarcomas. Prompt imaging and cytologic evaluation with appropriate ancillary studies is crucial in the management of these tumors.