HB‐EGF affects astrocyte morphology,proliferation, differentiation,and the expression of intermediate filament proteins

Heparin‐binding epidermal growth factor‐like growth factor (HB‐EGF), a vascular‐derived trophic factor, belongs to the epidermal growth factor (EGF) family of neuroprotective, hypoxia‐inducible proteins released by astrocytes in CNS injuries. It was suggested that HB–EGF can replace fetal calf serum (FCS) in astrocyte cultures. We previously demonstrated that in contrast to standard 2D cell culture systems, Bioactive3D culture system, when used with FCS, minimizes the baseline activation of astrocytes and preserves their complex morphology. Here, we show that HB‐EGF induced EGF receptor (EGFR) activation by Y1068 phosphorylation, Mapk/Erk pathway activation, and led to an increase in cell proliferation, more prominent in Bioactive3D than in 2D cultures. HB‐EGF changed morphology of 2D and Bioactive3D cultured astrocytes toward a radial glia‐like phenotype and induced the expression of intermediate filament and progenitor cell marker protein nestin. Glial fibrillary acidic protein (GFAP) and vimentin protein expression was unaffected. RT‐qPCR analysis demonstrated that HB‐EGF affected the expression of Notch signaling pathway genes, implying a role for the Notch signaling in HB‐EGF‐mediated astrocyte response. HB‐EGF can be used as a FCS replacement for astrocyte expansion and in vitro experimentation both in 2D and Bioactive3D culture systems; however, caution should be exercised since it appears to induce partial de‐differentiation of astrocytes.

     

Application of singular spectrum-based change-point analysis to EMG-onset detection

While many approaches have been proposed to identify the signal onset in EMG recordings, there is no standardized method for performing this task. Here, we propose to use a change-point detection procedure based on singular spectrum analysis to determine the onset of EMG signals. This method is suitable for automated real-time implementation, can be applied directly to the raw signal, and does not require any prior knowledge of the EMG signal’s properties. The algorithm proposed by Moskvina and Zhigljavsky (2003) was applied to EMG segments recorded from wrist and trunk muscles. Wrist EMG data was collected from 9 Parkinson’s disease patients with and without tremor, while trunk EMG data was collected from 13 healthy able-bodied individuals. Along with the change-point detection analysis, two threshold-based onset detection methods were applied, as well as visual estimates of the EMG onset by trained practitioners. In the case of wrist EMG data without tremor, the change-point analysis showed comparable or superior frequency and quality of detection results, as compared to other automatic detection methods. In the case of wrist EMG data with tremor and trunk EMG data, performance suffered because other changes occurring in these signals caused larger changes in the detection statistic than the changes caused by the initial muscle activation, suggesting that additional criteria are needed to identify the onset from the detection statistic other than its magnitude alone. Once this issue is resolved, change-point detection should provide an effective EMG-onset detection method suitable for automated real-time implementation.     

Complement: a novel factor in basal and ischemia-induced neurogenesis

Through its involvement in inflammation, opsonization, and cytolysis, the complement protects against infectious agents. Although most of the complement proteins are synthesized in the central nervous system (CNS), the role of the complement system in the normal or ischemic CNS remains unclear. Here we demonstrate for the first time that neural progenitor cells and immature neurons express receptors for complement fragments C3a and C5a (C3a receptor (C3aR) and C5a receptor). Mice that are deficient in complement factor C3 (C3(-/-)) lack C3a and are unable to generate C5a through proteolytic cleavage of C5 by C5-convertase. Intriguingly, basal neurogenesis is decreased both in C3(-/-) mice and in mice lacking C3aR or mice treated with a C3aR antagonist. The C3(-/-) mice had impaired ischemia-induced neurogenesis both in the subventricular zone, the main source of neural progenitor cells in adult brain, and in the ischemic region, despite normal proliferative response and larger infarct volumes. Thus, in the adult mammalian CNS, complement activation products promote both basal and ischemia-induced neurogenesis.     

Alloxan reduces amplitude of ventricular myocyte shortening and intracellular Ca2+ without altering L-type Ca2+ current, sarcoplasmic reticulum Ca2+ content or myofilament sensitivity to Ca2+ in Wistar rats

Alloxan is widely used to induce diabetes mellitus in experimental animals. Recent studies have provided evidence that alloxan has direct actions on cardiac muscle contraction. The aim of this study was to further investigate the mechanisms underlying the effects of alloxan on ventricular myocyte shortening and intracellular Ca²⁺ transport. Amplitude of myocyte shortening was reduced in a dose-dependent manner as the concentration of alloxan was increased in the range 10^?7–10^?4 M. Amplitude of shortening was reduced (56.8 ± 6.6%, n = 27) by 10^?6 M alloxan and was partially reversed during a 10 min washout. Amplitude of the Ca²⁺ transient was also reduced (79.7 ± 2.9%, n = 29) by 10^?6 M alloxan. Caffeine-evoked sarcoplasmic reticulum Ca²⁺ release, fractional release of Ca²⁺, assessed by comparing the amplitude of electrically evoked with that of caffeine-evoked Ca²⁺ transients, and fura-2-cell length trajectory during the late stages of relaxation of myocyte twitch contraction were not significantly altered by alloxan. The amplitude of L-type Ca²⁺ current was not altered by alloxan. Alterations in sarcoplasmic reticulum Ca²⁺ transport, myofilament sensitivity to Ca²⁺, and L-type Ca²⁺ current do not appear to underlie the negative inotropic effects of alloxan.     

High-sensitivity profiling of glycoproteins from human blood serum through multiple-lectin affinity chromatography and liquid chromatography/tandem mass spectrometry

We report here the use of high-performance lectin affinity enrichment of glycoproteins at microscale levels using a series of silica-bound lectins. The potential of this approach is being demonstrated for the glycoprotein enrichment from microliter volumes of human blood serum. Individual injections of sample to the affinity microcolumns packed with four lectin materials with different glycan specificities (Con A, SNA-I, UEA-I, PHA-L), followed by off-line reversed-phase pre-fractionation and nano-LC/MS/MS, permitted identification of 108 proteins in the lectin-bound fractions spanning a concentration dynamic range of 7-10 orders of magnitude. In contrast, multi-lectin microcolumn affinity chromatography, an alternative enrichment approach allowed identification of only 67 proteins. An attractive feature of high-performance lectin affinity chromatography at microscale levels is the substantial reduction of sample losses that are commonly experienced with extensive sample preparation needed for larger sample volumes.     

Tyramine-modified pectins via periodate oxidation for soybean hull peroxidase induced hydrogel formation and immobilization

Pectin was modified by oxidation with sodium periodate at molar ratios of 2.5, 5, 10, 15 and 20 mol% and reductive amination with tyramine and sodium cyanoborohydride afterwards. Concentration of tyramine groups within modified pectin ranged from 54.5 to 538 μmol/g of dry pectin while concentration of ionizable groups ranged from 3.0 to 4.0 mmol/g of dry polymer compared to 1.5 mmol/g before modification due to the introduction of amino group. All tyramine-pectins showed exceptional gelling properties and could form hydrogel both by cross-linking of carboxyl groups with calcium or by cross-linking phenol groups with peroxidase in the presence of hydrogen peroxide. These hydrogels were tested as carriers for soybean hull peroxidase (SHP) immobilization within microbeads formed in an emulsion based enzymatic polymerization reaction. SHP immobilized within tyramine-pectin microbeads had an increased thermal and organic solvent stability compared to the soluble enzyme. Immobilized SHP was more active in acidic pH region and had slightly decreased K _m value of 2.61 mM compared to the soluble enzyme. After 7 cycles of repeated use in batch reactor for pyrogallol oxidation microbeads, immobilized SHP retained half of the initial activity.

     

Loss of Small-RNA-Directed DNA Methylation in the Plant Cell Cycle Promotes Germline Reprogramming and Somaclonal Variation

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