Status of linusorbs in cold-pressed flaxseed oil during oxidation and their response toward antioxidants.

Linusorbs (LOs, cyclolinopeptides) are a class of naturally occurring cyclic hydrophobic peptides found in flaxseed oil, whose oxidation states indicate the oxidative stability and bitterness of flaxseed oil. Subjected to 63 °C accelerated oxidation, most Met-containing LOs in cold-pressed flaxseed oil entirely depleted by the 6th day except CLP, and MetO2-containing LOs became the dominant ones. However, no MetO2 form of Trp-containing LOs, such as CLD, CLF and CLG, were detected. Given their oxidative kinetics, methionine sulfoxide (MetO) residue in some LOs was less sensitive toward oxidation in the presence of Trp (Tryptophan) group, and the oxidative stability of Met-containing LOs was CLP < CLB < CLL ≈ CLM < CLO, as compared to MetO-containing LOs: CLD < CLE < CLC < CLF ≈ CLG. When antioxidant was added into cold-pressed flaxseed oil to assess the additives' antioxidant effect, no significant difference was observed on oil oxidative indices in early stage except α-tocopherol, where they vary dramatically in suppressing Met oxidation of LOs: L-AP (L-ascorbyl palmitate) > TBHQ (tert-butyl hydroquinone) > Î³-tocopherol > carnosic acid > α-tocopherol. Besides its ability to suppress oxidation of Trp-containing LOs, L-AP also exhibits superior antioxidant effect on non-Trp-containing LOs due to its amphiphilic property. Due to the prooxidative effects of both α- and γ-tocopherol on LOs that contain Trp, it has been suggested that tocopherols may repair Trp residue on LOs, leading to increased tendency of MetO residues to oxidize. The findings of this research are critical for elucidating the antioxidative mechanism of LOs, which can further lead to the establishment of strategies in suppressing bitter after taste to produce high-quality flaxseed oil.

A fine balance of hydrophobic-electrostatic communication pathways in a pH-switching protein.

Allostery is the phenomenon of coupling between distal binding sites in a protein. Such coupling is at the crux of protein function and regulation in a myriad of scenarios, yet determining the molecular mechanisms of coupling networks in proteins remains a major challenge. Here, we report mechanisms governing pH-dependent myristoyl switching in monomeric hisactophilin, whereby the myristoyl moves between a sequestered state, i.e., buried within the core of the protein, to an accessible state, in which the myristoyl has increased accessibility for membrane binding. Measurements of the pH and temperature dependence of amide chemical shifts reveal protein local structural stability and conformational heterogeneity that accompany switching. An analysis of these measurements using a thermodynamic cycle framework shows that myristoyl-proton coupling at the single-residue level exists in a fine balance and extends throughout the protein. Strikingly, small changes in the stereochemistry or size of core and surface hydrophobic residues by point mutations readily break, restore, or tune myristoyl switch energetics. Synthesizing the experimental results with those of molecular dynamics simulations illuminates atomistic details of coupling throughout the protein, featuring a large network of hydrophobic interactions that work in concert with key electrostatic interactions. The simulations were critical for discerning which of the many ionizable residues in hisactophilin are important for switching and identifying the contributions of nonnative interactions in switching. The strategy of using temperature-dependent NMR presented here offers a powerful, widely applicable way to elucidate the molecular mechanisms of allostery in proteins at high resolution.

Acute disseminated encephalomyelitis and routine childhood vaccinations - a self-controlled case series.

Acute disseminated encephalomyelitis (ADEM) is an autoimmune, central nervous system demyelinating disorder that follows antecedent immunologic challenges, such as infection or vaccination. This study aimed to investigate the potential association between routine childhood vaccinations and ADEM. Children under 7 years of age admitted to the two tertiary level pediatric hospitals in Victoria, Australia with ADEM from 2000-2015 had their clinical information linked to vaccination records from the Australian Childhood Immunization Register. Chart review was undertaken utilizing the Brighton Collaboration ADEM criteria. The self-controlled case-series (SCCS) methodology was employed to determine the relative incidences of ADEM post-vaccination in two risk intervals: 5-28 days and 2-42 days. Forty-six cases were eligible for SCCS analysis with a median age of 3.2 years. Of the forty-six cases, three were vaccine proximate cases and received vaccinations 23, 25 and 28 days before ADEM onset. Two vaccine proximate cases received their 4-year-old scheduled vaccinations (MMR and DTPa-IPV) and one vaccine proximate case the 1-year old scheduled vaccinations (MMR and Hib-MenC). The relative incidence of ADEM during the narrow and broad risk intervals were 1.041 (95% CI 0.323-3.356, p = 0.946) and 0.585 (95% CI 0.182-1.886, p = 0.370) respectively. Sensitivity analyses did not yield any substantial deviations. These results do not provide evidence of an association between vaccinations routinely provided to children aged under 7 years in Australia and the incidence of ADEM. However, these results should be interpreted with caution as the number of ADEM cases identified was limited and further research is warranted.

Antiresorptive and anabolic agents in the prevention and reversal of bone fragility.

Bone volume, microstructure and its material composition are maintained by bone remodelling, a cellular activity carried out by bone multicellular units (BMUs). BMUs are focally transient teams of osteoclasts and osteoblasts that respectively resorb a volume of old bone and then deposit an equal volume of new bone at the same location. Around the time of menopause, bone remodelling becomes unbalanced and rapid, and an increased number of BMUs deposit less bone than they resorb, resulting in bone loss, a reduction in bone volume and microstructural deterioration. Cortices become porous and thin, and trabeculae become thin, perforated and disconnected, causing bone fragility. Antiresorptive agents reduce fracture risk by reducing the rate of bone remodelling so that fewer BMUs are available to remodel bone. Bone fragility is not abolished by these drugs because existing microstructural deterioration is not reversed, unsuppressed remodelling continues producing microstructural deterioration and unremodelled bone that becomes more mineralized can become brittle. Anabolic agents reduce fracture risk by stimulating new bone formation, which partly restores bone volume and microstructure. To guide fracture prevention, this Review provides an overview of the structural basis of bone fragility, the mechanisms of remodelling and how anabolic and antiresorptive agents target remodelling defects.

High and low doses of cocaine intake are differentially regulated by dopamine D2 receptors in the ventral tegmental area and the nucleus accumbens.

Dopamine D2 receptors (D2Rs) in the ventral tegmental area (VTA) and the nucleus accumbens (NAc) are associated with vulnerability to addiction; however, whether D2Rs in these two brain regions play differential roles in regulation of drug intake is unknown. Here, we compared the effect of decreased mRNA level of Drd2 in each region on cocaine self-administration in a dose-response function. Drd2 mRNA levels in rat VTA or NAc were knocked down by bilateral microinjection of lentivirus coding shRNAs against rat Drd2 or scrambled shRNA. Drd2 knockdown was persistent and stable between 20 and 90 days after lentiviral infection. Animals were trained to self-administer cocaine 20 days after Drd2 shRNA treatment. Compared to scrambled shRNA treated rats, Drd2 knockdown in the VTA increased cocaine self-administration at all tested doses (0.02-0.56 mg/kg/infusion) producing an upward shift (both the ascending and descending limb) in the dose-response curve of cocaine self-administration. In contrast, intra-NAc knockdown increased cocaine self-administration only on the ascending limb of the dose-response curve (0.02-0.07 mg/kg/infusion). These data suggest that D2Rs in the VTA, not in the NAc, regulate high-dose cocaine intake. The present study not only demonstrates that low levels of D2Rs in either region increase low doses of cocaine intake, but also reveals for the first time their dissociable roles in limiting high doses of cocaine self-administration.

Bone corticalization requires local SOCS3 activity and is promoted by androgen action via interleukin-6.

Long bone strength is determined by its outer shell (cortical bone), which forms by coalescence of thin trabeculae at the metaphysis (corticalization), but the factors that control this process are unknown. Here we show that SOCS3-dependent cytokine expression regulates bone corticalization. Young male and female Dmp1Cre.Socs3 f/f mice, in which SOCS3 has been ablated in osteocytes, have high trabecular bone volume and poorly defined metaphyseal cortices. After puberty, male mice recover, but female corticalization is still impaired, leading to a lasting defect in bone strength. The phenotype depends on sex-steroid hormones: dihydrotestosterone treatment of gonadectomized female Dmp1Cre.Socs3 f/f mice restores normal cortical morphology, whereas in males, estradiol treatment, or IL-6 deletion, recapitulates the female phenotype. This suggests that androgen action promotes metaphyseal corticalization, at least in part, via IL-6 signaling.The strength of long bones is determined by coalescence of trabeculae during corticalization. Here the authors show that this process is regulated by SOCS3 via a mechanism dependent on IL-6 and expression of sex hormones.

Retinoic acid receptor signalling directly regulates osteoblast and adipocyte differentiation from mesenchymal progenitor cells.

Low and high serum retinol levels are associated with increased fracture risk and poor bone health. We recently showed retinoic acid receptors (RARs) are negative regulators of osteoclastogenesis. Here we show RARs are also negative regulators of osteoblast and adipocyte differentiation. The pan-RAR agonist, all-trans retinoic acid (ATRA), directly inhibited differentiation and mineralisation of early osteoprogenitors and impaired the differentiation of more mature osteoblast populations. In contrast, the pan-RAR antagonist, IRX4310, accelerated differentiation of early osteoprogenitors. These effects predominantly occurred via RARγ and were further enhanced by an RARα agonist or antagonist, respectively. RAR agonists similarly impaired adipogenesis in osteogenic cultures. RAR agonist treatment resulted in significant upregulation of the Wnt antagonist, Sfrp4. This accompanied reduced nuclear and cytosolic ß-catenin protein and reduced expression of the Wnt target gene Axin2, suggesting impaired Wnt/ß-catenin signalling. To determine the effect of RAR inhibition in post-natal mice, IRX4310 was administered to male mice for 10 days and bones were assessed by µCT. No change to trabecular bone volume was observed, however, radial bone growth was impaired. These studies show RARs directly influence osteoblast and adipocyte formation from mesenchymal cells, and inhibition of RAR signalling in vivo impairs radial bone growth in post-natal mice.

Increased miR-155-5p and reduced miR-148a-3p contribute to the suppression of osteosarcoma cell death.

Osteosarcoma (OS) is the most common cancer of bone and the 5th leading cause of cancer-related death in young adults. Currently, 5-year survival rates have plateaued at ~70% for patients with localized disease. Those with disseminated disease have an ~20% 5-year survival. An improved understanding of the molecular genetics of OS may yield new approaches to improve outcomes for OS patients. To this end, we applied murine models that replicate human OS to identify and understand dysregulated microRNAs (miRNAs) in OS. miRNA expression patterns were profiled in murine primary osteoblasts, osteoblast cultures and primary OS cell cultures (from primary and paired metastatic locations) isolated from two genetically engineered murine models of OS. The differentially expressed miRNA were further assessed by a cross-species comparison with human osteoblasts and OS cultures. We identified miR-155-5p and miR-148a-3p as deregulated in OS. miR-155-5p suppression or miR-148a-3p overexpression potently reduced proliferation and induced apoptosis in OS cells, yet strikingly, did not impact normal osteoblasts. To define how these miRNAs regulated OS cell fate, we used an integrated computational approach to identify putative candidate targets and then correlated these with the cell biological impact. Although we could not resolve the mechanism through which miR-148a-3p impacts OS, we identified that miR-155-5p overexpression suppressed its target Ripk1 (receptor (TNFRSF)-interacting serine-threonine kinase 1) expression, and miR-155-5p inhibition elevated Ripk1 levels. Ripk1 is directly involved in apoptosis/necroptosis. In OS cells, small interfering RNA against Ripk1 prevented cell death induced by the sequestration of miR-155-5p. Collectively, we show that miR-148a-3p and miR-155-5p are species-conserved deregulated miRNA in OS. Modulation of these miRNAs was specifically toxic to tumor cells but not normal osteoblasts, raising the possibility that these may be tractable targets for miRNA-based therapies for OS.

Paper-Based Electrical Respiration Sensor.

Current methods of monitoring breathing require cumbersome, inconvenient, and often expensive devices; this requirement sets practical limitations on the frequency and duration of measurements. This article describes a paper-based moisture sensor that uses the hygroscopic character of paper (i.e. the ability of paper to adsorb water reversibly from the surrounding environment) to measure patterns and rate of respiration by converting the changes in humidity caused by cycles of inhalation and exhalation to electrical signals. The changing level of humidity that occurs in a cycle causes a corresponding change in the ionic conductivity of the sensor, which can be measured electrically. By combining the paper sensor with conventional electronics, data concerning respiration can be transmitted to a nearby smartphone or tablet computer for post-processing, and subsequently to a cloud server. This means of sensing provides a new, practical method of recording and analyzing patterns of breathing.

Regional differences in mu and kappa opioid receptor G-protein activation in brain in male and female prairie voles.

Prairie voles are unusual mammals in that, like humans, they are capable of forming socially monogamous pair bonds, display biparental care, and engage in alloparental behaviors. Both mu and kappa opioid receptors are involved in behaviors that either establish and maintain, or result from pair bond formation in these animals. Mu and kappa opioid receptors both utilize inhibitory G-proteins in signal transduction mechanisms, however the efficacy by which these receptor subtypes stimulate G-protein signaling across the prairie vole neuraxis is not known. Utilizing [(35)S]GTPγS autoradiography, we characterized the efficacy of G-protein stimulation in coronal sections throughout male and female prairie vole brains by [D-Ala2,NMe-Phe4,Gly-ol5]-enkephalin (DAMGO) and U50,488H, selective mu and kappa opioid agonists, respectively. DAMGO stimulation was highest in the forebrain, similar to that found with other rodent species. U-50,488H produced greater stimulation in prairie voles than is typically seen in mice and rats, particularly in select forebrain areas. DAMGO produced higher stimulation in the core versus the shell of the nucleus accumbens (NAc) in females, while the distribution of U-50,488H stimulation was the opposite. There were no gender differences for U50,488H stimulation of G-protein activity across the regions examined, while DAMGO stimulation was greater in sections from females compared to those from males for NAc core, entopeduncular nucleus, and hippocampus. These data suggest that the kappa opioid system may be more sensitive to manipulation in prairie voles compared to mice and rats, and that female prairie voles may be more sensitive to mu agonists in select brain regions than males.

Incidence of symptomatic venous thromboembolism in oncological oral and maxillofacial operations: retrospective analysis.

We retrospectively analysed the incidence of symptomatic venous thromboembolism (VTE) and associated risk factors in operations under general anaesthesia for cancer of the oral cavity. To identify symptoms related to deep venous thrombosis (DVT) and pulmonary embolism (PE), together with associated risk factors, we reviewed medical records of patients operated on in the department of oral and maxillofacial surgery at the Queen Elizabeth Hospital, Birmingham, United Kingdom, between June 2007 and October 2012. All patients were categorised according to their level of risk of VTE. The incidence of VTE was calculated with univariate associations and odds ratios with related 95% confidence intervals, where possible. In total, 233 patients were included, comprising 244 operations (mean (SD) age at operation 60.9 (13) years). Almost all patients (97%) were classified as having the highest risk of VTE. Swelling of an extremity, expectoration of blood, and tightness of the chest were the most common symptoms for suspected cases. An incidence of 0.41% was found for symptomatic VTE; one man developed a PE 2 days after operation. Associations between the analysed factors and symptomatic VTE were not significant. The development of the complication in oncological oral and maxillofacial operations seems to be rare, even in patients with a high risk. We cannot recommend the use of routine thromboprophylaxis, but it could be advocated in patients with obvious serious risk factors.

Knockdown of PTHR1 in osteosarcoma cells decreases invasion and growth and increases tumor differentiation in vivo.

Osteosarcoma (OS) is the most common cancer of bone. Parathyroid hormone (PTH) regulates calcium homeostasis and bone development, while the paracrine/autocrine PTH-related protein (PTHrP) has central roles in endochondral bone formation and bone remodeling. Using a murine OS model, we found that OS cells express PTHrP and the common PTH/PTHrP receptor (PTHR1). To investigate the role of PTHR1 signaling in OS cell behavior, we used shRNA to reduce PTHR1 expression. This only mildly inhibited proliferation in vitro, but markedly reduced invasion through collagen and reduced expression of RANK ligand (RANKL). Administration of PTH(1-34) did not stimulate OS proliferation in vivo but, strikingly, PTHR1 knockdown resulted in a profound growth inhibition and increased differentiation/mineralization of the tumors. Treatment with neutralizing antibody to PTHrP did not recapitulate the knockdown of PTHR1. Consistent with this lack of activity, PTHrP was predominantly intracellular in OS cells. Knockdown of PTHR1 resulted in increased expression of late osteoblast differentiation genes and upregulation of Wnt antagonists. RANKL production was reduced in knockdown tumors, providing for reduced homotypic signaling through the receptor, RANK. Loss of PTHR1 resulted in the coordinated loss of gene signatures associated with the polycomb repressive complex 2 (PRC2). Using Ezh2 inhibitors, we demonstrate that the increased expression of osteoblast maturation markers is in part mediated by the loss of PRC2 activity. Collectively these results demonstrate that PTHR1 signaling is important in maintaining OS proliferation and undifferentiated state. This is in part mediated by intracellular PTHrP and through regulation of the OS epigenome.

Differential diagnosis of an unusual shoulder articular lesion in an ancient domestic dog (Canis lupus familiaris L., 1758).

A proximal humeral articular surface from an ancient domestic dog deliberate burial was examined during survey of small mammal bones from a prehistoric early Late Woodland archeological site. An unusual lesion on the caudolateral articular surface prompted micro-computed tomography to define detailed structure. Results indicate cortical or immature woven bone arising subchondrally, replacing normal trabeculae, extending through a breach in the cortical surface, and having sharp transition with surrounding normal bone. Organized bone within the lesion indicates that the dog lived for months-to-years following insult. Differential diagnoses initially included: sharp penetrating trauma; intrinsic or extrinsic blunt fracturing force; osteochondrosis or complication of an osteochondral lesion; unusual osteoarthritis; and neoplasia. Computed tomography ruled out normal or unusual osteoarthritis, and neoplasia. The nature and small size of the lesion, relatively small size of the dog, and lack of evidence for complicating infection, suggest against sharp penetrating trauma as a sole cause. The most plausible differential diagnoses include: uncommon fracture-producing force in a companion animal, and blunt intrinsic or extrinsic force causing fracture at a weak point, such as an early osteochondral lesion, that was obliterated by healing. Combined gross examination, micro-computed tomography, and archeological-anthropological influences facilitated refinement of differential diagnosis.

New Sources of Temperature-Sensitive Resistance to Wheat streak mosaic virus in Wheat.

Expressing temperature-sensitive resistance (TSR) protects wheat against yield losses from infection with Wheat streak mosaic virus (WSMV). In examining how 2,429 wheat accessions from the National Small Grains Collection responded to inoculation with the Sid81 isolate of WSMV, 20 candidate TSR sources were discovered. To differentiate their relative effectiveness, accession responses over 21 days to inoculation with GH95, Sid81, and PV57 virus isolates in regimes of 18 and 20°C were observed. At 18°C, all 20 candidate TSR sources were uniformly or nearly uniformly asymptomatic 21 days after inoculation with the PV57 isolate, resistance indistinguishable from resistant checks KS96HW10-3 and RonL. By contrast, the Sid81 isolate induced symptoms in low but significant proportions of plants of two candidates, and the GH95 isolate in high proportions for four candidates and low but significant proportions for two others. In the more stringent 20°C regime, the uniform or near-uniform induction of symptoms in response to inoculation with GH95 failed to differentiate among the 20 candidate TSR sources and two resistant checks, while PV57 and Sid81 identified several candidates that performed similarly to KS96HW10-3 and significantly better than RonL. By identifying new sources of resistance, this study contributes to the control of WSMV.

Temperature-Sensitive Resistance to Wheat streak mosaic virus in CO960333 and KS06HW79 Wheat.

Temperature-sensitive resistance (TSR) that can protect against losses to Wheat streak mosaic virus (WSMV) has been described in elite wheat germplasm. A TSR identified in the advanced breeding line CO960333 and its derivative KS06HW79 was examined in growth-chamber tests conducted under constant temperature regimes of 18, 21, and 24°C against an array of WSMV isolates. At 18°C, all tested isolates systemically infected the pedigree parents, while the progeny line CO960333 remained free of symptoms; at 24°C, all lines were susceptible. At the intermediate temperature of 21°C, the TSR of KS06HW79 was effective in contrast to the TSRs of KS03HW12 and 'RonL'. In field trials conducted in 2011 and 2012, the TSR expressed in KS06HW79 conferred complete protection against yield losses from inoculation with the Sidney 81 isolate of WSMV, while the TSR of RonL conferred similar protection in 2012 but allowed small losses in 2011. The resistance expressed by KS06HW79 is likely not due to the Wsm1 gene because it did not contain the tightly linked J15 sequence-characterized amplified region (SCAR) DNA marker. These findings suggest that KS06HW79 could be an additional TSR source of value to wheat-breeding programs seeking to control losses from WSMV.

Nonnative interactions regulate folding and switching of myristoylated protein.

We present an integrated experimental and computational study of the molecular mechanisms by which myristoylation affects protein folding and function, which has been little characterized to date. Myristoylation, the covalent linkage of a hydrophobic C14 fatty acyl chain to the N-terminal glycine in a protein, is a common modification that plays a critical role in vital regulated cellular processes by undergoing reversible energetic and conformational switching. Coarse-grained folding simulations for the model pH-dependent actin- and membrane-binding protein hisactophilin reveal that nonnative hydrophobic interactions of the myristoyl with the protein as well as nonnative electrostatic interactions have a pronounced effect on folding rates and thermodynamic stability. Folding measurements for hydrophobic residue mutations of hisactophilin and atomistic simulations indicate that the nonnative interactions of the myristoyl group in the folding transition state are nonspecific and robust, and so smooth the energy landscape for folding. In contrast, myristoyl interactions in the native state are highly specific and tuned for sensitive control of switching functionality. Simulations and amide hydrogen exchange measurements provide evidence for increases as well as decreases in stability localized on one side of the myristoyl binding pocket in the protein, implicating strain and altered dynamics in switching. The effects of folding and function arising from myristoylation are profoundly different from the effects of other post-translational modifications.

Dissecting the molecular determinants of ligand-binding-induced macromolecular switching using thermodynamic cycles.

The energetic networks that govern regulated switching processes in macromolecules are poorly understood at a molecular level. We illustrate a general methodology that uses thermodynamic cycles to measure the coupling energetics between specific groups in a macromolecule and ligand-binding-induced macromolecular switching. The approach is applied to new and published thermodynamic stability and/or binding data not previously analyzed in this way, for a wide range of switching systems, including H+ or Ca²+-binding-induced myristoyl switching, ion or peptide-binding-induced conformational switching in various proteins and small molecule binding to a ribo-switch. The results show how this powerful approach can be used to identify and dissect the molecular determinants of switching in macromolecules.