Absence of system xc⁻ on immune cells invading the central nervous system alleviates experimental autoimmune encephalitis

Background: Multiple sclerosis (MS) is an autoimmune demyelinating disease that affects the central nervous system (CNS), leading to neurodegeneration and chronic disability. Accumulating evidence points to a key role for neuroinflammation, oxidative stress, and excitotoxicity in this degenerative process. System x(c)- or the cystine/glutamate antiporter could tie these pathological mechanisms together: its activity is enhanced by reactive oxygen species and inflammatory stimuli, and its enhancement might lead to the release of toxic amounts of glutamate, thereby triggering excitotoxicity and neurodegeneration. Methods: Semi-quantitative Western blotting served to study protein expression of xCT, the specific subunit of system x(c)-, as well as of regulators of xCT transcription, in the normal appearing white matter (NAWM) of MS patients and in the CNS and spleen of mice exposed to experimental autoimmune encephalomyelitis (EAE), an accepted mouse model of MS. We next compared the clinical course of the EAE disease, the extent of demyelination, the infiltration of immune cells and microglial activation in xCT-knockout (xCT(-/-)) mice and irradiated mice reconstituted in xCT(-/-) bone marrow (BM), to their proper wild type (xCT(+/+)) controls. Results: xCT protein expression levels were upregulated in the NAWM of MS patients and in the brain, spinal cord, and spleen of EAE mice. The pathways involved in this upregulation in NAWM of MS patients remain unresolved. Compared to xCT(+/+) mice, xCT(-/-) mice were equally susceptible to EAE, whereas mice transplanted with xCT(-/-) BM, and as such only exhibiting loss of xCT in their immune cells, were less susceptible to EAE. In none of the above-described conditions, demyelination, microglial activation, or infiltration of immune cells were affected. Conclusions: Our findings demonstrate enhancement of xCT protein expression in MS pathology and suggest that system x(c)- on immune cells invading the CNS participates to EAE. Since a total loss of system x(c)- had no net beneficial effects, these results have important implications for targeting system x(c)- for treatment of MS

The role of time averaging of eddy covariance fluxes on water use efficiency dynamics of maize

Direct measurement of carbon and water fluxes at high frequencies make eddy covariance (EC) the technique most preferred to characterize water use efficiency (WUE). However, reliability of EC fluxes largely hinges on the energy balance ratio (EBR) and inclusion of low-frequency fluxes. This study is aimed at investigating the role of the averaging period in representing EC fluxes and its propagation into WUE dynamics. Carbon and water fluxes were monitored in a drip-irrigated maize field at 10 Hz frequency and were averaged over 1, 5, 10, 15, 30, 45, 60, and 120 min, considering daytime unstable conditions. The optimal averaging period to simulate WUE fluxes for each growth stage is obtained by considering cumulative frequency (Ogive) curves. A clear departure of EBR from unity was observed during the dough and maturity stages of the crop due to ignorance of canopy heat storage, low-frequency flux losses, and an inadequate averaging period. Deviations in representing water (carbon) fluxes relative to the conventional 30 min average are within ±3 % (±10 %) for 10–120 min averaging and beyond ±3 % (±10 %) for other time averages. Ogive plots show that the optimal averaging period to represent carbon, water, and WUE fluxes is 15–30 min for the sixth leaf and silking stages and is 45–60 min for the dough and maturity stages. Dynamics of WUE considering optimal averaging periods are in the range of μ ± σ: 1.49 ± 0.95, 1.37 ± 0.74, 1.39 ± 0.79, and 3.06 ± 0.69 µmol mmol−1 for the sixth leaf, silking, dough, and maturity stages, respectively. The error in representing WUE by conventional 30 min averaging is marginal (&lt; 1.5 %) throughout the crop period except for the dough stage (12.12 %). We conclude that the conventional 30 min averaging of EC fluxes is not appropriate for representing WUE throughout the crop period. Our findings can help to develop efficient water management strategies by accurately characterizing WUE fluxes from the EC measurements.</p

Resolving early mesoderm diversification through single-cell expression profiling.

In mammals, specification of the three major germ layers occurs during gastrulation, when cells ingressing through the primitive streak differentiate into the precursor cells of major organ systems. However, the molecular mechanisms underlying this process remain unclear, as numbers of gastrulating cells are very limited. In the mouse embryo at embryonic day 6.5, cells located at the junction between the extra-embryonic region and the epiblast on the posterior side of the embryo undergo an epithelial-to-mesenchymal transition and ingress through the primitive streak. Subsequently, cells migrate, either surrounding the prospective ectoderm contributing to the embryo proper, or into the extra-embryonic region to form the yolk sac, umbilical cord and placenta. Fate mapping has shown that mature tissues such as blood and heart originate from specific regions of the pre-gastrula epiblast, but the plasticity of cells within the embryo and the function of key cell-type-specific transcription factors remain unclear. Here we analyse 1,205 cells from the epiblast and nascent Flk1(+) mesoderm of gastrulating mouse embryos using single-cell RNA sequencing, representing the first transcriptome-wide in vivo view of early mesoderm formation during mammalian gastrulation. Additionally, using knockout mice, we study the function of Tal1, a key haematopoietic transcription factor, and demonstrate, contrary to previous studies performed using retrospective assays, that Tal1 knockout does not immediately bias precursor cells towards a cardiac fate.We thank M. de Bruijn, A. Martinez-Arias, J. Nichols and C. Mulas for discussion, the Cambridge Institute for Medical Research Flow Cytometry facility for their expertise in single-cell index sorting, and S. Lorenz from the Sanger Single Cell Genomics Core for supervising purification of Tal1−/− sequencing libraries. ChIP-seq reads were processed by R. Hannah. Research in the authors’ laboratories is supported by the Medical Research Council, Cancer Research UK, the Biotechnology and Biological Sciences Research Council, Bloodwise, the Leukemia and Lymphoma Society, and the Sanger-EBI Single Cell Centre, and by core support grants from the Wellcome Trust to the Cambridge Institute for Medical Research and Wellcome Trust - MRC Cambridge Stem Cell Institute and by core funding from Cancer Research UK and the European Molecular Biology Laboratory. Y.T. was supported by a fellowship from the Japan Society for the Promotion of Science. W.J. is a Wellcome Trust Clinical Research Fellow. A.S. is supported by the Sanger-EBI Single Cell Centre. This work was funded as part of Wellcome Trust Strategic Award 105031/D/14/Z ‘Tracing early mammalian lineage decisions by single-cell genomics’ awarded to W. Reik, S. Teichmann, J. Nichols, B. Simons, T. Voet, S. Srinivas, L. Vallier, B. Göttgens and J. Marioni.This is the author accepted manuscript. The final version is available from Nature Publishing Group via http://dx.doi.org/10.1038/nature1863

Antiinflammatory Therapy with Canakinumab for Atherosclerotic Disease

Background: Experimental and clinical data suggest that reducing inflammation without affecting lipid levels may reduce the risk of cardiovascular disease. Yet, the inflammatory hypothesis of atherothrombosis has remained unproved. Methods: We conducted a randomized, double-blind trial of canakinumab, a therapeutic monoclonal antibody targeting interleukin-1β, involving 10,061 patients with previous myocardial infarction and a high-sensitivity C-reactive protein level of 2 mg or more per liter. The trial compared three doses of canakinumab (50 mg, 150 mg, and 300 mg, administered subcutaneously every 3 months) with placebo. The primary efficacy end point was nonfatal myocardial infarction, nonfatal stroke, or cardiovascular death. RESULTS: At 48 months, the median reduction from baseline in the high-sensitivity C-reactive protein level was 26 percentage points greater in the group that received the 50-mg dose of canakinumab, 37 percentage points greater in the 150-mg group, and 41 percentage points greater in the 300-mg group than in the placebo group. Canakinumab did not reduce lipid levels from baseline. At a median follow-up of 3.7 years, the incidence rate for the primary end point was 4.50 events per 100 person-years in the placebo group, 4.11 events per 100 person-years in the 50-mg group, 3.86 events per 100 person-years in the 150-mg group, and 3.90 events per 100 person-years in the 300-mg group. The hazard ratios as compared with placebo were as follows: in the 50-mg group, 0.93 (95% confidence interval [CI], 0.80 to 1.07; P = 0.30); in the 150-mg group, 0.85 (95% CI, 0.74 to 0.98; P = 0.021); and in the 300-mg group, 0.86 (95% CI, 0.75 to 0.99; P = 0.031). The 150-mg dose, but not the other doses, met the prespecified multiplicity-adjusted threshold for statistical significance for the primary end point and the secondary end point that additionally included hospitalization for unstable angina that led to urgent revascularization (hazard ratio vs. placebo, 0.83; 95% CI, 0.73 to 0.95; P = 0.005). Canakinumab was associated with a higher incidence of fatal infection than was placebo. There was no significant difference in all-cause mortality (hazard ratio for all canakinumab doses vs. placebo, 0.94; 95% CI, 0.83 to 1.06; P = 0.31). Conclusions: Antiinflammatory therapy targeting the interleukin-1β innate immunity pathway with canakinumab at a dose of 150 mg every 3 months led to a significantly lower rate of recurrent cardiovascular events than placebo, independent of lipid-level lowering. (Funded by Novartis; CANTOS ClinicalTrials.gov number, NCT01327846.

Influence of socioeconomic factors on pregnancy outcome in women with structural heart disease

OBJECTIVE: Cardiac disease is the leading cause of indirect maternal mortality. The aim of this study was to analyse to what extent socioeconomic factors influence the outcome of pregnancy in women with heart disease.  METHODS: The Registry of Pregnancy and Cardiac disease is a global prospective registry. For this analysis, countries that enrolled ≥10 patients were included. A combined cardiac endpoint included maternal cardiac death, arrhythmia requiring treatment, heart failure, thromboembolic event, aortic dissection, endocarditis, acute coronary syndrome, hospitalisation for cardiac reason or intervention. Associations between patient characteristics, country characteristics (income inequality expressed as Gini coefficient, health expenditure, schooling, gross domestic product, birth rate and hospital beds) and cardiac endpoints were checked in a three-level model (patient-centre-country).  RESULTS: A total of 30 countries enrolled 2924 patients from 89 centres. At least one endpoint occurred in 645 women (22.1%). Maternal age, New York Heart Association classification and modified WHO risk classification were associated with the combined endpoint and explained 37% of variance in outcome. Gini coefficient and country-specific birth rate explained an additional 4%. There were large differences between the individual countries, but the need for multilevel modelling to account for these differences disappeared after adjustment for patient characteristics, Gini and country-specific birth rate.  CONCLUSION: While there are definite interregional differences in pregnancy outcome in women with cardiac disease, these differences seem to be mainly driven by individual patient characteristics. Adjustment for country characteristics refined the results to a limited extent, but maternal condition seems to be the main determinant of outcome

Machine Learning-Based Fraudulent and Harmful Link Detection System

Phishing sites which expects to take the victims confidential data by diverting them to surf a fake website page that resembles a honest to goodness one is another type of criminal acts through the internet and its one of the especially concerns toward numerous areas including e-managing an account and retailing. Phishing site detection is truly an unpredictable and element issue including numerous components and criteria that are not stable. On account of the last and in addition ambiguities in arranging sites because of the intelligent procedures programmers are utilizing, some keen proactive strategies can be helpful and powerful tools can be utilized, for example, fuzzy, neural system and data mining methods can be a successful mechanism in distinguishing phishing sites. We applied Random Forest (RF), one of the different types of machine learning based algorithms used for detection of Phishing websites. Finally we measured and compared the performance of the classifier in terms of accuracy

Inhibition of Reactive Gliosis Attenuates Excitotoxicity-Mediated Death of Retinal Ganglion Cells

Reactive gliosis is a hallmark of many retinal neurodegenerative conditions, including glaucoma. Although a majority of studies to date have concentrated on reactive gliosis in the optic nerve head, very few studies have been initiated to investigate the role of reactive gliosis in the retina. We have previously shown that reactive glial cells synthesize elevated levels of proteases, and these proteases, in turn, promote the death of retinal ganglion cells (RGCs). In this investigation, we have used two glial toxins to inhibit reactive gliosis and have evaluated their effect on protease-mediated death of RGCs. Kainic acid was injected into the vitreous humor of C57BL/6 mice to induce reactive gliosis and death of RGCs. C57BL/6 mice were also treated with glial toxins, alpha-aminoadipic acid (AAA) or Neurostatin, along with KA. Reactive gliosis was assessed by immunostaining of retinal cross sections and retinal flat-mounts with glial fibrillary acidic protein (GFAP) and vimentin antibodies. Apoptotic cell death was assessed by TUNEL assays. Loss of RGCs was determined by immunostaining of flat-mounted retinas with Brn3a antibodies. Proteolytic activities of matrix metalloproteinase-9 (MMP-9), tissue plasminogen activator (tPA), and urokinase plasminogen activator (uPA) were assessed by zymography assays. GFAP-immunoreactivity indicated that KA induced reactive gliosis in both retinal astrocytes and in Muller cells. AAA alone or in combination with KA decreased GFAP and vimentin-immunoreactivity in Mϋller cells, but not in astrocytes. In addition AAA failed to decrease KA-mediated protease levels and apoptotic death of RGCs. In contrast, Neurostatin either alone or in combination with KA, decreased reactive gliosis in both astrocytes and Mϋller cells. Furthermore, Neurostatin decreased protease levels and prevented apoptotic death of RGCs. Our findings, for the first time, indicate that inhibition of reactive gliosis decreases protease levels in the retina, prevents apoptotic death of retinal neurons, and provides substantial neuroprotection

Improved Metabolic Stability for 18 F PET Probes Rapidly Constructed via Tetrazine trans -Cyclooctene Ligation

The fast kinetics and bioorthogonal nature of the tetrazine trans-cyclooctene (TCO) ligation makes it a unique tool for PET probe construction. In this study, we report the development of an 18F-labeling system based on a CF3-substituted diphenyl-s-tetrazine derivative with the aim of maintaining high reactivity while increasing in vivo stability. c(RGDyK) was tagged by a CF3-substituted diphenyl-s-tetrazine derivative via EDC-mediated coupling. The resulting tetrazine-RGD conjugate was combined with a 19F-labeled TCO derivative to give HPLC standards. The analogous 18F-labeled TCO derivative was combined with the diphenyl-s-tetrazine-RGD at μM concentration. The resulting tracer was subjected to in vivo metabolic stability assessment, and microPET studies in murine U87MG xenograft models. The diphenyl-s-tetrazine-RGD combines with an 18F-labeled TCO in high yields (>97% decay-corrected on the basis of TCO) using only 4 equiv of tetrazine-RGD relative to the 18F-labeled TCO (concentration calculated based on product’s specific activity). The radiochemical purity of the 18F-RGD peptides was >95% and the specific activity was 111 GBq/μmol. Noninvasive microPET experiments demonstrated that 18F-RGD had integrin-specific tumor uptake in subcutaneous U87MG glioma. In vivo metabolic stability of 18F-RGD in blood, urine and major organs showed two major peaks: one corresponded to the Diels-Alder conjugate and the other was identified as the aromatized analog. A CF3-substituted diphenyl-s-tetrazine displays excellent speed and efficiency in 18F-PET probe construction, providing nearly quantitative 18F labeling within minutes at low micromolar concentrations. The resulting conjugates display improved in vivo metabolic stability relative to our previously described system

Targeting ανβ3 and ανβ5 inhibits photon-induced hypermigration of malignant glioma cells

Sublethal photon irradiation was recently suspected to increase tumor cell motility and promote locoregional recurrence of disease. This study was set up to describe mechanisms underlying increased glioma cell migration through photon irradiation and to analyse the modifiability of photon-altered glioma cell motility by integrin inhibition