Magnetic Resonance Spectroscopy discriminates the response to microglial stimulation of wild type and Alzheimer's disease models.

Microglia activation has emerged as a potential key factor in the pathogenesis of Alzheimers disease. Metabolite levels assessed by magnetic resonance spectroscopy (MRS) are used as markers of neuroinflammation in neurodegenerative diseases, but how they relate to microglial activation in health and chronic disease is incompletely understood. Using MRS, we monitored the brain metabolic response to lipopolysaccharides (LPS)-induced microglia activation in vivo in a transgenic mouse model of Alzheimers disease (APP/PS1) and healthy controls (wild-type (WT) littermates) over 4 hours. We assessed reactive gliosis by immunohistochemistry and correlated metabolic and histological measures. In WT mice, LPS induced a microglial phenotype consistent with activation, associated with a sustained increase in macromolecule and lipid levels (ML9). This effect was not seen in APP/PS1 mice, where LPS did not lead to a microglial response measured by histology, but induced a late increase in the putative inflammation marker myoinositol (mI) and metabolic changes in total creatine and taurine previously reported to be associated with amyloid load. We argue that ML9 and mI distinguish the response of WT and APP/PS1 mice to immune mediators. Lipid and macromolecule levels may represent a biomarker of activation of healthy microglia, while mI may not be a glial marker

Immunodeficiency, autoimmune thrombocytopenia and enterocolitis caused by autosomal recessive deficiency of PIK3CD-encoded phosphoinositide 3-kinase δ.

Phosphoinositide 3-kinase δ (PI3Kδ), a lipid kinase consisting of a catalytic (p110δ, encoded by PIK3CD) and a regulatory subunit (p85, encoded by PIK3R1), generates the second messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP3) in the plasma membrane of leukocytes downstream of antigen and cytokine receptors.1 Signaling via PDK1, AKT, mTOR and downstream targets such as FOXO1, contributes to the metabolic and transcriptional changes required for the expansion, differentiation and effector function of lymphocytes. Activating germline mutations in PIK3CD cause the immune dysregulatory disease activated PI3Kδ syndrome (APDS), usually presenting with recurrent sinopulmonary infections in childhood, herpesvirus infections and CD4+ lymphopenia, underscoring the important role of balanced p110δ activity in human adaptive immunity. Ablation of p110δ in mice leads to aberrant T cell responses and intestinal inflammation. In humans, immune dysregulation including severe colitis is present in many cancer patients who are treated with the p110δ-specific inhibitor Idelalisib. Recently, one patient with autosomal recessive deficiency of p85α and two patients with loss-of function mutations in p110δ have been described who developed humoral immunodeficiency and colitis

High-throughput mapping of regulatory DNA

Quantifying the effects of cis-regulatory DNA on gene expression is a major challenge. Here, we present the multiplexed editing regulatory assay (MERA), a high-throughput CRISPR-Cas9–based approach that analyzes the functional impact of the regulatory genome in its native context. MERA tiles thousands of mutations across ~40 kb of cis-regulatory genomic space and uses knock-in green fluorescent protein (GFP) reporters to read out gene activity. Using this approach, we obtain quantitative information on the contribution of cis-regulatory regions to gene expression. We identify proximal and distal regulatory elements necessary for expression of four embryonic stem cell–specific genes. We show a consistent contribution of neighboring gene promoters to gene expression and identify unmarked regulatory elements (UREs) that control gene expression but do not have typical enhancer epigenetic or chromatin features. We compare thousands of functional and nonfunctional genotypes at a genomic location and identify the base pair–resolution functional motifs of regulatory elements.National Institutes of Health (U.S.) (1U01HG007037

Regulation of rat intrapulmonary arterial tone by arachidonic acid and prostaglandin E2 during hypoxia

Aims Arachidonic acid (AA) and its metabolites, prostaglandins (PG) are known to be involved in regulation of vascular homeostasis including vascular tone and vessel wall tension, but their potential role in Hypoxic pulmonary vasoconstriction (HPV) remains unclear. In this study, we examined the effects of AA and PGE2 on the hypoxic response in isolated rat intrapulmonary arteries (IPAs). Methods and Results We carried out the investigation on IPAs by vessel tension measurement. Isotetrandrine (20 µM) significantly inhibited phase I, phase IIb and phase IIc of hypoxic vasoconstriction. Both indomethacin (100 µM) and NS398 attenuated KPSS-induced vessel contraction and phase I, phase IIb and phase IIc of HPV, implying that COX-2 plays a primary role in the hypoxic response of rat IPAs. PGE2 alone caused a significant vasoconstriction in isolated rat IPAs. This constriction is mediated by EP4. Blockage of EP4 by L-161982 (1 µM) significantly inhibited phase I, phase IIb and phase IIc of hypoxic vasoconstriction. However, AH6809 (3 µM), an antagonist of EP1, EP2, EP3 and DP1 receptors, exerted no effect on KPSS or hypoxia induced vessel contraction. Increase of cellular cAMP by forskolin could significantly reduce KPSS-induced vessel contraction and abolish phase I, phase II b and phase II c of HPV. Conclusion Our results demonstrated a vasoconstrictive effect of PGE2 on rat IPAs and this effect is via activation of EP4. Furthermore, our results suggest that intracellular cAMP plays dual roles in regulation of vascular tone, depending on the spatial distribution of cAMP and its coupling with EP receptor and Ca2+ channels

CRL4 antagonizes SCFFbxo7-mediated turnover of cereblon and BK channel to regulate learning and memory

Intellectual disability (ID), one of the most common human developmental disorders, can be caused by genetic mutations in Cullin 4B (Cul4B) and cereblon (CRBN). CRBN is a substrate receptor for the Cul4A/B-DDB1 ubiquitin ligase (CRL4) and can target voltage- and calcium-activated BK channel for ER retention. Here we report that ID-associated CRL4CRBNmutations abolish the interaction of the BK channel with CRL4, and redirect the BK channel to the SCFFbxo7ubiquitin ligase for proteasomal degradation. Glioma cell lines harbouring CRBN mutations record density-dependent decrease of BK currents, which can be restored by blocking Cullin ubiquitin ligase activity. Importantly, mice with neuron-specific deletion of DDB1 or CRBN express reduced BK protein levels in the brain, and exhibit similar impairment in learning and memory, a deficit that can be partially rescued by activating the BK channel. Our results reveal a competitive targeting of the BK channel by two ubiquitin ligases to achieve exquisite control of its stability, and support changes in neuronal excitability as a common pathogenic mechanism underlying CRL4CRBN–associated ID

Three types of K(+) currents in murine osteocyte-like cells (MLO-Y4).

Metadata onlyOsteocytes play an important role in signaling within bone. Communication of osteocytes with each other and with bone lining cells may have a function in mineral homeostasis and mechanotransduction. However, very little is known of the expression of ion channels in these cells. Using the whole-cell patch-clamp technique, we have detected three types of K(+) currents in the mouse osteocyte-like cell line MLO-Y4. The most commonly observed current (48% of cells) activated rapidly (20 msec) in response to depolarizing steps from -40 mV and exhibited voltage-dependent inactivation. The current was inhibited by 20 mmol/L tetraethyl ammonium (TEA) and abolished by intracellular 2 mmol/L 4-aminopyridine (4-AP). Biophysical and pharmacological characteristics of the current differed from those of inactivating K(+) currents in osteoblastic cells. In 22% of cells, a slowly activating, voltage-activated current was observed (threshold at 20-30 mV). This current was TEA insensitive, was abolished by intracellular application of 2 mmol/L 4-AP, and was strongly inhibited by apamin, a selective inhibitor of small conductance (SK) Ca(2+)-activated K(+) channels. A third current developed during whole-cell dialysis (37% of cells). This current showed little voltage sensitivity. It was abolished by intracellular application of 2 mmol/L 4-AP, high-extracellular Ba(2+) (108 mmol/L), or by inclusion of ATP in the intracellular solution, but was insensitive to TEA, apamin, Cs(+), and glibenclamide. None of these currents was affected by replacement of chloride with acetate in the bath or pipette salines. Reverse-transcription polymerase chain reaction confirmed the presence of mRNA for the types 1 and 2 SK channels, but message for the large conductance (BK) Ca(2+)-activated K(+) channel was not detected in these cells. Message for the sulphonylurea receptor SUR2, a subunit of glibenclamide-insensitive ATP-dependent K(+) channels (K(ATP)), was also detected, but the glibenclamide-sensitive SUR1 subunit was not. These data are the first descriptions of SK- and ATP-sensitive, glibenclamide-insensitive channels in cells of osteoblastic lineage. Our findings are consistent with a change in K(+) channel expression during differentiation from osteoblast to osteocyte. K(+) channels of osteocytes will contribute to maintenance of the cell membrane potential and thus may participate in mechanosensitivity and osteocyte intercellular communication. In addition, they may be involved in homeostatic maintenance of the extracellular fluid occupying the periosteocytic space

Cost-Efficient Materials in Aerospace: Composite vs Aluminium,

Aluminium alloys are a series of traditional materials applied in aerospace industry. Composite materials have been being used to replace some aluminium alloys in airframe structures. To select a cost-efficient material in aerospace design, a framework for systematic assessment of composites and aluminium is therefore suggested. Through evaluation of 3Ps which represent prices, properties and processing of material, life cycle assessment of aluminium and composite could be made. By comparison of and trade-off the listed baselines of 3Ps, an economic, rational, available and useful material can be logically selected. © 2008 Springer-Verlag London Limited