Generation and Characterization of Specific Monoclonal Antibodies and Nanobodies Directed Against the ATP-Gated Channel P2X4

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A cDNA Immunization Strategy to Generate Nanobodies against Membrane Proteins in Native Conformation

Nanobodies (Nbs) are soluble, versatile, single-domain binding modules derived from the VHH variable domain of heavy-chain antibodies naturally occurring in camelids. Nbs hold huge promise as novel therapeutic biologics. Membrane proteins are among the most interesting targets for therapeutic Nbs because they are accessible to systemically injected biologics. In order to be effective, therapeutic Nbs must recognize their target membrane protein in native conformation. However, raising Nbs against membrane proteins in native conformation can pose a formidable challenge since membrane proteins typically contain one or more hydrophobic transmembrane regions and, therefore, are difficult to purify in native conformation. Here, we describe a highly efficient genetic immunization strategy that circumvents these difficulties by driving expression of the target membrane protein in native conformation by cells of the immunized camelid. The strategy encompasses ballistic transfection of skin cells with cDNA expression plasmids encoding one or more orthologs of the membrane protein of interest and, optionally, other costimulatory proteins. The plasmid is coated onto 1 µm gold particles that are then injected into the shaved and depilated skin of the camelid. A gene gun delivers a helium pulse that accelerates the DNA-coated particles to a velocity sufficient to penetrate through multiple layers of cells in the skin. This results in the exposure of the extracellular domains of the membrane protein on the cell surface of transfected cells. Repeated immunization drives somatic hypermutation and affinity maturation of target-specific heavy-chain antibodies. The VHH/Nb coding region is PCR-amplified from B cells obtained from peripheral blood or a lymph node biopsy. Specific Nbs are selected by phage display or by screening of Nb-based heavy-chain antibodies expressed as secretory proteins in transfected HEK cells. Using this strategy, we have successfully generated agonistic and antagonistic Nbs against several cell surface ecto-enzymes and ligand-gated ion channels

The Mw7.8 2016 Pedernales, Ecuador earthquake aftershock sequence: a detailed spatio-temporal analysis of the rupture processes, stress patterns and slip behavior

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Antihypertensive Therapy Is Associated with Reduced Rate of Conversion to Alzheimer's Disease in Midregional Proatrial Natriuretic Peptide Stratified Subjects with Mild Cognitive Impairment

Background: Hypertension is a major risk factor of Alzheimer's disease (AD); however, controlled studies on the effect of antihypertensive treatment on the risk of dementia are inconclusive. Therefore, a biological marker that predicts individual response to antihypertensive treatment would be of high clinical relevance. Midregional proatrial natriuretic peptide (MR-proANP), an inactive surrogate molecule of the mature atrial natriuretic peptide, is related to circulatory function and hypertension. Methods: A sample population of 134 subjects with mild cognitive impairment (MCI) was followed for up to 6 years. Multivariable Cox regression analysis was conducted to predict conversion to AD based on all relevant variables. Results: Baseline MR-proANP was significantly increased in the AD converter group (p 74 pmol/L. Further subgrouping by age (>/<= 72 years at baseline) increased the positive correlation of antihypertensive treatment and MCI outcome for patients below the age of 72 years (conversion rate reduced by 74%, p = .016). Conclusions: These data seem to support the notion of a potential impact of circulatory function for the prognosis of AD at a prodromal stage. The MR-proANP levels may be useful to predict the effect of antihypertensive treatment on conversion rates to AD in subjects with MCI

Increased surface P2X4 receptor regulates anxiety and memory in P2X4 internalization-defective knock-in mice

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