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Background Due to unmet need for bone augmentation, our aim was to promote osteogenic differentiation of human adipose stem cells (hASCs) encapsulated in gellan gum (GG) or collagen type I (COL) hydrogels with bioactive glass (experimental glass 2-06 of composition [wt-%]: Na2O 12.1, K2O 14.0, CaO 19.8, P2O5 2.5, B2O3 1.6, SiO2 50.0) extract based osteogenic medium (BaG OM) for bone construct development. GG hydrogels were crosslinked with spermidine (GG-SPD) or BaG extract (GG-BaG). Methods Mechanical properties of cell-free GG-SPD, GG-BaG, and COL hydrogels were tested in osteogenic medium (OM) or BaG OM at 0, 14, and 21 d. Hydrogel embedded hASCs were cultured in OM or BaG OM for 3, 14, and 21 d, and analyzed for viability, cell number, osteogenic gene expression, osteocalcin production, and mineralization. Hydroxyapatite-stained GG-SPD samples were imaged with Optical Projection Tomography (OPT) and Selective Plane Illumination Microscopy (SPIM) in OM and BaG OM at 21 d. Furthermore, Raman spectroscopy was used to study the calcium phosphate (CaP) content of hASC-secreted ECM in GG-SPD, GG-BaG, and COL at 21 d in BaG OM. Results The results showed viable rounded cells in GG whereas hASCs were elongated in COL. Importantly, BaG OM induced significantly higher cell number and higher osteogenic gene expression in COL. In both hydrogels, BaG OM induced strong mineralization confirmed as CaP by Raman spectroscopy and significantly improved mechanical properties. GG-BaG hydrogels rescued hASC mineralization in OM. OPT and SPIM showed homogeneous 3D cell distribution with strong mineralization in BaG OM. Also, strong osteocalcin production was visible in COL. Conclusions Overall, we showed efficacious osteogenesis of hASCs in 3D hydrogels with BaG OM with potential for bone-like grafts.

Hops are a major component of beer that is added during brewing. In addition to its wide range of bioactivity, it exhibits neuroactive properties as a sedative and sleeping aid. The compounds responsible for this activity are yet to be revealed and understood in terms of their pharmacological properties. Here we evaluated the potential of several hops flavonoids in modulating the GABAergic activity and assessed their selectivity to GABAA receptors subtypes. GABA-potentiating effects were measured using [3H]ethynylbicycloorthobenzoate (EBOB) radioligand binding assay in native and recombinant α1β3γ2, α2β3γ2 and α6β3δ receptors expressed in HEK293 cells. Flumazenil sensitivity of GABA-potentiating effects and [3H]Ro 15-4513 binding assay were used to examine the flavonoids binding to benzodiazepine site. The prenylflavonoids xanthohumol (XN), isoxanthohumol (IXN) and 8-prenylnaringenin (8PN) potentiated GABA-induced displacement of [3H]EBOB binding in a concentration-dependent manner. The IC50 for this potentiation in native GABAA receptors were 29.7 µM, 11.6 µM, 7.3 µM, respectively. In recombinant receptors, the sensitivity to prenylflavonoid potentiation of GABA-induced displacement of [3H]EBOB binding followed the order α6β3δ > α2β3γ2 > α1β3γ2 with the strongest inhibition observed by 8PN in α6β3δ (IC50 = 3.6 μM). Flumazenil had no significant effect on the prenylflavonoid-induced displacement of [3H]EBOB binding and [3H]Ro 15-4513 displacement from native GABAA receptors was only detected at high micromolar concentrations (100 µM). We identified potent prenylflavonoids in hops that positively modulate GABA-induced responses in native and αβγ/δ recombinant GABAA receptors at low micromolar concentrations. These GABAergic modulatory effects were not mediated via the high-affinity benzodiazepine binding site.

Human genetic evidence demonstrates that WNT1 mutations cause osteogenesis imperfecta (OI) and early‐onset osteoporosis, implicating WNT1 as a major regulator of bone metabolism. However, its main cellular source and mechanisms of action in bone remain elusive. We generated global and limb bud mesenchymal cell–targeted deletion of Wnt1 in mice. Heterozygous deletion of Wnt1 resulted in mild trabecular osteopenia due to decreased osteoblast function. Targeted deletion of Wnt1 in mesenchymal progenitors led to spontaneous fractures due to impaired osteoblast function and increased bone resorption, mimicking the severe OI phenotype in humans with homozygous WNT1 mutations. Importantly, we showed for the first time that Wnt1 signals strictly in a juxtacrine manner to induce osteoblast differentiation and to suppress osteoclastogenesis, in part via canonical Wnt signaling. In conclusion, mesenchymal cell‐derived Wnt1, acting in short range, is an essential regulator of bone homeostasis and an intriguing target for therapeutic interventions for bone diseases. © 2019 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals, Inc.

Most clear cell renal cell carcinomas (ccRCCs) have inactivation of the von Hippel-Lindau tumor suppressor protein (pVHL), resulting in the accumulation of hypoxia-inducible factor -subunits (HIF-) and their downstream targets. HIF-2 expression is particularly high in ccRCC and is associated with increased ccRCC growth and aggressiveness. In the canonical HIF signaling pathway, HIF-prolyl hydroxylase 3 (PHD3) suppresses HIF-2 protein by post-translational hydroxylation under sufficient oxygen availability. Here, using immunoblotting and immunofluorescence staining, qRT-PCR, and siRNA-mediated gene silencing, we show that unlike in the canonical pathway, PHD3 silencing in ccRCC cells leads to down-regulation of HIF-2 protein and mRNA. Depletion of other PHD family members had no effect on HIF-2 expression, and PHD3 knockdown in non-RCC cells resulted in the expected increase in HIF-2 protein expression. Accordingly, PHD3 knockdown decreased HIF-2 target gene expression in ccRCC cells and expression was restored upon forced HIF-2 expression. The effect of PHD3 depletion was pinpointed to HIF2A mRNA stability. In line with these in vitro results, a strong positive correlation of PHD3 and HIF2A mRNA expression in ccRCC tumors was detected. Our results suggest that in contrast to the known negative regulation of HIF-2 in most cell types, high PHD3 expression in ccRCC cells maintains elevated HIF-2 expression and that of its target genes, which may enhance kidney cancer aggressiveness.

Decidual stromal cells differentiate from endometrial stromal fibroblasts (ESFs) under the influence of progesterone and cyclic adenosine monophosphate (cAMP) and are essential for implantation and the maintenance of pregnancy. They evolved in the stem lineage of placental (eutherian) mammals coincidental with the evolution of implantation. Here we use the well-established in vitro decidualization protocol to compare early (3 days) and late (8 days) gene transcription patterns in immortalized human ESF. We document extensive, dynamic changes in the early and late decidual cell transcriptomes. The data suggest the existence of an early signal transducer and activator of transcription (STAT) pathway dominated state and a later nuclear factor κB (NFKB) pathway regulated state. Transcription factor expression in both phases is characterized by putative or known progesterone receptor ( PGR) target genes, suggesting that both phases are under progesterone control. Decidualization leads to proliferative quiescence, which is reversible by progesterone withdrawal after 3 days but to a lesser extent after 8 days of decidualization. In contrast, progesterone withdrawal induces cell death at comparable levels after short or long exposure to progestins and cAMP. We conclude that decidualization is characterized by a biphasic gene expression dynamic that likely corresponds to different phases in the establishment of the fetal-maternal interface.

Bacteria of the genus Borrelia cause vector-borne infections like the most important hard tick-borne disease in the northern hemisphere, Lyme borreliosis (LB), and soft tick or louse transmitted relapsing fevers (RF), prevalent in temperate and tropical areas. Borrelia burgdorferi sensu lato (s.l.) includes several genospecies and causes LB in humans. In infected patients, Borrelia burgdorferi sensu stricto (s.s.) expresses the BmpA, BmpB, BmpC and BmpD proteins. The role of these proteins in the pathogenesis of LB remains incompletely characterized, but they are, however, closely related to Treponema pallidum PnrA (Purine nucleoside receptor A), a substrate-binding lipoprotein of the ATP-binding cassette (ABC) transporter family preferentially binding purine nucleosides. Based on 3D homology modeling, the Bmp proteins share the typical fold of the substrate-binding protein family and the ligand-binding properties of BmpA, BmpB and BmpD are highly similar, whereas those of BmpC differ markedly. Nevertheless, these residues are highly conserved within the genus Borrelia and the inferred phylogenetic tree also reveals that the RF Borrelia lack BmpB proteins but has an additional Bmp protein (BmpA2) missing in LB-causing Borrelia burgdorferi s.l. Our results indicate that the Bmp proteins could bind nucleosides, although BmpC might have a different ligand-binding specificity and, therefore, a distinct function. Furthermore, the work provides a means for classifying the Bmp proteins and supports further elucidation of the roles of these proteins.

Colorectal carcinomas that are mismatch repair (MMR)‐deficient in the absence of MLH1 promoter methylation or germline mutations represent Lynch‐like syndrome (LLS). Double somatic events inactivating MMR genes are involved in the etiology of LLS tumors. Our purpose was to define the clinical and broader molecular hallmarks of LLS tumors and the population incidence of LLS, which remain poorly characterized. We investigated 762 consecutive colorectal carcinomas operated in Central Finland in 2000–2010. LLS cases were identified by a stepwise protocol based on MMR protein expression, MLH1 methylation and MMR gene mutation status. LLS tumors were profiled for CpG Island Methylator Phenotype (CIMP) and somatic mutations in 578 cancer‐relevant genes. Among 107 MMR‐deficient tumors, 81 (76%) were attributable to MLH1 promoter methylation and 9 (8%) to germline mutations (Lynch syndrome, LS), leaving 14 LLS cases (13%) (3 remained unclassified). LLS carcinomas were diagnosed at a mean age of 65 years (vs. 44 years in LS, p < 0.001), had a proximal to distal ratio of 1:1, and all were BRAF V600E‐negative. Two somatic events in MMR genes were identifiable in 11 tumors (79%). As novel findings, the tumors contained an average of 31 nonsynonymous somatic mutations/Mb and 13/14 were CIMP‐positive. In conclusion, we establish the epidemiological, clinical and molecular characteristics of LLS in a population‐based study design. Significantly more frequent CIMP‐positivity and lower rates of somatic mutations make a distinction to LS. The absence of BRAF V600E mutation separates LLS colorectal carcinomas from MLH1‐methylated colorectal carcinomas with CIMP‐positive phenotype. peerReviewed

The aim of this study was to investigate the short-term reproducibility of aortic pulse wave velocity (PWVao) and augmentation index (AIx%) assessed by the non-invasive oscillometric device. Altogether of 55 (19 boys, 36 girls) adolescents 16-19-years-of-age participated in the study. PWVao and AIx% were measured during the same laboratory visit at 2 min intervals using the Arteriograph™ device. Peak oxygen uptake (VO2peak ) was assessed by the maximal exercise test on a cycle ergometer and body fat percentage by bioelectrical impedance analysis. We studied reproducibility using intraclass correlation coefficients (ICC), coefficient variation with the root-means-square method expressed as percentages (CV%), and 95% limit of agreement (95% LA) and coefficient repeatability of the Bland-Altman plot. ICC for PWVao was 0·90, CV% 3·7 and the limits of agreement were -0·70 and 0·58 with a coefficient repeatability of 0·65. ICC for AIx% was 0·88, CV% 29·1%, and the limits of agreement were -6·8 and 7·1 with a coefficient repeatability of 6·9. No relevant differences in ICC, CV%, and coefficient repeatability for PWVao between adolescents with higher or lower VO2peak or body fat percentage were observed. For AIx%, ICC was lower, CV% higher and coefficient repeatability higher in adolescents with higher VO2peak or lower body fat percentage than in adolescents with lower VO2peak or higher body fat percentage. Short-term reproducibility of Arteriograph™-derived PWVao was relatively good and was not affected by VO2peak or adiposity. However, the reproducibility of AIx% was modest especially among adolescents with higher VO2peak and lower body fat percentage.

Muscimol, the major psychoactive ingredient in the mushroom Amanita muscaria, has been regarded as a universal non-selective GABA-site agonist. Deletion of the GABAA receptor (GABAA R) δ subunit in mice (δKO) leads to a drastic reduction in high-affinity muscimol binding in brain sections and to a lower behavioral sensitivity to muscimol than their wild type counterparts. Here, we use forebrain and cerebellar brain homogenates from WT and δKO mice to show that deletion of the δ subunit leads to a > 50% loss of high-affinity 5 nM [3 H]muscimol-binding sites despite the relatively low abundance of δ-containing GABAA Rs (δ-GABAA R) in the brain. By subtracting residual high-affinity binding in δKO mice and measuring the slow association and dissociation rates we show that native δ-GABAA Rs in WT mice exhibit high-affinity [3 H]muscimol-binding sites (KD ~1.6 nM on α4βδ receptors in the forebrain and ~1 nM on α6βδ receptors in the cerebellum at 22°C). Co-expression of the δ subunit with α6 and β2 or β3 in recombinant (HEK 293) expression leads to the appearance of a slowly dissociating [3 H]muscimol component. In addition, we compared muscimol currents in recombinant α4β3δ and α4β3 receptors and show that δ subunit co-expression leads to highly muscimol-sensitive currents with an estimated EC50 of around 1-2 nM and slow deactivation kinetics. These data indicate that δ subunit incorporation leads to a dramatic increase in GABAA R muscimol sensitivity. We conclude that biochemical and behavioral low-dose muscimol selectivity for δ-subunit-containing receptors is a result of low nanomolar-binding affinity on δ-GABAA Rs.

This study describes a validated LC–MS/MS method for assaying 23 steroids within a single run from 150 μl of human plasma, serum or prostatic tissue homogenate. Isotope-labeled steroids were used as internal standards. Samples were extracted with toluene, and ketosteroids were derivatized with hydroxylamine prior to LC–MS/MS analysis. The steroids were separated on a C18 column and methanol was used as an organic solvent with the addition of 0.2 mM ammonium fluoride to improve underivatized estradiol (E2) ionization. Certified reference serums as well as plasma samples, and homogenates of prostate tissue were utilized in the method validation. The specificity of the method was inspected with a total of 27 steroids. The validation proved that the method was suitable for the quantitative analysis of a wide panel of androgens (testosterone, T (3.3 pM-13 nM); androstenedione, A4 (3.3 pM-13 nM); 5α-androstanedione, DHA4 (13 pM-13 nM); dehydroepiandrosterone, DHEA (67 pM-133 nM); dihydrotestosterone, DHT (33 pM-33 nM); 11-ketodihydrotestosterone, 11KDHT (13 pM-13nM); 11-ketotestosterone, 11KT (33 pM-6.7 nM); 11β-hydroxyandrostenedione, 11bOHA4 (33 pM-13 nM); 11β-hydroxytestosterone, 11OHT (13 pM-33 nM)), as well as estrogens (estrone, E1 (3.3 pM-13 nM)), progestagens (17α-hydroxypregnenolone, 17OHP5 (32 pM-127 nM); 17α-hydroxyprogesterone, 17OHP4 (67 pM-133 nM); progesterone, P4 (3.3 pM-13 nM); pregnenolone, P5 (6.6 pM-13 nM)), and glucocorticoids (cortisol, F (33 pM-134 nM); cortisone E (66 pM-131 nM); corticosterone, B (33 pM-67 nM); 11-deoxycortisol, S (33 pM-66 nM); 21-hydroxyprogesterone, 21OHP4 (32 pM-13 nM)). Furthermore, E2 (335 pM-134 nM) and 11α-hydroxyandrostenedione, 11aOHA4 (33 pM-33 nM) could be analyzed if the concentration in the sample was high enough. In addition, aldosterone, A (128 pM-64 nM) and 11-ketoandrostenedione, 11KA4 (33 pM-13 nM) could be analyzed semiquantitatively. The limits of quantification for all compounds ranged from 0.9 to 91 pg/ml, and from 0.009 to 0.9 pg/mg tissue. Compared to our previous method, this new method also permits the analysis of the more challenging steroids, like DHT, DHEA and P5, and a panel of 11-ketosteroids.

The correlation between the Phosphoinositide 3-kinase (PI3K) axis and crucial mechanisms involved in the maintenance of the neoplastic nature of multiple myeloma (MM) has recently evolved a general agreement that PI3K inhibition-based therapies could construct an exciting perspective for the future treatment strategies. Our results outlined that abrogation of PI3K using pan-PI3K inhibitor BKM120 decreased survival of MM cells through induction of a caspase-3-dependent apoptosis coupled with SIRT1-mediated G2/M arrest in both KMM-1 and RPMI 8226 cell lines; however, the cell responses to the inhibitor was quite different, introducing wild-type PTEN-expressing RPMI 8226 as less sensitive cells. By investigating the sensitivity extent of a panel of hematological cell lines to BKM120, we found no significant association with respect to PTEN status. As far as we are aware, the results of the present study propose for the first time that the inhibitory effect of BKM120 was overshadowed, at least partially, through over-expression of either c-Myc or nuclear factor (NF)-κB in less sensitive MM cells. While there was no significant effect of the inhibitor on the expression of c-Myc in RPMI 8226, we found an enhanced cytotoxic effect when BKM120 was used in combination with a small molecule inhibitor of c-Myc. Noteworthy, the results of the synergistic experiments also revealed that BKM120 could produce a synergistic anti-cancer effect with carfilzomib (CFZ) and provided an enhanced therapeutic efficacy in MM cells, highlighting that PI3K inhibition might be a befitting approach in MM both in mono and combined therapy.

The aim of this study was to evaluate the DNA damage in peripheral lymphocytes and the frequencies of CD8+ T cells expressing CD25, CD28 and CD45ro in aged individuals with inverted CD4:CD8 ratio. Blood samples of elderly individuals (aged >65) with normal CD4:CD8 ratio (n = 8) and inverted CD4:CD8 ratio (n = 8) were collected to identify the expression of CD25+, CD28+ and CD45ro+ in CD8+ T cells. DNA damage index was evaluated by the alkaline comet assay which was performed in lymphocytes treated with different concentrations of methyl methanesulfonate (MMS) (control non-treatment, 2 × 10−5 M, 4 × 10−5 M) for 1, 2 or 24 h. Elderly individuals with inverted CD4:CD8 ratio presented low frequency of CD8+ CD28+. Moreover, low DNA damage was observed in lymphocytes of elderly with inverted CD4:CD8 ratio in different doses of MMS. Aged individuals with inverted CD4:CD8 ratio presented lower DNA damage events in peripheral lymphocytes, suggesting a resistance for cell death in T cells of individuals with immune risk profile.

The estimation of the correct binding mode and affinity of a ligand into a target protein using computational methods is challenging. However, docking can introduce poses from which the correct binding mode could be identified using other methods. Here, we analyzed the reliability of binding energy estimation using the molecular mechanics-generalized Born surface area (MMGBSA) method without and with energy minimization to identify the likely ligand binding modes within docking results. MMGBSA workflow (a) outperformed docking in recognizing the correct binding modes of androgen receptor ligands and (b) improved the correlation coefficient of computational and experimental results of rescored docking poses to phosphodiesterase 4B. Combined with stability and atomic distance analysis, MMGBSA helped to (c) identify the binding modes and sites of metabolism of cytochrome P450 2A6 substrates. The standard deviation of estimated binding energy within one simulation was lowered by minimization in all three example cases. Minimization improved the identification of the correct binding modes of androgen receptor ligands. Although only three case studies are shown, the results are analogous and indicate that these behaviors could be generalized. Such identified binding modes could be further used, for example, with free energy perturbation methods to understand binding energetics more accurately.

Myeloid cell leukemia 1 (Mcl1), is an antiapoptotic member of the Bcl-2 family proteins, has gained considerable importance due to its overexpression activity prevents the oncogenic cells to undergo apoptosis. This overexpression activity of Mcl1 eventually develops strong resistance to a wide variety of anticancer agents. Therefore, designing novel inhibitors with potentials to elicit higher binding affinity and specificity to inhibit Mcl1 activity is of greater importance. Thus, Mcl1 acts as an attractive cancer target. Despite recent experimental advancement in the identification and characterization of benzothiophene and benzofuran scaffold-merged compounds, the molecular mechanisms of their binding to Mcl1 are yet to be explored. The current study demonstrates an integrated approach – pharmacophore-based 3D-QSAR, docking, molecular dynamics (MD) simulation and free-energy estimation – to access the precise and comprehensive effects of current inhibitors targeting Mcl1 together with its known activity values. The pharmacophore – ANRRR.240 – based 3D-QSAR model from the current study provided high confidence (R2=0.9154, Q2=0.8736 and RMSE = 0.3533) values. Furthermore, the docking correctly predicted the binding mode of highly active compound 42. Additionally, the MD simulation for docked complex under explicit-solvent conditions together with free-energy estimation exhibited stable interaction and binding strength over the time period. Also, the decomposition analysis revealed potential energy contributing residues – M231, M250, V253, R265, L267 and F270 – to the complex stability. Overall, the current investigation might serve as a valuable insight, either to (i) improve the binding affinity of the current compounds or (ii) discover new generation anticancer agents that can effectively downregulate Mcl1 activity. Communicated by Ramaswamy H. Sarma

The discovery of genetic mechanisms for resistance to obesity and diabetes may illuminate new therapeutic strategies for the treatment of this global health challenge. We used the polygenic 'lean' mouse model, which has been selected for low adiposity over 60 generations, to identify mitochondrial thiosulfate sulfurtransferase (Tst; also known as rhodanese) as a candidate obesity-resistance gene with selectively increased expression in adipocytes. Elevated adipose Tst expression correlated with indices of metabolic health across diverse mouse strains. Transgenic overexpression of Tst in adipocytes protected mice from diet-induced obesity and insulin-resistant diabetes. Tst-deficient mice showed markedly exacerbated diabetes, whereas pharmacological activation of TST ameliorated diabetes in mice. Mechanistically, TST selectively augmented mitochondrial function combined with degradation of reactive oxygen species and sulfide. In humans, TST mRNA expression in adipose tissue correlated positively with insulin sensitivity in adipose tissue and negatively with fat mass. Thus, the genetic identification of Tst as a beneficial regulator of adipocyte mitochondrial function may have therapeutic significance for individuals with type 2 diabetes N. Morton was supported by a Career Development Fellowship, Institutional Strategic Support Fund award and a New Investigator Award from the Wellcome Trust (100981/Z/13/Z), a Research Councils UK Fellowship and a British Heart Foundation Centre of Research Excellence exchange award. We thank the Slovenian Research Agency (core funding P4-0220, project N5-0003 Syntol and J4-6804 to S.H.); and a Young Scientist Fellowship to J. Beltram. We acknowledge support of the British Heart Foundation Research Excellence Award in support of the Bioinformatics Core contribution. T. Stulnig received funding from the Federal Ministry of Economy, Family and Youth and the Austrian National Foundation for Research, Technology and Development. G. Churchill was supported by the US National Institutes of Health grant R01GM 070683. J.M. Fernandez-Real acknowledges funding from FIS PI11/00214. A. Vidal-Puig was funded by the UK Medical Research Council (MRC) MDU, an MRC Programme grant, MRC DMC Core and MITIN (HEALTH-F4-2008-223450)

Objective— Palmitoylethanolamide is an endogenous fatty acid mediator that is synthetized from membrane phospholipids by N -acyl phosphatidylethanolamine phospholipase D. Its biological actions are primarily mediated by PPAR-α (peroxisome proliferator-activated receptors α) and the orphan receptor GPR55. Palmitoylethanolamide exerts potent anti-inflammatory actions but its physiological role and promise as a therapeutic agent in chronic arterial inflammation, such as atherosclerosis remain unexplored. Approach and Results— First, the polarization of mouse primary macrophages towards a proinflammatory phenotype was found to reduce N -acyl phosphatidylethanolamine phospholipase D expression and palmitoylethanolamide bioavailability. N -acyl phosphatidylethanolamine phospholipase D expression was progressively downregulated in the aorta of apolipoprotein E deficient (ApoE −/− ) mice during atherogenesis. N -acyl phosphatidylethanolamine phospholipase D mRNA levels were also downregulated in unstable human plaques and they positively associated with smooth muscle cell markers and negatively with macrophage markers. Second, ApoE − /− mice were fed a high-fat diet for 4 or 16 weeks and treated with either vehicle or palmitoylethanolamide (3 mg/kg per day, 4 weeks) to study the effects of palmitoylethanolamide on early established and pre-established atherosclerosis. Palmitoylethanolamide treatment reduced plaque size in early atherosclerosis, whereas in pre-established atherosclerosis, palmitoylethanolamide promoted signs of plaque stability as evidenced by reduced macrophage accumulation and necrotic core size, increased collagen deposition and downregulation of M1-type macrophage markers. Mechanistically, we found that palmitoylethanolamide, by activating GPR55, increases the expression of the phagocytosis receptor MerTK (proto-oncogene tyrosine-protein kinase MER) and enhances macrophage efferocytosis, indicative of proresolving properties. Conclusions— The present study demonstrates that palmitoylethanolamide protects against atherosclerosis by promoting an anti-inflammatory and proresolving phenotype of lesional macrophages, representing a new therapeutic approach to resolve arterial inflammation.

As more information is needed about the health aspects of milk processing; we classified milk products based on their homogenisation and heat-treatment history in the following inclusive classes: (i) homogenised, (ii) non-homogenised, (iii) fat-free; and (i) low-pasteurised or less heat-treated, (ii) high-pasteurised at = 100 degrees C or sterilised. Milk product consumption of Finnish children was studied at the age of 6 months (n = 1305), 1 y (n = 1513), and 3 y (n = 1328) both using conventional food grouping and the novel processing-based grouping. At 6 months, more than three quarters of the children consumed cows' milk products (median consumption 511 g d(-1)); at 3 y most of the consumed milk products were low-pasteurised or less heat-treated and homogenised. In contrast to children aged 3 y, almost all milk products consumed by infants aged 6 months were pasteurised at high temperature or sterilised. (C) 2018 The Authors. Published by Elsevier Ltd.

Background: Epidemiologic evidence suggests that diets rich in whole grains are associated with a reduced risk of developing chronic diseases and all-cause mortality. However, the molecular mechanisms behind these beneficial metabolic effects are poorly understood. Objective: Our aim was to investigate novel trimethylated (betainized) compounds from mice and humans, and their association with whole grain-rich diets and insulin resistance and insulin secretion. Design: Fasting plasma samples were obtained in a mouse (C57BL/6J male) feeding trial and a controlled dietary intervention. The mouse trial involved feeding the mice a rye and wheat bran-enriched feed which was compared with a high-fat diet. In the human trial, participants recruited from Kuopio, Finland (n = 69) and Naples, Italy (n = 54) with characteristics of the metabolic syndrome were randomly assigned to either a whole grain-enriched diet or a control diet for 12 wk. Plasma concentrations of betainized compounds were analyzed with the use of liquid chromatography-tandem mass spectrometry. Insulin resistance and insulin secretion were assessed in an oral-glucose-tolerance test and a meal-glucose-tolerance test. Results: The betaines that were increased in mouse plasma after bran-enriched feeding were identified de novo via chemical synthesis and liquid chromatography-tandem mass spectrometry, and confirmed to be associated with an increased intake of whole-grain products in humans. In particular, the concentrations of pipecolic acid betaine were increased at the end of the whole-grain intervention in both the Kuopio cohort (P

Direct oral anticoagulants provide an alternative to vitamin K antagonists for the anticoagulation therapy in atrial fibrillation (AF). The availability of several treatment options with different attributes makes shared decision-making appropriate for the choice of anticoagulation therapy. The aim of this study was to understand how physicians choose an oral anticoagulant (OAC) for patients with AF and how physicians view patients' participation in this decision. Semi-structured interviews with 17 Finnish physicians (eight general practitioners and nine specialists) working in the public sector were conducted. An interview guide on experience, prescribing and opinions about oral anticoagulants was developed based on previous literature. The data were thematically analysed using deductive and inductive approaches. Based on the interviews, patient's opinion was the most influential factor in decision-making when there were no clinical factors limiting the choice between OACs. Of patient's preferences, the most important was the attitude towards co-payments of OACs. Patients' opinions on monitoring of treatment, dosing and antidote availability were also mentioned by the interviewees. The choice of an OAC in AF was patient-centred as all interviewees expressed that patient's opinion affects the choice.

BACKGROUND There are few prospective studies on the associations of changes in objectively measured vigorous physical activity (VPA∆ ), moderate-to-vigorous physical activity (MVPA∆ ), light physical activity (LPA∆ ), and sedentary time (ST∆ ) with changes in cardiometabolic risk factors (∆ ) in children. We therefore investigated these relationships among children. METHODS The participants were a population sample of 258 children aged 6-8 years followed for 2 years. We assessed PA and ST by a combined heart rate and movement sensor; computed continuous age- and sex-adjusted z-scores for waist circumference, blood pressure, and fasting insulin, glucose, triglycerides, and high-density lipoprotein (HDL) cholesterol; and constructed a cardiometabolic risk score (CRS) of these risk factors. Data were analyzed using linear regression models adjusted for age, sex, the explanatory and outcome variables at baseline, and puberty. RESULTS VPA∆ associated inversely with CRS∆ (β = -0.209, P = 0.001), body fat percentage (BF%)∆ (β = -0.244, P = 0.001), insulin∆ (β = -0.220, P = 0.001), and triglycerides∆ (β = -0.164, P = 0.012) and directly with HDL cholesterol∆ (β = 0.159, P = 0.023). MVPA∆ associated inversely with CRS∆ (β = -0.178, P = 0.012), BF%∆ (β = -0.298, P =

Background: Prenatal environment has been shown to influence child's risk of atopic diseases. Laboratory-confirmed data about the role of maternal infections during pregnancy is scarce. Objective: The aim of this study was to determine the associations between serologically confirmed maternal infections during pregnancy and atopic disease in the offspring. Methods: This was a nested case-control study within a prospective birth cohort study. Altogether 202 atopic case children and 333 matched non-atopic control children were included. Atopic outcome was defined as having an atopic disease and IgE sensitization by the age of 5 years. We analysed serologically acute enterovirus (EV), influenza virus A (IAV) and Mycoplasma pneumoniae (M. pneumoniae) infections during pregnancy, and mother's seropositivity against human cytomegalovirus (CMV) and Helicobacter pylori. Results: Maternal EV infection during pregnancy was inversely associated with atopic outcome in the offspring (odds ratio 0.43; 95% confidence interval: 0.23-0.80, P = 0.008). Acute IAV or M. pneumoniae infections or seropositivity against CMV or Helicobacter pylori were not associated with the atopic outcome. Conclusions and Clinical Relevance: Our results suggest that maternal EV infections during pregnancy are inversely associated with atopic disease in the offspring. Our finding provides further support to the previous studies suggesting an important role of the in utero environment in the development of atopic diseases.

The aim of this study is to investigate the expression of the luteinizing hormone/choriogonadotropin (LHCG) receptor in the human penis to see, if the luteinizing hormone (LH) effects are possible in the spongious and cavernous tissue of the penis. The number of men with erection disturbances increases significantly simultaneously with the elevated LH concentrations between 40 and 70 years. It is possible that the elevated LH concentrations may influence locally the erectile mechanisms. The precondition for this is the expression of LHCG receptors in the penis. Penile tissue was obtained from three patients undergoing total or partial penectomy due to a rectal cancer with secondary penile metastasis or squamous cell carcinoma of the penis. Immunohistochemistry was used for the detection of the LHCG receptor. Positive immunoreaction for LHCG receptors was discovered in the endothelial cells of cavernous spaces in the corpus cavernosum and corpus spongiosum penis, also in the endothelial cells of the capillary walls in all patients. Our results show that LHCG receptor is expressed in the spongious and cavernous tissue of the human penis. This finding suggests that LH can affect the spongious and cavernous tissue in human and play a significant role in the development of erectile dysfunction among the aging men.

Background Viral infection is implicated in development of autoimmunity. Parvovirus B19 (B19V) nonstructural protein, NS1, a helicase, covalently modifies self double-stranded deoxyribonucleic acid (dsDNA) and induces apoptosis. This study tested whether resulting apoptotic bodies (ApoBods) containing virally modified dsDNA could induce autoimmunity in an animal model. Methods BALB/c mice were inoculated with (1) pristane-induced, (2) B19V NS1-induced, or (3) staurosporine-induced ApoBods. Serum was tested for dsDNA autoantibodies by Crithidia luciliae staining and enzyme-linked immunosorbent assay. Brain, heart, liver, and kidney pathology was examined. Deposition of self-antigens in glomeruli was examined by staining with antibodies to dsDNA, histones H1 and H4, and TATA-binding protein. Results The B19V NS1-induced ApoBod inoculation induced dsDNA autoantibodies in a dose-dependent fashion. Histopathological features of immune-mediated organ damage were evident in pristane-induced and NS1-induced ApoBod groups; severity scores were higher in these groups than in staurosporine-treated groups. Tissue damage was dependent on NS1-induced ApoBod dose. Nucleosomal antigens were deposited in target tissue from pristane-induced and NS1-induced ApoBod inoculated groups, but not in the staurosporine-induced ApoBod inoculated group. Conclusions This study demonstrated proof of principle in an animal model that virally modified dsDNA in apoptotic bodies could break tolerance to self dsDNA and induce dsDNA autoantibodies and end-organ damage., Inoculation of normal mice with apoptotic bodies, generated by parvovirus B19V helicase NS1 expression, induces dsDNA antibodies and organ damage. This study provides proof of concept that viral modification of host cell DNA can lead to autoimmunity.

A Gram-stain-positive, catalase-positive and pleomorphic rod organism was isolated from malted barley in Finland, classified initially by partial 16S rRNA gene sequencing and originally deposited in the VTT Culture Collection as a strain of Propionibacterium acidipropionici (currently Acidipropionibacterium acidipropionici). The subsequent comparison of the whole 16S rRNA gene with other representatives of the genus Acidipropionibacterium revealed that the strain belongs to a novel species, most closely related to Acidipropionibacterium microaerophilum and Acidipropionibacterium acidipropionici, with similarity values of 98.46 and 98.31 %, respectively. The whole genome sequencing using PacBio RS II platform allowed further comparison of the genome with all of the other DNA sequences available for the type strains of the Acidipropionibacterium species. Those comparisons revealed the highest similarity of strain JS278T to A. acidipropionici, which was confirmed by the average nucleotide identity analysis. The genome of strain JS278T is intermediate in size compared to the A. acidipropionici and Acidipropionibacterium jensenii at 3 432 872 bp, the G+C content is 68.4 mol%. The strain fermented a wide range of carbon sources, and produced propionic acid as the major fermentation product. Besides its poor ability to grow at 37° C and positive catalase reaction, the observed phenotype was almost indistinguishable from those of A. acidipropionici and A. jensenii. Based on our findings, we conclude that the organism represents a novel member of the genus Acidipropionibacterium, for which we propose the name Acidipropionibacterium virtanenii sp. nov. The type strain is JS278T (=VTT E-113202T =DSM 106790T ).

Objective The study aim was to compare the performance of interleukin-1 receptor antagonist (IL-1Ra) to C-reactive protein (CRP) and procalcitonin (PCT) in early prediction of the clinical course of febrile neutropenia. Methods The study population consisted of 86 consecutive patients with febrile neutropenia who received intensive chemotherapy for haematological malignancy between November 2009 and November 2012 at the adult haematology ward of Kuopio University Hospital. Twenty-three (27%) patients had acute myeloid leukaemia and 63 (73%) patients were autologous stem cell transplant recipients. IL-1Ra, CRP and procalcitonin were measured at the onset of fever (d0), on day 1 (d1) and on day 2 (d2). Results Eight patients developed severe sepsis, including three patients with septic shock. Eighteen patients had bacteraemia. After the onset of febrile neutropenia Youden´s indices (with their 95% confidence intervals) to identify severe sepsis were for IL-1Ra on d0 0.57 (0.20-0.71) and on d1 0.65 (0.28-0.78), for CRP on d0 0.41 (0.04-0.61) and on d1 0.47 (0.11-0.67) and for PCT on d0 0.39 (0.05-0.66) and on d1 0.52 (0.18-0.76). Conclusions In haematological patients, IL-1Ra has a comparable capacity with CRP and PCT to predict severe sepsis at the early stages of febrile neutropenia.

BRAF V600E is the most common mutation in conventional ameloblastoma (AM) of the mandible. In contrast, maxillary AMs appear to harbor more frequently RAS, FGFR2, or SMO mutations. Unicystic ameloblastoma (UAM) is considered a less aggressive variant of ameloblastoma, amenable to more conservative treatment, and classified as a distinct entity. The aim of this study was to characterize the mutation profile of UAM ( n = 39) and to compare it to conventional AM ( n = 39). The associations between mutation status and recurrence probability were also analyzed. In the mandible, 94% of UAMs (29/31, including 8/8 luminal, 6/8 intraluminal, and 15/15 mural subtypes) and 74% of AMs (28/38) revealed BRAF V600E mutations. Among the BRAF wild-type cases, 1 UAM showed a missense SMO mutation (p.L412F), whereas 2 NRAS (p.Q61R), 2 HRAS (p.Q61R), and 2 FGFR2 (p.C383R) activating mutations were identified in AM. Of the 3 maxillary UAMs, only 1 revealed a BRAF V600E mutation. Taken together, our findings demonstrate high frequency of activating BRAF V600E mutations in both UAM and AM of the mandible. In maxillary UAMs, the BRAF V600E mutation prevalence appears to be lower as was shown for AM previously. It could therefore be argued that UAM and AM are part of the spectrum of the same disease. AMs without BRAF V600E mutations were associated with an increased rate of local recurrence ( P = 0.0003), which might indicate that routine mutation testing also has an impact on prognosis.

OBJECTIVE To evaluate effects of the peripherally acting α2-adrenoceptor antagonist MK-467 on cardiopulmonary function in sheep sedated with medetomidine and ketamine. ANIMALS 9 healthy adult female sheep. PROCEDURES Each animal received an IM injection of a combination of medetomidine (30 μg/kg) and ketamine (1 mg/kg; Med-Ket) alone and Med-Ket and 3 doses of MK-467 (150, 300, and 600 μg/kg) in a randomized blinded 4-way crossover study. Atipamezole (150 μg/kg, IM) was administered 60 minutes later to reverse sedation. Cardiopulmonary variables and sedation scores were recorded, and drug concentrations in plasma were analyzed. Data were analyzed with a repeated-measures ANCOVA and 1-way ANOVA. Reference limits for the equivalence of sedation scores were set at 0.8 and 1.25. RESULTS Heart rate, cardiac output, and Pao2 decreased and mean arterial blood pressure, central venous pressure, and systemic vascular resistance increased after Med-Ket alone. Administration of MK-467 significantly alleviated these effects, except for the decrease in cardiac output. After sedation was reversed with atipamezole, no significant differences were detected in cardiopulmonary variables among the treatments. Administration of MK-467 did not significantly alter plasma concentrations of medetomidine, ketamine, norketamine, or atipamezole. Sedation as determined on the basis of overall sedation scores was similar among treatments. CONCLUSIONS AND CLINICAL RELEVANCE Concurrent administration of MK-467 alleviated cardiopulmonary effects in sheep sedated with Med-Ket without affecting sedation or reversal with atipamezole.

The aim of this study was to develop a rapid single-tube two-color flow cytometric method to distinguish between febrile bacterial and viral infections. In this prospective comparative study, the quantitative flow cytometric analysis of CD35 and CD64 on isolated human leukocytes was obtained from 286 hospitalized febrile patients, of which 197 patients were found to have either a bacterial (n = 136) or viral (n = 61) infection. The data from infected patients was compared to 49 healthy controls and 23 patients in an inflammatory state. We developed a flow cytometric marker for bacterial infections, defined as the two-color bacterial infection index (TC-BI-index), by incorporating the quantitative analysis of CD35 and CD64 on isolated neutrophils, monocytes, and B-lymphocytes, which displayed 90% sensitivity and specificity in distinguishing between microbiologically confirmed bacterial (n = 77) and viral infections (n = 61) within 45 min. We propose that TC-BI-index test will be useful in assisting physicians to ascertain whether antibiotic treatment is required, thus limiting unnecessary antimicrobial usage.

Contradictory findings on the role of the type 1 cannabinoid receptor (CB1R) during the pathogenesis of Alzheimer’s disease (AD) have been reported. Here, we evaluated the CB1R brain profile in an AD mouse model using longitudinal positron emission tomography with an inverse agonist for CB1R, [18F]FMPEP-d2. APP/PS1-21 and wild-type (n 1⁄4 8 in each group) mice were repeatedly imaged between 6 to 15 months of age, accompanied by brain autoradiography, western blot, and CB1R immunohistochemistry with additional mice. [18F]FMPEP-d2 positron emission tomography demonstrated lower (p < 0.05) binding ratios in the parietotemporal cortex and hippocampus of APP/PS1-21 mice compared with age-matched wild-type mice. Western blot demonstrated no differences between APP/PS1-21 and wild-type mice in the CB1R abundance, whereas significantly lower (p < 0.05) receptor expression was observed in male than female mice. The results provide the first demonstration that [18F]FMPEP-d2 is a promising imaging tool for AD research in terms of CB1R availability, but not expression. This finding may further facilitate the development of novel therapeutic approaches based on endocannabinoid regulation.