GRK5-mediated exacerbation of pathological cardiac hypertrophy involves facilitation of nuclear NFAT activity.
G protein-coupled receptor kinases (GRKs) acting in the cardiomyocyte regulate important signaling events that control cardiac function. Both GRK2 and GRK5, the predominant GRKs expressed in the heart, have been shown to be upregulated in failing human myocardium. Although the canonical role of GRKs is to desensitize G protein-coupled receptors via phosphorylation, it has been demonstrated that GRK5, unlike GRK2, can reside in the nucleus of myocytes and exert G protein-coupled receptor-independent effects that promote maladaptive cardiac hypertrophy and heart failure. To explore novel mechanisms by which GRK5 acting in the nucleus of cardiomyocytes participates in pathological cardiac hypertrophy. In this study, we have found that GRK5-mediated pathological cardiac hypertrophy involves the activation of the nuclear factor of activated T cells (NFAT) because GRK5 causes enhancement of NFAT-mediated hypertrophic gene transcription. Transgenic mice with cardiomyocyte-specific GRK5 overexpression activate an NFAT-reporter in mice basally and after hypertrophic stimulation, including transverse aortic constriction and phenylephrine treatment. Complimentary to this, GRK5 null mice exhibit less NFAT transcriptional activity after transverse aortic constriction. Furthermore, the loss of NFATc3 expression in the heart protected GRK5 overexpressing transgenic mice from the exaggerated hypertrophy and early progression to heart failure seen after transverse aortic constriction. Molecular studies suggest that GRK5 acts in concert with NFAT to increase hypertrophic gene transcription in the nucleus via GRK5's ability to bind DNA directly without a phosphorylation event. GRK5, acting in a kinase independent manner, is a facilitator of NFAT activity and part of a DNA-binding complex responsible for pathological hypertrophic gene transcription.


Proteomic analysis of naphthalene-induced airway epithelial injury and repair in a cystic fibrosis mouse model.
Combined results from laser capture microdissection of mouse airway epithelial cells followed by high power (MALDI-FTICR) MS, and fluorescent two-dimensional gel elctrophoresis (2D-DIGE) of the whole lung, allowed us to identify proteins differentially expressed after naphthalene induced airway injury. Further, we discovered several novel aspects of Cystic Fibrosis (CF) lung pathology in an F508del-Cftr mouse model using this approach. The combined MALDI-FTICR-MS and 2D-DIGE data show that lung carbonyl reductase (CBR2), involved in prostaglandin metabolism, converting PGE2 to PGF2alpha, is localized to airway cells and is reduced 2-fold in mutant mice compared to normal, both before and after challenge. Further, we observe a downregulation of two key enzymes of retinoic acid metabolism after injury, which is more pronounced in CF mutant mice. These data show that state-of-the-art proteomics can be used to evaluate airway injury in small cell samples. Further, the results suggest the involvement of prostaglandin and retinoic acid metabolism in the abnormal responses of CF mutant mice to injury.


Dissociation of cohesin from chromosome arms and loss of arm cohesion during early mitosis depends on phosphorylation of SA2.
Cohesin is a protein complex that is required to hold sister chromatids together. Cleavage of the Scc1 subunit of cohesin by the protease separase releases the complex from chromosomes and thereby enables the separation of sister chromatids in anaphase. In vertebrate cells, the bulk of cohesin dissociates from chromosome arms already during prophase and prometaphase without cleavage of Scc1. Polo-like kinase 1 (Plk1) and Aurora-B are required for this dissociation process, and Plk1 can phosphorylate the cohesin subunits Scc1 and SA2 in vitro, consistent with the possibility that cohesin phosphorylation by Plk1 triggers the dissociation of cohesin from chromosome arms. However, this hypothesis has not been tested yet, and in budding yeast it has been found that phosphorylation of Scc1 by the Polo-like kinase Cdc5 enhances the cleavability of cohesin, but does not lead to separase-independent dissociation of cohesin from chromosomes. To address the functional significance of cohesin phosphorylation in human cells, we have searched for phosphorylation sites on all four subunits of cohesin by mass spectrometry. We have identified numerous mitosis-specific sites on Scc1 and SA2, mutated them, and expressed nonphosphorylatable forms of both proteins stably at physiological levels in human cells. The analysis of these cells lines, in conjunction with biochemical experiments in vitro, indicate that Scc1 phosphorylation is dispensable for cohesin dissociation from chromosomes in early mitosis but enhances the cleavability of Scc1 by separase. In contrast, our data reveal that phosphorylation of SA2 is essential for cohesin dissociation during prophase and prometaphase, but is not required for cohesin cleavage by separase. The similarity of the phenotype obtained after expression of nonphosphorylatable SA2 in human cells to that seen after the depletion of Plk1 suggests that SA2 is the critical target of Plk1 in the cohesin dissociation pathway.


Activation of a MnO2 cathode by water-stimulated Mg(2+) insertion for a magnesium ion battery.
Magnesium batteries have been considered to be one of the promising beyond lithium ion technologies due to magnesium's abundance, safety, and high volumetric capacity. However, very few materials show reversible performance as a cathode in magnesium ion systems. We present herein the best reported cycling performances of MnO2 as a magnesium battery cathode material. We show that the previously reported poor Mg(2+) insertion/deinsertion capacities in MnO2 can be greatly improved by synthesizing self-standing nanowires and introducing a small amount of water molecules into the electrolyte. Electrochemical and elemental analysis results revealed that the magnitude of Mg(2+) insertion into MnO2 highly depends on the ratio between water molecules and Mg(2+) ions present in the electrolyte and the highest Mg(2+) insertion capacity was observed at a ratio of 6H2O/Mg(2+) in the electrolyte. We demonstrate for the first time, that MnO2 nanowire electrode can be "activated" for Mg(2+) insertion/deinsertion by cycling in water containing electrolyte resulting in enhanced reversible Mg(2+) insertion/deinsertion even with the absence of water molecules. The MnO2 nanowire electrode cycled in dry Mg electrolyte after activation in water-containing electrolyte showed an initial capacity of 120 mA h g(-1) at a rate of 0.4 C and maintained 72% of its initial capacity after 100 cycles.


The neural basis of conceptual-emotional integration and its role in major depressive disorder.
The importance of differentiating between social concepts when appraising actions (e.g., understanding behavior as critical vs. fault-finding) and its contribution to vulnerability to major depressive disorder (MDD) is unknown. We predicted poor integration of differentiated conceptual knowledge when people with MDD appraise their social actions, contributing to their tendency to grossly overgeneralize self-blame (e.g., "I am unlikable rather than critical"). To test this hypothesis, we used a neuropsychological test measuring social conceptual differentiation and its relationship with emotional biases in a remitted MDD and a control group. During fMRI, guilt- and indignation-evoking sentences were presented. As predicted, conceptual overgeneralization was associated with increased emotional intensity when appraising social actions. Interdependence of conceptual overgeneralization and negative emotional biases was stronger in MDD (reproducible in the subgroup without medication) and was associated with overgeneralized self-blame. This high conceptual-emotional interdependence was associated with functional disconnection between the right superior anterior temporal lobe (ATL) and right dorsolateral prefrontal cortex (PFC) as well as a septal region across groups when experiencing guilt (SPM8). Strong coupling of conceptual information (ATL) with information about the context of actions and emotions (frontal-subcortical regions) is thus associated with appraisal being less dependent on conceptual overgeneralization, thereby protecting against excessive self-blame.


Development of an acute model of inhalational melioidosis in the common marmoset (Callithrix jacchus).
Studies of inhalational melioidosis were undertaken in the common marmoset (Callithrix jacchus). Following exposure to an inhaled challenge with aerosolized Burkholderia pseudomallei, lethal infection was observed in marmosets challenged with doses below 10 cfu; a precise LD(50) determination was not possible. The model was further characterized using a target challenge dose of approximately 10(2) cfu. A separate pathogenesis time-course experiment was also conducted. All animals succumbed, between 27 and 78 h postchallenge. The challenge dose received and the time to the humane endpoint (1 °C below normal body temperature postfever) were correlated. The first indicator of disease was an increased core body temperature (T(c) ), at 22 h postchallenge. This coincided with bacteraemia and bacterial dissemination. Overt clinical signs were first observed 3-5 h later. A sharp decrease (typically within 3-6 h) in the T(c) was observed prior to humanely culling the animals in the lethality study. Pathology was noted in the lung, liver and spleen. Disease progression in the common marmoset appears to be consistent with human infection in terms of bacterial spread, pathology and physiology. The common marmoset can therefore be considered a suitable animal model for further studies of inhalational melioidosis.


Pheochromocytomas and paragangliomas in humans and dogs.
Pheochromocytomas (PCCs) and paragangliomas (PGLs) are described in several species. In humans and dogs they have many similarities: the excessive catecholamine release in hormonally active PCC causes similar clinical signs, the frequency of metastasis is similar, and they are histopathologically almost identical. Surgery is curative when PCC and PGL have not metastasized, while only palliative treatment is possible for patients with metastatic disease. Mutations in succinate dehydrogenase subunit B (SDHB) are associated with metastatic behaviour in human PCC/PGL and the same mutation has been described in dogs. The dog might therefore be a suitable model for study of the pathogenesis of metastatic PCC and PGL in humans. Further molecular studies of common tumourigenic pathways and comparative studies of histopathology of human and canine PCC and PGL are warranted.


Rotational dynamics of the single tryptophan of porcine pancreatic phospholipase A2, its zymogen, and an enzyme/micelle complex. A steady-state and time-resolved anisotropy study.
The rotational dynamics of the single tryptophan of porcine pancreatic phospholipase A2 and its zymogen (prophospholipase A2) have been studied by polarized fluorescence using steady-state and time-resolved single-photon counting techniques. The motion of Trp-3 in phospholipase A2 consists of a rapid subnanosecond wobble of the indole ring with an amplitude of about +/- 20 degrees accompanied by slower isotropic rotation of the entire protein. The rotational correlation times for overall particle rotational diffusion are consistent with conventional hydrodynamic theory. When phospholipase A2 binds to micelles of n-hexadecylphosphocholine, the amplitude of the fast ring rotation decreases. The whole particle rotational correlation time of the enzyme/micelle complex is smaller than the minimum value calculated from hydrodynamic theory. A similar result is obtained for the micelle itself by using the lipophilic probe transparinaric acid. These low values for the particle correlation times can be understood by postulating that an isotropic motion of the fluorophore in the small detergent particles contributes to the angular reorientation of the fluorophore. The internal reorientational motion of the tryptophan in the zymogen, prophospholipase A2, is of larger amplitude than that observed for the enzyme; specifically, the proenzyme exhibits a motion with a significant amplitude on the nanosecond time scale. This additional freedom of motion is attributed to segmental mobility of the N-terminal residues of prophospholipase A2. This demonstrates that this region of the protein is flexible in the zymogen but not in the processed enzyme. The implications of these findings for the mechanism of surface activation of phospholipase A2 are discussed by analogy with a trypsinogen-trypsin activation model.


A Proposal to Address NFL Club Doctors' Conflicts of Interest and to Promote Player Trust.
How can we ensure that players in the National Football League receive excellent health care they can trust from providers who are as free from conflicts of interest as realistically possible? NFL players typically receive care from the club's own medical staff. Club doctors are clearly important stakeholders in player health. They diagnose and treat players for a variety of ailments, physical and mental, while making recommendations to the player concerning those ailments. At the same time, club doctors have obligations to the club, namely to inform and advise clubs about the health status of players. While players and clubs share an interest in player health-both of them want players to be healthy so they can play at peak performance-there are several areas where their interests can diverge, and the divergence presents legal and ethical challenges. The current structure forces club doctors to have obligations to two parties-the club and the player-and to make difficult judgments about when one party's interests must yield to another's. None of the three parties involved should prefer this conflicted approach. We propose to resolve the problem of dual loyalty by largely severing the club doctor's ties with the club and refashioning that role into one of singular loyalty to the player-patient. The main idea is to separate the roles of serving the player and serving the club and replace them with two distinct sets of medical professionals: the Players' Medical Staff (with exclusive loyalty to the player) and the Club Evaluation Doctor (with exclusive loyalty to the club). We begin by explaining the broad ethical principles that guide us and that help shape our recommendation. We then provide a description of the role of the club doctor in the current system. After explaining the concern about the current NFL player health care structure, we provide a recommendation for improving this structure. We then discuss how the club medical staff fits into the broader microenvironment affecting player health.


High-numerical-aperture focusing of radially polarized doughnut beams with a parabolic mirror and a flat diffractive lens.
Recently, Dorn et al. [Phys. Rev. Lett. 91, 233901 (2003)] demonstrated the significance of radially polarized doughnut beams in obtaining very small focal spots (with an area of approximately 0.26 lambda2) with high-numerical-aperture (NA) aplanatic microscope objectives. We propose two simple alternative ways to focus such radially polarized beams: a parabolic mirror and a flat diffractive lens. Because of their large apodization factor for a high NA, a significant further reduction in spot area (up to a factor of 1.76 at a NA of 1) compared with the aplanatic system can be achieved.


Indications for vena caval fenestration.
A retrospective study of 20 patients who underwent vena caval fenestration showed that in 50% of the patients the procedure was done for prophylaxis and in 50% it was done for therapeutic reasons. After this procedure five patients had persistent leg swelling, two had deep venous thrombosis, two had pulmonary emboli and one died of a respiratory arrest. We recommend limiting the use of vena caval fenestration to those patients who have verified pulmonary embolism while adequately anticoagulated or patients who have pulmonary embolism and a major contraindication to anticoagulation.


Longitudinal change in food habits between adolescence (11-12 years) and adulthood (32-33 years): the ASH30 Study.
Links between diet in childhood and the prevention of disease in adulthood have been established. This longitudinal dietary survey provided quantitative evidence of dietary change from adolescence to adulthood, in Northumberland, England. To report longitudinal dietary change in 198 respondents between the ages of 12 and 33 years, to explore dietary 'tracking' between the same time points and to describe the effects of gender, socio-economic status and location on dietary change. A longitudinal study recorded dietary change from adolescence to adulthood. Two 3-day food diaries were collected in 1980 and 2000 from the same 198 respondents. Foods consumed were assigned to the five categories in the Balance of Good Health (BGH). Demographic and socio-economic information were obtained in 1980 and 2000. Intakes of foods containing fat and/ or sugar and milk and dairy foods decreased (p < 0.01 and p < 0.031, respectively), while intakes of fruits and vegetables increased (p < 0.01). Intakes of bread, other cereals and potatoes (p = 0.002, r = +0.219); fruits and vegetables (p < 0.01, r = +0.256) and meat, fish and alternatives (p = 0.026, r = +0.158) 'tracked' from adolescence to adulthood. Men had increased intake from meat, fish and alternatives and decreased milk and dairy foods more than female respondents (p = 0.003 and p = 0.019). Respondents who had moved away from Northumberland had a greater increase in intake of fruits and vegetables compared with those who remained in the local (p = 0.010). Individuals who had moved to a lower socio-economic group had increased their intake of bread, other cereals and potatoes (p = 0.040). Food intake changed considerably in a direction more in the line with current dietary recommendations. Food intake in adolescence was a significant, but not strong, predictor of intake in adulthood. Dietary change is influenced by variables including gender, location and socio-economic status.


IMPROVE trial: a randomized controlled trial of patient-controlled analgesia for sickle cell painful episodes: rationale, design challenges, initial experience, and recommendations for future studies.
The hallmark of sickle cell disease (SCD) is pain from a vaso-occlusive crisis. Although ambulatory pain accounts for most days in pain, pain is also the most common cause of hospitalization and is typically treated with parenteral opioids. The evidence base is lacking for most analgesic practice in SCD, particularly for the optimal opioid dosing for patient-controlled analgesia (PCA), in part because of the challenges of the trial design and conduct for this rare disease. The purpose of this report is to describe our Network's experiences with protocol development, implementation, and analysis, including overall study design, the value of pain assessments rather than 'crisis' resolution as trial endpoints, and alternative statistical analysis strategies. The Improving Pain Management and Outcomes with Various Strategies (IMPROVE) PCA trial was a multisite inpatient randomized controlled trial comparing two PCA-dosing strategies in adults and children with SCD and acute pain conducted by the SCD Clinical Research Network. The specified primary endpoint was a 25-mm change in a daily average pain intensity using a Visual Analogue Scale, and a number of related pain intensity and pain interference measures were selected as secondary efficacy outcomes. A time-to-event analysis strategy was planned for the primary endpoint. Of 1116 individuals admitted for pain at 31 participating sites over a 6-month period, 38 were randomized and 4 withdrawn. The trial was closed early due to poor accrual, reflecting a substantial number of challenges encountered during trial implementation. While some of the design issues were unique to SCD or analgesic studies, many of the trial implementation challenges reflected the increasing complexity of conducting clinical trials in the inpatient setting with multiple care providers and evolving electronic medical record systems, particularly in the context of large urban academic medical centers. Complicated clinical organization of many sites likely slowed study initiation. More extensive involvement of research staff and site principal investigator in the clinical care operations improved site performance. During the subsequent data analysis, alternative statistical approaches were considered, the results of which should inform future efficacy assessments and increase future trial recruitment success by allowing substantial reductions in target sample size. A complex randomized analgesic trial was initiated within a multisite disease network seeking to provide an evidence base for clinical care. A number of design considerations were shown to be feasible in this setting, and several pain intensity and pain interference measures were shown to be sensitive to time- and treatment-related improvements. While the premature closure and small sample size precluded definitive conclusions regarding treatment efficacy, this trial furnishes a template for design and implementation considerations that should improve future SCD analgesic trials.


Genetic basis for the hierarchical interaction between Tobamovirus spp. and L resistance gene alleles from different pepper species.
The pepper L gene conditions the plant's resistance to Tobamovirus spp. Alleles L(1), L(2), L(3), and L(4) confer a broadening spectra of resistance to different virus pathotypes. In this study, we report the genetic basis for the hierarchical interaction between L genes and Tobamovirus pathotypes. We cloned L(3) using map-based methods, and L(1), L(1a), L(1c), L(2), L(2b), and L(4) using a homology-based method. L gene alleles encode coiled-coil, nucleotide-binding, leucine-rich repeat (LRR)-type resistance proteins with the ability to induce resistance response to the viral coat protein (CP) avirulence effectors by themselves. Their different recognition spectra in original pepper species were reproduced in an Agrobacterium tumefaciens-mediated transient expression system in Nicotiana benthamiana. Chimera analysis with L(1), which showed the narrowest recognition spectrum, indicates that the broader recognition spectra conferred by L(2), L(2b), L(3), and L(4) require different subregions of the LRR domain. We identified a critical amino acid residue for the determination of recognition spectra but other regions also influenced the L genes' resistance spectra. The results suggest that the hierarchical interactions between L genes and Tobamovirus spp. are determined by the interaction of multiple subregions of the LRR domain of L proteins with different viral CP themselves or some protein complexes including them.


Safety and Efficacy of Pembrolizumab in Combination with Acalabrutinib in Advanced Head and Neck Squamous Cell Carcinoma: Phase 2 Proof-of-Concept Study.
Programmed cell death-1 (PD-1) receptor inhibitors have shown efficacy in head and neck squamous cell carcinoma (HNSCC), but treatment failure or secondary resistance occurs in most patients. In preclinical murine carcinoma models, inhibition of Bruton's tyrosine kinase (BTK) induces myeloid cell reprogramming that subsequently bolsters CD8+ T cell responses, resulting in enhanced antitumor activity. This phase 2, multicenter, open-label, randomized study evaluated pembrolizumab (anti-PD-1 monoclonal antibody) plus acalabrutinib (BTK inhibitor) in recurrent or metastatic HNSCC. Patients received pembrolizumab 200 mg intravenously every 3 weeks, alone or in combination with acalabrutinib 100 mg orally twice daily. Safety and overall response rate (ORR) were co-primary objectives. The secondary objectives were progression-free survival (PFS) and overall survival. Seventy-six patients were evaluated (pembrolizumab, n = 39; pembrolizumab + acalabrutinib, n = 37). Higher frequencies of grade 3-4 treatment-emergent adverse events (AE; 65% vs. 39%) and serious AEs (68% vs. 31%) were observed with combination therapy versus monotherapy. ORR was 18% with monotherapy versus 14% with combination therapy. Median PFS was 2.7 [95% confidence interval (CI), 1.4-6.8] months in the combination arm and 1.7 (95% CI, 1.4-4.0) months in the monotherapy arm. The study was terminated due to lack of clinical benefit with combination treatment. To assess how tumor immune contexture was affected by therapy in patients with pre- and post-treatment biopsies, spatial proteomic analyses were conducted that revealed a trend toward increased CD45+ leukocyte infiltration of tumors from baseline at day 43 with pembrolizumab (monotherapy, n = 5; combination, n = 2), which appeared to be higher in combination-treated patients; however, definitive conclusions could not be drawn due to limited sample size. Despite lack of clinical efficacy, immune subset analyses suggest that there are additive effects of this combination; however, the associated toxicity limits the feasibility of combination treatment with pembrolizumab and acalabrutinib in patients with recurrent or metastatic HNSCC.


Association of polymorphisms in the ALOX15B gene with coronary artery disease.
Atherosclerosis is a multifactorial disease and the underlying cause of coronary artery disease (CAD), myocardial infarction and stroke. Two main features are involved in the progression of atherosclerosis, lipid retention and inflammation. 12/15-lipoxygenases are involved in inflammation and have been implicated in atherosclerosis. Genetic association studies of the 15-lipoxygenase 1 (ALOX15) in humans revealed a neutral to atheroprotective role of the enzyme. Recently the epidermis-type 15-lipoxygenase 2 (ALOX15B) has been identified in human atherosclerotic plaques but its role in human atherosclerosis is still unclear. We screened the ALOX15B gene for polymorphisms and investigated the association of 18 detected polymorphisms with angiographically documented CAD in a case-control study (n=496). In addition, we measured in vitro the enzyme activity and Michaelis-Menten kinetics of the detected non-synonymous polymorphic variants p.Arg486His (c.1457G>A), p.Gln656Arg (c.1967A>G) and p.Ile676Val (c.2026A>G). We found that the linked polymorphisms at position c.1458-38G>C, c.1579+71C>T and c.1656G>A are associated with CAD (OR: 0.51 (0.27-0.94), p-value: 0.03). In addition, we show that the activity and the kinetics of the three non-synonymous ALOX15B enzyme variants (p.Arg486His, p.Gln656Arg and p.Ile676Val) are similar to the wild-type enzyme. Our data indicate that the ALOX15B gene may be associated with coronary artery disease. However, larger studies would be necessary to confirm the association of these polymorphisms with CAD. In contrast, our study did not find frequent non-synonymous polymorphisms in ALOX15B altering enzyme activity in Europeans.


Diagnosing Topological Edge States via Entanglement Monogamy.
Topological phases of matter possess intricate correlation patterns typically probed by entanglement entropies or entanglement spectra. In this Letter, we propose an alternative approach to assessing topologically induced edge states in free and interacting fermionic systems. We do so by focussing on the fermionic covariance matrix. This matrix is often tractable either analytically or numerically, and it precisely captures the relevant correlations of the system. By invoking the concept of monogamy of entanglement, we show that highly entangled states supported across a system bipartition are largely disentangled from the rest of the system, thus, usually appearing as gapless edge states. We then define an entanglement qualifier that identifies the presence of topological edge states based purely on correlations present in the ground states. We demonstrate the versatility of this qualifier by applying it to various free and interacting fermionic topological systems.


The hypoxic tumor microenvironment and gene expression.
Solid tumors are not static entities but are constantly responding to environmental signals as they grow and develop. One mechanism by which they respond to the adverse conditions of the tumor microenvironment is through coordinated changes in gene expression. The synchronized turning of genes on and off leads to biologic adaption to the adverse oxygen-poor environment. Because tumor hypoxia can be found in almost every solid tumor, it represents one of the most pervasive microenvironmental stresses that can impact malignant progression and therapeutic response. Interestingly, tumors that exhibit robust induction of hypoxia-responsive gene expression networks show a clinically more aggressive natural history. The contribution of hypoxia-responsive gene networks to malignant response is currently under investigation. An understanding of the coordinated functions of hypoxia induced and repressed genes can lead to a better understanding of the clinical significance of the hypoxic tumor phenotype.


Isoreticular expansion of polyMOFs achieves high surface area materials.
The concept of isoreticular chemistry has become a core principle in metal-organic framework (MOF) materials. Isoreticular chemistry has shown that organic ligands of different sizes, but with a common geometry/symmetry can be used to generate MOFs of related topologies, but with expanded pore sizes and volumes. In this report, polymer-MOF hybrid materials (polyMOFs) with a UiO (UiO = University of Oslo) architecture are shown to adhere to the principle of isoreticular expansion, generating polyMOFs with large surface areas and enhanced stability.


Reciprocal regulation of c-Src and STAT3 in non-small cell lung cancer.
Signal transducer and activator of transcription-3 (STAT3) is downstream of growth factor and cytokine receptors, and regulates key oncogenic pathways in non-small cell lung cancer (NSCLC). Activation of STAT3 by cellular Src (c-Src) promotes tumor progression. We hypothesized that c-Src inhibition could activate STAT3 by inducing a homeostatic feedback loop, contributing to c-Src inhibitor resistance. The effects of c-Src inhibition on total and phosphorylated STAT3 were measured in NSCLC cell lines and in murine xenograft models by Western blotting. c-Src and STAT3 activity as indicated by phosphorylation was determined in 46 human tumors and paired normal lung by reverse phase protein array. Modulation of dasatinib (c-Src inhibitor) cytotoxicity by STAT3 knockdown was measured by MTT, cell cycle, and apoptosis assays. Depletion of c-Src by small interfering RNA or sustained inhibition by dasatinib increased pSTAT3, which could be blocked by inhibition of JAK. Similarly, in vivo pSTAT3 levels initially decreased but were strongly induced after sustained dasatinib treatment. In human tumors, phosphorylation of the autoinhibitory site of c-Src (Y527) correlated with STAT3 phosphorylation (r = 0.64; P = 2.5 x 10(-6)). STAT3 knockdown enhanced the cytotoxicity of dasatinib. c-Src inhibition leads to JAK-dependent STAT3 activation in vitro and in vivo. STAT3 knockdown enhances the cytotoxicity of dasatinib, suggesting a compensatory pathway that allows NSCLC survival. Data from human tumors showed a reciprocal regulation of c-Src and STAT3 activation, suggesting that this compensatory pathway functions in human NSCLC. These results provide a rationale for combining c-Src and STAT3 inhibition to improve clinical responses.


Hybrid incompatibility arises in a sequence-based bioenergetic model of transcription factor binding.
Postzygotic isolation between incipient species results from the accumulation of incompatibilities that arise as a consequence of genetic divergence. When phenotypes are determined by regulatory interactions, hybrid incompatibility can evolve even as a consequence of parallel adaptation in parental populations because interacting genes can produce the same phenotype through incompatible allelic combinations. We explore the evolutionary conditions that promote and constrain hybrid incompatibility in regulatory networks using a bioenergetic model (combining thermodynamics and kinetics) of transcriptional regulation, considering the bioenergetic basis of molecular interactions between transcription factors (TFs) and their binding sites. The bioenergetic parameters consider the free energy of formation of the bond between the TF and its binding site and the availability of TFs in the intracellular environment. Together these determine fractional occupancy of the TF on the promoter site, the degree of subsequent gene expression and in diploids, and the degree of dominance among allelic interactions. This results in a sigmoid genotype-phenotype map and fitness landscape, with the details of the shape determining the degree of bioenergetic evolutionary constraint on hybrid incompatibility. Using individual-based simulations, we subjected two allopatric populations to parallel directional or stabilizing selection. Misregulation of hybrid gene expression occurred under either type of selection, although it evolved faster under directional selection. Under directional selection, the extent of hybrid incompatibility increased with the slope of the genotype-phenotype map near the derived parental expression level. Under stabilizing selection, hybrid incompatibility arose from compensatory mutations and was greater when the bioenergetic properties of the interaction caused the space of nearly neutral genotypes around the stable expression level to be wide. F2's showed higher hybrid incompatibility than F1's to the extent that the bioenergetic properties favored dominant regulatory interactions. The present model is a mechanistically explicit case of the Bateson-Dobzhansky-Muller model, connecting environmental selective pressure to hybrid incompatibility through the molecular mechanism of regulatory divergence. The bioenergetic parameters that determine expression represent measurable properties of transcriptional regulation, providing a predictive framework for empirical studies of how phenotypic evolution results in epistatic incompatibility at the molecular level in hybrids.


Dissecting MHC class II export, B cell maturation, and DM stability defects in invariant chain mutant mice.
Invariant (Ii) chain loss causes defective class II export, B cell maturation, and reduced DM stability. In this study, we compare Ii chain and class II mutant mouse phenotypes to dissect these disturbances. The present results demonstrate that ER retention of alphabeta complexes, and not beta-chain aggregates, disrupts B cell development. In contrast, we fail to detect class II aggregates in Ii chain mutant thymi. Ii chain loss in NOD mice leads to defective class II export and formation of alphabeta aggregates, but in this background, downstream signals are misregulated and mature B cells develop normally. Finally, Ii chain mutant strains all display reduced levels of DM, but mice expressing either p31 or p41 alone, and class II single chain mutants, are indistinguishable from wild type. We conclude that Ii chain contributions as a DM chaperone are independent of its role during class II export. This Ii chain/DM partnership favors class II peptide loading via conventional pathway(s).


Viable rabbits derived from reconstructed oocytes by germinal vesicle transfer after intracytoplasmic sperm injection (ICSI).
Abnormal oocyte spindle due to the improper function of ooplasm is associated with female infertility of advanced maternal age. A possible way to overcome this problem is to transfer an oocyte germinal vesicle (GV) which contains genetic materials of a patient with a history of poor embryo development to the cytoplast from a donor oocyte. Here we demonstrate that GV transfer is feasible using a rabbit model. When the GVs were transferred to auto- or hetero-cytoplasts of GV stage oocytes, around 80% of the reconstructed oocytes could mature in vitro and 7.1-9.4% of the oocytes developed to blastocyst stage after intracytoplasmic sperm injection (ICSI). Transfer of 93 fertilized eggs reconstructed via GV transfer into six recipients resulted in two live offspring. Results of this experiment indicate that GV transfer can potentially become a new approach in treatment of infertility because of advanced maternal age.


Boosting BCG with MVA85A: the first candidate subunit vaccine for tuberculosis in clinical trials.
There is an urgent need for an improved vaccine against tuberculosis. Heterologous prime-boost immunization regimes induce higher levels of cellular immunity than homologous boosting with the same vaccine. Using BCG as the priming immunization in such a regime allows for the retention of the beneficial protective effects of BCG against disseminated disease in childhood. Recombinant poxviruses are powerful boosting agents, for both CD4+ and CD8+ T cells. Here we review the preclinical data from a BCG prime-recombinant modified vaccinia virus Ankara expressing antigen 85A (MVA85A) boost strategy. MVA85A is now in clinical trials in the UK and Africa and the design of these trials, including the ethical and regulatory issues are discussed.


Angiotensin extraction by trout tissues in vivo and metabolism by the perfused gill.
Plasma clearance and tissue accumulation of 125I-angiotensin I, [Asp1, Ile5]ANG I, and [14C]sucrose, an inert volume reference, were measured after a bolus injection into the dorsal aorta of rainbow trout, Salmo gairdneri. Retention and metabolism of ANG I to angiotensin II (ANG II) and their constituent 1-4 peptide by the gill were examined using an isolated perfused arch preparation in which outflow from the respiratory and central filamental (venous) pathways was separated. Clearance of ANG I from plasma is multiexponential, reflecting dilution and tissue extraction. Liver, bile, gonads, corpuscles of Stannius, and white skeletal muscle accumulate more 125I than 14C; gill tissue accumulates less 125I than 14C. ANG I and II are retained by the perfused gill longer than the inert vascular marker sucrose, even though the distribution volumes of the former are less. The gill respiratory pathway converts ANG I to ANG II whereas the venous pathway metabolizes either ANG I or II to the 1-4 peptide and other metabolites. The gill respiratory pathway is in series with the systemic vasculature, has a large blood-cell contact area, and, like the mammalian lung, is ideally suited to activate ANG I. The gill venous pathway is in parallel with the systemic vasculature and removes ANG II from the circulation. During stress, elevated plasma catecholamines may reduce venous perfusion and thereby help maintain elevated circulating ANG II levels through reduced venous metabolism.


SEPT9_i1 and genomic instability: mechanistic insights and relevance to tumorigenesis.
Septins are highly conserved cytoskeletal GTP-binding proteins implicated in numerous cellular processes from apoptosis to vesicle trafficking. Septins have been associated with leukemia and solid tumor malignancies, including breast, ovarian, and prostate. We previously reported that high SEPT9_i1 expression in human mammary epithelial cell lines (HMECs) led to malignant cellular phenotypes such as increased cell proliferation, invasiveness, motility, and genomic instability. Our goal here was to better understand how SEPT9_i1 expression might contribute to genomic instability and malignant progression. First, we confirmed that even transient expression of SEPT9_i1 was sufficient to increase aneuploidy in HMECs. We then analyzed SEPT9_i1 by immunoprecipitation and immunofluorescence studies and found that SEPT9_i1 interacts with both α and γ tubulin. SEPT9_i1 expressing cells demonstrated dramatic chromosome segregation defects, centrosome amplification and cytokinesis defects, suggesting two possible molecular mechanisms contributing to the development of genomic instability. This suggests that SEPT9_i1 may promote genomic instability through both cytokinesis and mitotic spindle defects which lead to chromosome missegregation.


Strategies for evaluating clinical change: implications for practice and research.
The methods and procedures of evaluation research using single-system or small-N designs are defined and described. Various components of single-system evaluation research, including specifying the problem, measuring and recording the data, selecting an appropriate design, and analyzing the data, are presented and discussed. The argument is made that single-system evaluation research methods are ideally suited to document clinical change on an individual basis and can provide a mechanism for establishing therapeutic accountability. The relationship between single-system evaluation research and the more traditional experimental procedures is briefly explored, and the implications for establishing an empirically derived basis for clinical practice are discussed.


Resistant starch as a prebiotic and synbiotic: state of the art.
Non-infectious diseases such as CHD and certain cancers have become major causes of death and disability in affluent countries. Probiotics (principally lactic acid bacteria; LAB) may assist in lowering the risk of these diseases. Experimental studies with probiotics have given generally inconclusive outcomes for infectious disease and for biomarkers for non-infectious disease. In part this situation may reflect their inability to colonise the adult human gut effectively. Prebiotics can assist in promoting colonisation, and resistant starch (RS), as a high-amylose starch, is a prebiotic and synbiotic. This starch exerts its synbiotic action through adhesion of the bacteria to the granule surface. Consumption of RS assists in recovery from infectious diarrhoea in man and animals. A rice porridge, high in RS, appears to modify the autochthonous porcine large-bowel microflora favourably through lowering Escherichia coli and coliform numbers. Many of the beneficial effects of RS on large-bowel function appear to be exerted through short-chain fatty acids (SCFA)formed by bacterial fermentation. In man LAB are found in relatively highest numbers in milk-fed infants where the profile of fermentation products differs quite markedly from that in adults. It appears unlikely that ingestion of current probiotics will alter either total SCFA or the proportions of the major acids. More emphasis needs to be given to the investigation of the effects of complex carbohydrates, including RS, on the autochthonous microflora of the human large bowel.


Technological Advancements in Echocardiographic Assessment of Thoracic Aortic Dilatation: Head to Head Comparison Among Multidetector Computed Tomography, 2-Dimensional, and 3-Dimensional Echocardiography Measurements.
The aim of this study was to evaluate the feasibility and accuracy of 2-dimensional (2D) and 3-dimensional (3D) transthoracic echocardiography (2DTTE, 3DTTE) versus multidetector computed tomography (MDCT) in patients with ascending aortic (AA) dilation. Fifty consecutive patients with AA dilation were evaluated by 2DTTE, X-plane (XP) 3DTTE, and MDCT. Aorta diameters were measured at aortic annulus, aortic root (SIN), sinotubular junction, AA, aortic arch before the prebrachiocephalic artery (PRE), and before left subclavian artery (INTRA). Leading edge-to-leading edge (L-L) and inner-to-inner (I-I) measurements were compared with MDCT data. Feasibility, quality of imaging, and accuracy was high with all echocardiographic methods. Specifically for MDCT maximum SIN diameter, the best correlation and agreement was obtained using XP maximum diameter at 3DTTE (MDCT: 44.8±7.4 mm vs. XP: 44.4±7.4 mm; r=0.975; bias=-0.4 mm). The same was true for AA maximum diameter at MDCT (MDCT: 46.6±8.1 mm vs. XP: 47.5±8.1 mm; r=0.991; bias=0.1 mm). For aortic arch the best correlation and agreement with MDCT were as follows: 2DTTE L-L diameter for arch PRE (MDCT: 37.9±5.3 mm vs. TTE: 36.6±4.5 mm; r=0.927; bias=-0.9 mm) and MDCT minimum diameter with XP minimum diameter for arch INTRA (MDCT: 28.2±5.0 mm vs. TTE 28.8±4.7 mm; r=0.939; bias=-0.3 mm). In patients with aortic dilatation or aneurysm, new techniques (mainly 2D-3D probes allowing XP views) facilitate accuracy of aortic measurements at different sites of the vessel and allow standardization of analysis to better compare with MDCT.


Standard-dose vs. low-dose CT protocols in the evaluation of localized lung lesions: Capability for lesion characterization-iLEAD study.
To determine the lesion characterization capability by low dose CT for localized lung lesions in comparison with standard dose CT. Approval for this study was granted by our Institutional Review Board. Fifty-two consecutive patients (36 males and 16 females, median age of 71 years.) who had CT examinations for evaluation of lung lesions comprise the study population. Two chest CT scans were performed with current time product of 50 and 150 mAs at 120 kVp, with the same scan length with a 16 detector-row CT scanner. Three readers evaluated 52 target lesions and assigned an overall impression score to each target lesion, using a 5 point scale from 1 (definitely benign) to 5 (definitely malignant). Six features of the lesions including lesion type, margin characteristics, calcification, lobulation, speculation, and pleural indentation were also reported with 5-point scales. The weighted kappa analyses and receiver operating characteristic analysis were used for analysis. The mean kappa value between low-and standard-dose CT was 0.82 for overall impression of the lesions, showing almost perfect agreement. Area under the curve of low-dose CT (Az = 0.74) had no significant difference from that of standard-dose CT (Az = 0.74) (p = 0.61). The kappa values for six lesion features ranged from 0.45 to 0.83, showing moderate to almost perfect agreement. Lesion characterization capability by low-dose CT images was comparable to that by standard-dose CT images and therefore sufficient for evaluation of localized lung lesions.


Treatment of aggressive pituitary tumours and carcinomas: results of a European Society of Endocrinology (ESE) survey 2016.
To collect outcome data in a large cohort of patients with aggressive pituitary tumours (APT)/carcinomas (PC) and specifically report effects of temozolomide (TMZ) treatment. Electronic survey to ESE members Dec 2015-Nov 2016. Reports on 166 patients (40 PC, 125 APT, 1 unclassified) were obtained. Median age at diagnosis was 43 (range 4-79) years. 69% of the tumours were clinically functioning, and the most frequent immunohistochemical subtype were corticotroph tumours (45%). Ki-67 index did not distinguish APT from PC, median 7% and 10% respectively. TMZ was first-line chemotherapy in 157 patients. At the end of the treatment (median 9 cycles), radiological evaluation showed complete response (CR) in 6%, partial response (PR) in 31%, stable disease (SD) in 33% and progressive disease in 30%. Response was more frequent in patients receiving concomitant radiotherapy and TMZ. CR was seen only in patients with low MGMT expression. Clinically functioning tumours were more likely to respond than non-functioning tumours, independent of MGMT status. Of patients with CR, PR and SD, 25, 40 and 48% respectively progressed after a median of 12-month follow-up. Other oncological drugs given as primary treatment and to TMZ failures resulted in PR in 20%. This survey confirms that TMZ is established as first-line chemotherapeutic treatment of APT/PC. Clinically functioning tumours, low MGMT and concurrent radiotherapy were associated with a better response. The limited long-term effect of TMZ and the poor efficacy of other drugs highlight the need to identify additional effective therapies.


Slowed Metabolic Decline After 1 Year of Oral Insulin Treatment Among Individuals at High Risk for Type 1 Diabetes in the Diabetes Prevention Trial-Type 1 (DPT-1) and TrialNet Oral Insulin Prevention Trials.
We assessed whether oral insulin slowed metabolic decline after 1 year of treatment in individuals at high risk for type 1 diabetes. Two oral insulin trials that did not show efficacy overall and had type 1 diabetes as the primary end point were analyzed: the Diabetes Prevention Trial-Type 1 (DPT-1) and the TrialNet oral insulin trials. Oral glucose tolerance tests at baseline and after 1 year of treatment were analyzed. Among those at high risk (with a Diabetes Prevention Trial-Type 1 Risk Score [DPTRS] ≥6.75), the area under the curve (AUC) C-peptide increased significantly from baseline to 1 year in each oral insulin group, whereas the AUC glucose increased significantly in each placebo group. At 1 year, the AUC C-peptide/AUC glucose (AUC Ratio) was significantly higher in the oral insulin group than in the placebo group in each trial (P < 0.05; P = 0.057 when adjusted for age in the TrialNet trial) and in both trials combined (P < 0.01 with or without adjustment for age). For a DPTRS <6.75, oral insulin groups did not differ from placebo groups in the AUC Ratio. The findings suggest that 1 year of treatment with oral insulin slows metabolic deterioration in individuals at high risk for type 1 diabetes. Moreover, the findings further suggest that metabolic end points can be useful adjuncts to the diagnostic end point in assessments of preventive treatments for the disorder.


Prolonged postdiapause: influence on some indicators of carbohydrate and lipid metabolism of the red mason bee, Osmia rufa.
Bees of the genus Osmia are being used in crop pollination at an increasing rate. However, a short life expectancy of adult individuals limits the feasibility of their use. Cocoons of the red mason bee, Osmia rufa L. (Hymenoptera: Megachilidae), can be stored at 4° C in a postdiapause state, and adult bees can be used for pollination outside their natural flight period. The period of storage in this form has an unfavorable influence on the survival rate, life expectancy, and fertility of the bee. It was suggested that the negative results are connected with exhaustion of energy reserves. To test this hypothesis, the present study examined the contents of protein, carbohydrates, lipids, and the activities of some enzymes, and their degradation in red mason bees that emerged in spring according to their biological clock and in summer after elongated diapause. It was found that postdiapause artificially elongated by 3 months caused significant decreases in body weight, total sugar, glycogen, lipids, and protein content in O. rufa. Glucose level was highest in bees that emerged in the summer, which was coincident with increased activities of maltase and trehalase. The activities of sucrase and cellobiase were not changed, while amylase activity was considerably decreased. The activities of triacylglycerols lipase and C2, C4, C10 carboxylesterases were highest in bees that emerged in July. Low temperatures restrict O. rufa emergence, and during prolonged postdiapause, metabolic processes lead to significant reductions of structural and energetic compounds.


SU-E-I-23: Effective Estimation of Focal Spot Isocenter for Sinograms Obtained with Flying Focal Spot Scans for Filtered Back Projection.
To validate the determination of parameters required to preprocess sinograms obtained by fan beam CT with flying focal spot (FFS) scans, such that a combined data set could be used with existing filtered back projection (FBP) reconstruction software based on single focal spot (SFS) geometry. The preprocessing step requires determination of the isocenter for each FFS in the target reconstruction geometry. A Philips Brilliance Big Bore CT was used to obtain FFS sinograms, which double samples the number of gantry positions relative to SFS sinograms, having the same number of detector measurements for each gantry position. Mass weighting of sinograms of a Teflon rod scan and curve fitting were used to determine isocenters for focal spots. FFS sinograms were reformatted before FBP such that each pair of adjacent gantry-step measurements were combined by interleaving detector readings. The interleaved measurements were assigned to the average gantry angle. Image transition sharpness was compared for 33 images of the Teflon rod reconstructed with 1/8 focal spot deflection difference to test the isocenter Result. SFS and FFS Sinograms of a high- resolution bar pattern phantom were reconstructed by an in-house reconstruction code package for resolution comparison. In-house reconstructed images of the Teflon rod were compared with scanner images for verifying the effectiveness of this sinogram reformatting method. Image profile was the sharpest when the Teflon rod sinogram was reconstructed with isocenter parameters found by the curve fitting experiment. It was comparable to the sharpness of scanner image profile when the in-house image was reconstructed with resolution of 1.0mm FWHM. The good clinical resolution of images reconstructed with investigated FFS isocenters suggests that the experiment of estimating focal spot centers and reformatting sinograms is effective for utilizing reconstruction software that does not support FFS geometry. Supported by grant R01 CA149305 and a grant from Varian Medical Systems.


3D molecular network and magnetic ordering, formed by multi-dentate magnetic couplers, bis(benzene)chromium(i) and [1,2,5]thiadiazolo[3,4-c][1,2,5]thiadiazolidyl.
The reaction between bis(benzene)chromium(0), Cr0(C6H6)2, and [1,2,5]thiadiazolo[3,4-c][1,2,5]thiadiazole (abbreviated as TDTD) formed single crystals of the 1 : 1 salt, [CrI(C6H6)2]+[TDTD]-. The crystal structure of [Cr(C6H6)2][TDTD] belongs to the monoclinic P21/c space group, and involves a CdSO4-type network (or quartz dual net), which is formed by CHN hydrogen bonds between [Cr(C6H6)2]+ (S = 1/2) and [TDTD]- (S = 1/2). In addition to this network, the two components form an alternating chain crystal with a π-π overlap along the [110] and [11[combining macron]0] directions. The theoretical calculations for the pairwise intermolecular magnetic exchange interactions in [Cr(C6H6)2][TDTD] reveal the presence of 3D interactions, ranging from an antiferromagnetic interaction of -8.96 cm-1 to a ferromagnetic one of 1.70 cm-1. The temperature dependence of the paramagnetic susceptibility χp indicates the dominance of an antiferromagnetic interaction with a negative Weiss constant of -4.8 K and a magnetic ordering at 8 K, which can be characterized in terms of weak ferromagnetism.


Brain network analysis: separating cost from topology using cost-integration.
A statistically principled way of conducting brain network analysis is still lacking. Comparison of different populations of brain networks is hard because topology is inherently dependent on wiring cost, where cost is defined as the number of edges in an unweighted graph. In this paper, we evaluate the benefits and limitations associated with using cost-integrated topological metrics. Our focus is on comparing populations of weighted undirected graphs that differ in mean association weight, using global efficiency. Our key result shows that integrating over cost is equivalent to controlling for any monotonic transformation of the weight set of a weighted graph. That is, when integrating over cost, we eliminate the differences in topology that may be due to a monotonic transformation of the weight set. Our result holds for any unweighted topological measure, and for any choice of distribution over cost levels. Cost-integration is therefore helpful in disentangling differences in cost from differences in topology. By contrast, we show that the use of the weighted version of a topological metric is generally not a valid approach to this problem. Indeed, we prove that, under weak conditions, the use of the weighted version of global efficiency is equivalent to simply comparing weighted costs. Thus, we recommend the reporting of (i) differences in weighted costs and (ii) differences in cost-integrated topological measures with respect to different distributions over the cost domain. We demonstrate the application of these techniques in a re-analysis of an fMRI working memory task. We also provide a Monte Carlo method for approximating cost-integrated topological measures. Finally, we discuss the limitations of integrating topology over cost, which may pose problems when some weights are zero, when multiplicities exist in the ranks of the weights, and when one expects subtle cost-dependent topological differences, which could be masked by cost-integration.


Earlier development of the accumbens relative to orbitofrontal cortex might underlie risk-taking behavior in adolescents.
Adolescence has been characterized by risk-taking behaviors that can lead to fatal outcomes. This study examined the neurobiological development of neural systems implicated in reward-seeking behaviors. Thirty-seven participants (7-29 years of age) were scanned using event-related functional magnetic resonance imaging and a paradigm that parametrically manipulated reward values. The results show exaggerated accumbens activity, relative to prefrontal activity in adolescents, compared with children and adults, which appeared to be driven by different time courses of development for these regions. Accumbens activity in adolescents looked like that of adults in both extent of activity and sensitivity to reward values, although the magnitude of activity was exaggerated. In contrast, the extent of orbital frontal cortex activity in adolescents looked more like that of children than adults, with less focal patterns of activity. These findings suggest that maturing subcortical systems become disproportionately activated relative to later maturing top-down control systems, biasing the adolescent's action toward immediate over long-term gains.


Advanced modality imaging evaluation in acute ischemic stroke may lead to delayed endovascular reperfusion therapy without improvement in clinical outcomes.
Advanced neuroimaging techniques may improve patient selection for endovascular stroke treatment but may also delay time to reperfusion. We studied the effect of advanced modality imaging with CT perfusion (CTP) or MRI compared with non-contrast CT (NCT) in a multicenter cohort. This is a retrospective study of 10 stroke centers who select patients for endovascular treatment using institutional protocols. Approval was obtained from each institution's review board as only de-identified information was used. We collected demographic and radiographic data, selected time intervals, and outcome data. ANOVA was used to compare the groups (NCT vs CTP vs MRI). Binary logistic regression analysis was performed to determine factors associated with a good clinical outcome. 556 patients were analyzed. Mean age was 66 ± 15 years and median National Institutes of Health Stroke Scale score was 18 (IQR 14-22). NCT was used in 286 (51%) patients, CTP in 190 (34%) patients, and MRI in 80 (14%) patients. NCT patients had significantly lower median times to groin puncture (61 min, IQR (40-117)) compared with CTP (114 min, IQR (81-152)) or MRI (124 min, IQR (87-165)). There were no differences in clinical outcomes, hemorrhage rates, or final infarct volumes among the groups. The current retrospective study shows that multimodal imaging may be associated with delays in treatment without reducing hemorrhage rates or improving clinical outcomes. This exploratory analysis suggests that prospective randomised studies are warranted to support the hypothesis that advanced modality imaging is superior to NCT in improving clinical outcomes.


Ultrasonic defect characterization using parametric-manifold mapping.
The aim of ultrasonic non-destructive evaluation includes the detection and characterization of defects, and an understanding of the nature of defects is essential for the assessment of structural integrity in safety critical systems. In general, the defect characterization challenge involves an estimation of defect parameters from measured data. In this paper, we explore the extent to which defects can be characterized by their ultrasonic scattering behaviour. Given a number of ultrasonic measurements, we show that characterization information can be extracted by projecting the measurement onto a parametric manifold in principal component space. We show that this manifold represents the entirety of the characterization information available from far-field harmonic ultrasound. We seek to understand the nature of this information and hence provide definitive statements on the defect characterization performance that is, in principle, extractable from typical measurement scenarios. In experiments, the characterization problem of surface-breaking cracks and the more general problem of elliptical voids are studied, and a good agreement is achieved between the actual parameter values and the characterization results. The nature of the parametric manifold enables us to explain and quantify why some defects are relatively easy to characterize, whereas others are inherently challenging.


Use of hydrogen/deuterium exchange mass spectrometry and mutagenesis as a tool to identify the binding region of inhibitors targeting the human mitotic kinesin Eg5.
An experimental procedure associating both hydrogen/deuterium exchange mass spectrometry (H/D-MS) and mutagenesis was developed to identify the protein-binding region of small inhibitors targeting the motor domain of the human mitotic kinesin Eg5. All the tested inhibitors decrease the deuterium incorporation rate of the same peptides corresponding to the following secondary structure elements: loop L5/helix alpha2 (region Tyr125-Glu145) and strand beta5/helix alpha3 (region Ile202-Leu227). Replacement of these two regions by the equivalent ones from N. crassa conventional kinesin heavy chain completely abolishes the modification of the deuterium incorporation rate by the inhibitors as well as their effects on the basal ATPase activity. The six tested inhibitors thus share a common binding site on Eg5. The strategy reported here allows the regions of a protein involved in ligand binding to be rapidly pinpointed and can be applied to other proteins and used as a general in vitro screening procedure to identify compounds targeting specific binding regions.


Genetic Variability Under the Seedbank Coalescent.
We analyze patterns of genetic variability of populations in the presence of a large seedbank with the help of a new coalescent structure called the seedbank coalescent. This ancestral process appears naturally as a scaling limit of the genealogy of large populations that sustain seedbanks, if the seedbank size and individual dormancy times are of the same order as those of the active population. Mutations appear as Poisson processes on the active lineages and potentially at reduced rate also on the dormant lineages. The presence of "dormant" lineages leads to qualitatively altered times to the most recent common ancestor and nonclassical patterns of genetic diversity. To illustrate this we provide a Wright-Fisher model with a seedbank component and mutation, motivated from recent models of microbial dormancy, whose genealogy can be described by the seedbank coalescent. Based on our coalescent model, we derive recursions for the expectation and variance of the time to most recent common ancestor, number of segregating sites, pairwise differences, and singletons. Estimates (obtained by simulations) of the distributions of commonly employed distance statistics, in the presence and absence of a seedbank, are compared. The effect of a seedbank on the expected site-frequency spectrum is also investigated using simulations. Our results indicate that the presence of a large seedbank considerably alters the distribution of some distance statistics, as well as the site-frequency spectrum. Thus, one should be able to detect from genetic data the presence of a large seedbank in natural populations.


Phosphotyrosine profiling of human cerebrospinal fluid.
Cerebrospinal fluid (CSF) is an important source of potential biomarkers that affect the brain. Biomarkers for neurodegenerative disorders are needed to assist in diagnosis, monitoring disease progression and evaluating efficacy of therapies. Recent studies have demonstrated the involvement of tyrosine kinases in neuronal cell death. Thus, neurodegeneration in the brain is related to altered tyrosine phosphorylation of proteins in the brain and identification of abnormally phosphorylated tyrosine peptides in CSF has the potential to ascertain candidate biomarkers for neurodegenerative disorders. In this study, we used an antibody-based tyrosine phosphopeptide enrichment method coupled with high resolution Orbitrap Fusion Tribrid Lumos Fourier transform mass spectrometer to catalog tyrosine phosphorylated peptides from cerebrospinal fluid. The subset of identified tyrosine phosphorylated peptides was also validated using parallel reaction monitoring (PRM)-based targeted approach. To date, there are no published studies on global profiling of phosphotyrosine modifications of CSF proteins. We carried out phosphotyrosine profiling of CSF using an anti-phosphotyrosine antibody-based enrichment and analysis using high resolution Orbitrap Fusion Lumos mass spectrometer. We identified 111 phosphotyrosine peptides mapping to 66 proteins, which included 24 proteins which have not been identified in CSF previously. We then validated a set of 5 tyrosine phosphorylated peptides in an independent set of CSF samples from cognitively normal subjects, using a PRM-based targeted approach. The findings from this deep phosphotyrosine profiling of CSF samples have the potential to identify novel disease-related phosphotyrosine-containing peptides in CSF.


Exercise training increases size of hippocampus and improves memory.
The hippocampus shrinks in late adulthood, leading to impaired memory and increased risk for dementia. Hippocampal and medial temporal lobe volumes are larger in higher-fit adults, and physical activity training increases hippocampal perfusion, but the extent to which aerobic exercise training can modify hippocampal volume in late adulthood remains unknown. Here we show, in a randomized controlled trial with 120 older adults, that aerobic exercise training increases the size of the anterior hippocampus, leading to improvements in spatial memory. Exercise training increased hippocampal volume by 2%, effectively reversing age-related loss in volume by 1 to 2 y. We also demonstrate that increased hippocampal volume is associated with greater serum levels of BDNF, a mediator of neurogenesis in the dentate gyrus. Hippocampal volume declined in the control group, but higher preintervention fitness partially attenuated the decline, suggesting that fitness protects against volume loss. Caudate nucleus and thalamus volumes were unaffected by the intervention. These theoretically important findings indicate that aerobic exercise training is effective at reversing hippocampal volume loss in late adulthood, which is accompanied by improved memory function.


Cholecystectomy and the risk of alimentary tract cancers: a systematic review.
To investigate the association between cholecystectomy and gastro-intestinal tract (GIT) cancers. We conducted a systematic review according to the PRISMA guidelines. A MEDLINE search was performed with predefined search criteria for English Language articles on the association between cholecystectomy and GIT cancers. Additional articles were retrieved by manual search of references. All relevant articles were accessed in full text. Data on study type; cases; controls; country; effect estimate; adjustments for confounders and quality of publication were extracted. The quality of the publications were scored by adherence to the STROBE checklist. The data for each part of the GIT were presented in separate tables. Seventy-five studies and 5 meta-analyses satisfied the predefined criteria for inclusion and were included in this review. There were inconsistent reports and no strong evidence of an association between cholecystectomy and cancers of the oesophagus (Adenocarcinoma), pancreas, small bowel and right-sided colon cancers. In squamous cancer of the oesophagus, cancers of the stomach, liver, bile ducts, small bowel and left sided colon cancers, good quality studies suggested a lack of association with cholecystectomy. Equally, distal colon and rectal cancers were found not to be associated with cholecystectomy. Several mechanisms for carcinogenesis/promotion of carcinogensis have been proposed. These have focused on a role for bile salts in carcinogenesis with several potential mutagenic molecular events and gut metabolic hormones signaling cell proliferation or initiation of carcinogenesis. This is a comprehensive review of the association between GIT cancers and cholecystectomy. This review found no clear association between cholecystectomy and GIT cancers.


Mechanisms, diagnosis and management of hepatic encephalopathy.
Hepatic encephalopathy (HE) is a serious neuropsychiatric complication of both acute and chronic liver disease. Symptoms of HE can include confusion, disorientation and poor coordination. A general consensus exists that the synergistic effects of excess ammonia and inflammation cause astrocyte swelling and cerebral edema; however, the precise molecular mechanisms that lead to these morphological changes in the brain are unclear. Cerebral edema occurs to some degree in all patients with HE, regardless of its grade, and could underlie the pathogenesis of this disorder. The different grades of HE can be diagnosed by a number of investigations, including neuropsychometric tests (such as the psychometric hepatic encephalopathy score), brain imaging and clinical scales (such as the West Haven criteria). HE is best managed by excluding other possible causes of encephalopathy alongside identifying and the precipitating cause, and confirming the diagnosis by a positive response to empiric treatment. Empiric therapy for HE is largely based on the principle of reducing the production and absorption of ammonia in the gut through administration of pharmacological agents such as rifaximin and lactulose, which are approved by the FDA for the treatment of HE.


Monounsaturated fatty acid, carbohydrate intake, and diabetes status are associated with arterial pulse pressure.
Diabetes is a global epidemic. Cardiovascular disease (CVD) is one of the most prevalent consequences of diabetes. Nutrition is considered a modifiable risk factor for CVD, particularly for individuals with diabetes; albeit, there is little consensus on the role of carbohydrates, proteins and fats for arterial health for persons with or without diabetes. In this study, we examined the association of macronutrients with arterial pulse pressure (APP), a surrogate measure of arterial health by diabetes status and race. Participants were 892 Mexican Americans (MA), 1059 Black, non-Hispanics (BNH) and 2473 White, non-Hispanics (WNH) with and without diabetes of a weighted sample from the National Nutrition and Health Examination Survey (NHANES) 2007-2008. The cross-sectional analysis was performed with IBM-SPSS version 18 with the complex sample analysis module. The two-year sample weight for the sub-sample with laboratory values was applied to reduce bias and approximate a nationally, representative sample. Arterial stiffness was assessed by arterial pulse pressure (APP). APP was higher for MA [B = 0.063 (95% CI 0.015 to 0.111), p = 0.013] and BNH [B = 0.044 (95% CI 0.006 to 0.082), p = 0.018] than WNH, controlling for diabetes, age, gender, body mass index (BMI), fiber intake, energy intake (Kcal) and smoking. A two-way interaction of diabetes by carbohydrate intake (grams) was inversely associated with APP [B = -1.18 (95% CI -0.178 to -0.058), p = 0.001], controlling for race, age, gender, BMI, Kcal and smoking. BNH with diabetes who consumed more mono-unsaturated fatty acids (MUFA) than WNH with diabetes had lower APP [B = -0.112 (95%CI-0.179 to -0.045), p = 0.003] adjusting for saturated fatty acids, Kcal, age, gender, BMI and smoking. Higher MUFA and carbohydrate intake for persons with diabetes reflecting lower APP may be due to replacement of saturated fats with CHO and MUFA. The associations of APP with diabetes, race and dietary intake need to be confirmed with intervention and prospective studies. Confirmation of these results would suggest that dietary interventions for minorities with diabetes may improve arterial health.


The association between early arterial oxygenation and mortality in ventilated patients with acute ischaemic stroke.
There are conflicting data that suggest that hyperoxia may be associated with either worse or better outcomes in patients suffering a stroke. To investigate the association between PaO(2) in the first 24 hours in the intensive care unit and mortality among ventilated patients with acute ischaemic stroke. Retrospective cohort study. Data were extracted from the Australian and New Zealand Intensive Care Society Adult Patient Database. Adults ventilated for ischaemic stroke in 129 ICUs in Australia and New Zealand, 2000-2009. The primary outcome was the odds ratio for in hospital mortality associated with "worst" PaO(2) considered as a categorical variable, with data divided into deciles and compared with the mortality of the 10th decile. For patients on an FiO(2) of _50% at any time in the first 24 hours, "worst" PaO(2) was defined as the PaO(2) associated with the highest alveolar-arterial (A-a) gradient. For patients on an FiO(2) of <50%, it was defined as the lowest PaO(2). Secondary outcomes were ICU and hospital length of stay and the proportion of patients in each decile discharged home. Of the 2643 patients eligible for study inclusion, 1507 (57%) died in hospital. The median "worst" PaO(2) was 117mmHg (interquartile range, 87-196mmHg). There was no association between worst PaO(2) and mortality, length of stay or likelihood of discharge home. We found no association between worst arterial oxygen tension in the first 24 hours in ICU and outcome in ventilated patients with ischaemic stroke.


Chronic melatonin consumption prevents obesity-related metabolic abnormalities and protects the heart against myocardial ischemia and reperfusion injury in a prediabetic model of diet-induced obesity.
Obesity, a major risk factor for ischemic heart disease, is associated with increased oxidative stress and reduced antioxidant status. Melatonin, a potent free radical scavenger and antioxidant, has powerful cardioprotective effects in lean animals but its efficacy in obesity is unknown. We investigated the effects of chronic melatonin administration on the development of the metabolic syndrome as well as ischemia-reperfusion injury in a rat model of diet-induced obesity (DIO). Male Wistar rats received a control diet, a control diet with melatonin, a high-calorie diet, or a high-calorie diet with melatonin (DM). Melatonin (4 mg/kg/day) was administered in the drinking water. After 16 wk, biometric and blood metabolic parameters were measured. Hearts were perfused ex vivo for the evaluation of myocardial function, infarct size (IFS) and biochemical changes [activation of PKB/Akt, ERK, p38 MAPK, AMPK, and glucose transporter (GLUT)-4 expression). The high-calorie diet caused increases in body weight (BW), visceral adiposity, serum insulin and triglycerides (TRIG), with no change in glucose levels. Melatonin treatment reduced the BW gain, visceral adiposity, blood TRIG, serum insulin, homeostatic model assessment index and thiobarbituric acid reactive substances in the DIO group. Melatonin reduced IFS in DIO and control groups and increased percentage recovery of functional performance of DIO hearts. During reperfusion, hearts from melatonin-treated rats had increased activation of PKB/Akt, ERK42/44 and reduced p38 MAPK activation. Chronic melatonin treatment prevented the metabolic abnormalities induced by DIO and protected the heart against ischemia-reperfusion injury. These beneficial effects were associated with activation of the reperfusion injury salvage kinases pathway.


A prospective study of the immediate and delayed adverse events following intravenous immunoglobulin infusions.
To document the incidence of immediate and delayed adverse events (AE) following intravenous immunoglobulin (IVIG) infusion in children. Immediate and delayed adverse events were prospectively recorded for 345 infusions in 58 children receiving IVIG for immunodeficiency (n = 33) or immunomodulation (n = 25). For each infusion adverse events were documented during the infusion and by follow up interview 4-7 days later. Immediate adverse events occurred in 10.3% and delayed adverse events in 41.4% of children treated during the study period. Three and a half per cent of the infusions were associated with immediate AE and 20.9% with delayed adverse events. Headache was the most common delayed AE, occurring in 24.1% of patients and 12.8% of infusions. Delayed adverse events to IVIG infusions are common in children. They occur more frequently than immediate adverse events and are the cause of significant morbidity. Recognition of the high frequency of delayed adverse events is important in the care of children receiving IVIG therapy.


A community-engaged randomized controlled trial of an integrative intervention with HIV-positive, methamphetamine-using men who have sex with men.
Contingency management (CM) is an evidence-based intervention providing tangible rewards as positive reinforcement for abstinence from stimulants such as methamphetamine. Integrative approaches targeting affect regulation could boost the effectiveness of CM in community-based settings and optimize HIV/AIDS prevention efforts. This randomized controlled trial with HIV-positive, methamphetamine-using men who have sex with men (MSM) is examining the efficacy of a 5-session, individually delivered positive affect regulation intervention - Affect Regulation Treatment to Enhance Methamphetamine Intervention Success (ARTEMIS). ARTEMIS is designed to sensitize individuals to non-drug-related sources of reward as well as assist with managing depression and other symptoms of stimulant withdrawal during CM. HIV-positive, methamphetamine-using MSM who are enrolled in a community-based, 12-week CM program are randomized to receive ARTEMIS or an attention-matched control condition. Follow-up assessments are conducted at 3, 6, 12, and 15 months after enrollment in CM. Four peripheral venous blood samples are collected over the 15-month follow-up with specimen banking for planned biomarker sub-studies. The primary outcome is mean HIV viral load. Secondary outcomes include: sustained HIV viral suppression, T-helper cell count, psychological adjustment, stimulant use, and potentially amplified transmission risk behavior. Implementation of this randomized controlled trial highlights the importance of delineating boundaries between research activities and community-based service provision. It also provides insights into best practices for integrating the distinct agendas of academic and community partners in clinical research. This trial is currently enrolling and data collection is anticipated to be completed in September of 2018. This trial was registered on clinicaltrials.gov ( NCT01926184 ) on August 16, 2013.


Lactate dehydrogenase C and energy metabolism in mouse sperm.
We demonstrated previously that disruption of the germ cell-specific lactate dehydrogenase C gene (Ldhc) led to male infertility due to defects in sperm function, including a rapid decline in sperm ATP levels, a decrease in progressive motility, and a failure to develop hyperactivated motility. We hypothesized that lack of LDHC disrupts glycolysis by feedback inhibition, either by causing a defect in renewal of the NAD(+) cofactor essential for activity of glyceraldehyde 3-phosphate dehydrogenase, sperm (GAPDHS), or an accumulation of pyruvate. To test these hypotheses, nuclear magnetic resonance analysis was used to follow the utilization of labeled substrates in real time. We found that in sperm lacking LDHC, glucose consumption was disrupted, but the NAD:NADH ratio and pyruvate levels were unchanged, and pyruvate was rapidly metabolized to lactate. Moreover, the metabolic disorder induced by treatment with the lactate dehydrogenase (LDH) inhibitor sodium oxamate was different from that caused by lack of LDHC. This supported our earlier conclusion that LDHA, an LDH isozyme present in the principal piece of the flagellum, is responsible for the residual LDH activity in sperm lacking LDHC, but suggested that LDHC has an additional role in the maintenance of energy metabolism in sperm. By coimmunoprecipitation coupled with mass spectrometry, we identified 27 proteins associated with LDHC. A majority of these proteins are implicated in ATP synthesis, utilization, transport, and/or sequestration. This led us to hypothesize that in addition to its role in glycolysis, LDHC is part of a complex involved in ATP homeostasis that is disrupted in sperm lacking LDHC.


Counting-backward test for executive function in idiopathic normal pressure hydrocephalus.
The aim of this study was to develop and validate a bedside test for executive function in patients with idiopathic normal pressure hydrocephalus (INPH). Twenty consecutive patients with INPH and 20 patients with Alzheimer's disease (AD) were enrolled in this study. We developed the counting-backward test for evaluating executive function in patients with INPH. Two indices that are considered to be reflective of the attention deficits and response suppression underlying executive dysfunction in INPH were calculated: the first-error score and the reverse-effect index. Performance on both the counting-backward test and standard neuropsychological tests for executive function was assessed in INPH and AD patients. The first-error score, reverse-effect index and the scores from the standard neuropsychological tests for executive function were significantly lower for individuals in the INPH group than in the AD group. The two indices for the counting-backward test in the INPH group were strongly correlated with the total scores for Frontal Assessment Battery and Phonemic Verbal Fluency. The first-error score was also significantly correlated with the error rate of the Stroop colour-word test and the score of the go/no-go test. In addition, we found that the first-error score highly distinguished patients with INPH from those with AD using these tests. The counting-backward test is useful for evaluating executive dysfunction in INPH and for differentiating between INPH and AD patients. In particular, the first-error score may reflect deficits in the response suppression related to executive dysfunction in INPH.


Regulation of arylamine N-acetyltransferases.
Acetylation catalysed by the arylamine N-acetyltransferases (NATs; 126.96.36.199) is a major biotransformation pathway for arylamine and hydrazine drugs, as well as many carcinogens that we are exposed to on a daily basis. These compounds can either be detoxified by NATs or bioactivated to metabolites that have the potential to cause toxicity such as cancer. As a result, the levels of NATs in the body have clinical importance with regard to drug effect and individual susceptibility to toxicity. Like many other drug metabolising enzymes, the activity of NATs varies considerably between individuals, due in part to genetic polymorphisms. However, it is becoming increasingly evident that non-genetic factors also play an important role in regulating NAT activity in vivo. This review focuses on the non-genetic control of NAT expression, including transcriptional, post-transcriptional/translational, and post-translational regulation. In addition, the dysregulation of NAT1 expression in cancer cells is reviewed, as this is an emerging area that may provide insight into a role for NAT1 in cancer biology.


Polycomb Repressive Complex 2 regulates lineage fidelity during embryonic stem cell differentiation.
Polycomb Repressive Complex 2 (PRC2) catalyzes histone H3 lysine 27 tri-methylation (H3K27me3), an epigenetic modification associated with gene repression. H3K27me3 is enriched at the promoters of a large cohort of developmental genes in embryonic stem cells (ESCs). Loss of H3K27me3 leads to a failure of ESCs to properly differentiate, making it difficult to determine the precise roles of PRC2 during lineage commitment. Moreover, while studies suggest that PRC2 prevents DNA methylation, how these two epigenetic regulators coordinate to regulate lineage programs is poorly understood. Using several PRC2 mutant ESC lines that maintain varying levels of H3K27me3, we found that partial maintenance of H3K27me3 allowed for proper temporal activation of lineage genes during directed differentiation of ESCs to spinal motor neurons (SMNs). In contrast, genes that function to specify other lineages failed to be repressed in these cells, suggesting that PRC2 is also necessary for lineage fidelity. We also found that loss of H3K27me3 leads to a modest gain in DNA methylation at PRC2 target regions in both ESCs and in SMNs. Our study demonstrates a critical role for PRC2 in safeguarding lineage decisions and in protecting genes against inappropriate DNA methylation.


Genistein potentiates the effect of arsenic trioxide against human hepatocellular carcinoma: role of Akt and nuclear factor-κB.
Hepatocellular carcinoma (HCC) is a highly lethal malignancy mostly because of de novo and acquired resistance to conventional chemotherapy. Constitutive activation of Akt and nuclear factor-κB (NF-κB) represent major cellular abnormalities associated with both the pathogenesis and therapeutic resistance of HCC. The aim of the present study was to determine whether genistein, a natural Akt/NF-κB inhibitor, could enhance the anti-HCC efficacy of ATO both in vitro and in vivo. Our results demonstrated that genistein not only potentiated the proliferation-inhibiting and apoptosis-inducing effect of ATO on human HCC cell lines in vitro, but also dramatically augmented its suppressive effect on both tumor growth and angiogenesis in nude mice. The mechanism is at least partially due to the suppressive effect of genistein both on the proper and ATO-induced Akt activation, and on the activity of NF-κB, and the latter correlated with the suppression of NF-κB regulated gene products, including cyclin D1, Bcl-xL, Bcl-2, c-myc, COX-2, and VEGF. These data suggest that the combination of ATO with genistein presents a promising therapeutic approach for the treatment of HCC.


Medicinal cannabis for psychiatric disorders: a clinically-focused systematic review.
Medicinal cannabis has received increased research attention over recent years due to loosening global regulatory changes. Medicinal cannabis has been reported to have potential efficacy in reducing pain, muscle spasticity, chemotherapy-induced nausea and vomiting, and intractable childhood epilepsy. Yet its potential application in the field of psychiatry is lesser known. The first clinically-focused systematic review on the emerging medical application of cannabis across all major psychiatric disorders was conducted. Current evidence regarding whole plant formulations and plant-derived cannabinoid isolates in mood, anxiety, sleep, psychotic disorders and attention deficit/hyperactivity disorder (ADHD) is discussed; while also detailing clinical prescription considerations (including pharmacogenomics), occupational and public health elements, and future research recommendations. The systematic review of the literature was conducted during 2019, assessing the data from all case studies and clinical trials involving medicinal cannabis or plant-derived isolates for all major psychiatric disorders (neurological conditions and pain were omitted). The present evidence in the emerging field of cannabinoid therapeutics in psychiatry is nascent, and thereby it is currently premature to recommend cannabinoid-based interventions. Isolated positive studies have, however, revealed tentative support for cannabinoids (namely cannabidiol; CBD) for reducing social anxiety; with mixed (mainly positive) evidence for adjunctive use in schizophrenia. Case studies suggest that medicinal cannabis may be beneficial for improving sleep and post-traumatic stress disorder, however evidence is currently weak. Preliminary research findings indicate no benefit for depression from high delta-9 tetrahydrocannabinol (THC) therapeutics, or for CBD in mania. One isolated study indicates some potential efficacy for an oral cannabinoid/terpene combination in ADHD. Clinical prescriptive consideration involves caution in the use of high-THC formulations (avoidance in youth, and in people with anxiety or psychotic disorders), gradual titration, regular assessment, and caution in cardiovascular and respiratory disorders, pregnancy and breast-feeding. There is currently encouraging, albeit embryonic, evidence for medicinal cannabis in the treatment of a range of psychiatric disorders. Supportive findings are emerging for some key isolates, however, clinicians need to be mindful of a range of prescriptive and occupational safety considerations, especially if initiating higher dose THC formulas.


The role of vitamers and dietary-based metabolites of vitamin D in prevention of vitamin D deficiency.
There is little doubt that vitamin D deficiency across all age groups in Europe is a problem. Low vitamin D status arises due to limited, if any, dermal synthesis during the winter months at latitudes above 40°N, putting increased importance on dietary supply of the vitamin. However, dietary intakes by most populations are low due to the limited supply of vitamin D-rich foods in the food chain. Thus strategies that effectively address this public health issue are urgently required. It has been emphasized and re-emphasized that there are only a limited number of public health strategies available to correct low dietary vitamin D intake: (1) improving intake of naturally occurring vitamin D-rich foods, (2) vitamin D fortification (mandatory or voluntarily) of food, and (3) vitamin D supplementation. Recent evidence suggests that the levels of vitamin D added to food would need to be high so as to ensure dietary requirements are met and health outcomes optimized. In addition, knowledge of the most effective forms of vitamin D to use in some of these preventative approaches is important. There is still uncertainty in relation to the relative efficacy of vitamin D(2) versus D(3), the two main food derived forms and those used in vitamin D supplements. The major metabolite of vitamin D with biological activity is 1,25(OH)(2)D; however, this is usually used for pharmacological purposes and is not typically used in normal, healthy people. The other major metabolite, 25(OH)D, which has also been used for pharmacological purposes is present in certain foods such as meat and meat products (particularly offal) as well as eggs. This metabolite may have the potential to boost vitamin D status up to five times more effectively that native vitamin D(3) in foods. However, the exact bioactivity of this compound needs to be established.


Iron deficiency, prolonged bottle-feeding, and racial/ethnic disparities in young children.
Childhood iron deficiency is associated with behavioral and cognitive delays. Few studies have explored the relationship between prolonged bottle-feeding and iron-deficiency anemia among toddlers. To examine the association between prolonged bottle-feeding and iron deficiency in a nationally representative sample of children ages 1 to 3 years. The National Health and Nutrition Examination Survey III provides data on the feeding practices of children 1 to 3 years old and contains measures of iron status including transferrin saturation, free erythrocyte protoporphyrin, and serum ferritin. The prevalence of iron deficiency and duration of bottle-feeding were determined for black, white, and Mexican American toddlers. Bivariate and multivariate analyses were performed to examine the association between bottle-feeding duration and iron deficiency. Among 2121 children ages 1 to 3 years, the prevalence of iron deficiency was 6% among whites, 8% among blacks, and 17% among Mexican Americans (P< .001). With increasing duration of bottle-feeding, the prevalence of iron deficiency among all children increased (3.8%, bottle-fed < or =12 months; 11.5%, bottle-fed 13-23 months; and 12.4%, bottle-fed 24-48 months [P< .001]). At 24 to 48 months of age, 36.8% of Mexican American children were still bottle-fed, compared with 16.9% of white and 13.8% of black children. In multivariate analyses, bottle-feeding for 24 to 48 months and Mexican ethnicity were associated with iron deficiency (odds ratio, 2.8; 95% confidence interval, 1.3-6.0; and odds ratio, 2.9; 95% confidence interval, 1.5-5.6, respectively). Children with prolonged bottle-feeding and Mexican American children are at higher risk for iron deficiency. Screening practices and nutritional counseling should be targeted at these high-risk groups.


Roles of amino acids in preventing and treating intestinal diseases: recent studies with pig models.
Animal models are needed to study and understand a human complex disease. Because of their similarities in anatomy, structure, physiology, and pathophysiology, the pig has proven its usefulness in studying human gastrointestinal diseases, such as inflammatory bowel disease, ischemia/reperfusion injury, diarrhea, and cancer. To understand the pathogenesis of these diseases, a number of experimental models generated in pigs are available, for example, through surgical manipulation, chemical induction, microbial infection, and genetic engineering. Our interests have been using amino acids as therapeutics in pig and human disease models. Amino acids not only play an important role in protein biosynthesis, but also exert significant physiological effects in regulating immunity, anti-oxidation, redox regulation, energy metabolism, signal transduction, and animal behavior. Recent studies in pigs have shown that specific dietary amino acids can improve intestinal integrity and function under normal and pathological conditions that protect the host from different diseases. In this review, we summarize several pig models in intestinal diseases and how amino acids can be used as therapeutics in treating pig and human diseases.


Evidence for functional, inhibitory, histamine H3 receptors in rat carotid body Type I cells.
The Type I cells are the sensory elements of the carotid bodies and play a critical role in defining the ventilatory response to hypoxia and hypercapnia. Type I cells release multiple neurotransmitters during a chemosensory stimulus resulting in increased firing of the carotid sinus nerve and modification of the breathing pattern. While much is known about the actions of individual neurotransmitters in this system, very little is known about how multiple neurotransmitters may integrate to shape the output of the carotid body. Recent data has indicated that the neurotransmitter histamine does not excite isolated Type I cells despite being released during hypoxia and its receptors being present on the Type I cells. Here the hypothesis that histamine might modulate an excitatory neurotransmitter such as acetylcholine was tested. Using calcium imaging techniques it was found that histamine attenuated calcium signaling events initiated by the muscarinic acetylcholine receptor agonist acetyl-beta-methylcholine via an H3 receptor mediated mechanism. In summary, these results suggest that when acetylcholine and histamine are co-released from Type I cells in response to chemostimuli, histamine may attenuate or modulate the excitatory presynaptic actions of acetylcholine.


Fifteen-minute consultation: Not the whole story-considering children's spirituality and advance care planning.
In a society of diverse views, faiths and beliefs, what can paediatric palliative care contribute to our understanding of children's spirituality? By failing to recognise and respond to their spirituality in this work, we risk missing something of profound importance to children and their families. We overlook their search for wholeness in the absence of cure and fall short of offering truly holistic care. This paper explores how developments in advance care planning and related documentation are addressing these issues. Since children's spirituality is elusive and rarely explored in practice, it aims to clarify our understanding of it with a variety of examples and contains suggestions for hearing the voice of the child amid the needs of parents and professionals.


Mutagenicity and mutational spectrum of N-methyl-N'-nitro-N-nitrosoguanidine in the hprt gene in G1-S and late S phase of diploid human fibroblasts.
To investigate the effect of DNA replication on the mutation spectrum induced in diploid human fibroblasts by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), cells were synchronized and exposed to MNNG either at the G1-S border or in late S phase, and the mutations in the hypoxanthine (guanine) phosphoribosyltransferase (hprt) gene were examined. The coding regions of 92 independent mutants were characterized by direct sequencing of mRNA-polymerase chain reaction-amplified complementary DNA. While there was little difference in the sensitivity of the two populations to the cytotoxic effects of MNNG, the frequency of mutants induced in late S populations was significantly lower than that induced in G1-S populations. The majority of induced complementary DNA mutations were single base substitutions (54%) and splice site mutations (43%). Analysis of the intron-exon boundaries of more than one-half of the splicing mutants showed that almost all contained base substitutions in the hprt gene. A broad mutational spectrum was observed in low-dose (4, 6, or 8 microM) treatments; only 27% were G to A transitions, whereas 80% of base substitutions derived from the high-dose (10 or 12 microM) treatments were G to A transitions in G1-S populations. An intermediate frequency (64%) of G to A transitions was observed in late S populations exposed to MNNG. When the causative premutation lesion was O6-methylguanine, 75% of G to A transitions that were observed in G1-S populations clustered on both the transcribed and the nontranscribed strands of the 5' half of the hprt gene. In contrast, 50% of G to A transitions were located only on the nontranscribed strand of this region in late S populations. The results indicate that O(6)-alkylguanine-DNA-alkyltransferase may not efficiently remove O(6)-methylguanine from the 5' half of the gene but can repair lesions far away from this region during initiation of replication. Our results are consistent with the notion that the putative origin of replication is located at intron 1 of the hprt gene.