Cx43 carboxyl terminal domain determines AQP4 and Cx30 endfoot organization and blood brain barrier permeability

The neurovascular unit (NVU) consists of cells intrinsic to the vessel wall, the endothelial cells and pericytes, and astrocyte endfeet that surround the vessel but are separated from it by basement membrane. Endothelial cells are primarily responsible for creating and maintaining blood–brain-barrier (BBB) tightness, but astrocytes contribute to the barrier through paracrine signaling to the endothelial cells and by forming the glia limitans. Gap junctions (GJs) between astrocyte endfeet are composed of connexin 43 (Cx43) and Cx30, which form plaques between cells. GJ plaques formed of Cx43 do not diffuse laterally in the plasma membrane and thus potentially provide stable organizational features to the endfoot domain, whereas GJ plaques formed of other connexins and of Cx43 lacking a large portion of its cytoplasmic carboxyl terminus are quite mobile. In order to examine the organizational features that immobile GJs impose on the endfoot, we have used super-resolution confocal microscopy to map number and sizes of GJ plaques and aquaporin (AQP)-4 channel clusters in the perivascular endfeet of mice in which astrocyte GJs (Cx30, Cx43) were deleted or the carboxyl terminus of Cx43 was truncated. To determine if BBB integrity was compromised in these transgenic mice, we conducted perfusion studies under elevated hydrostatic pressure using horseradish peroxide as a molecular probe enabling detection of micro-hemorrhages in brain sections. These studies revealed that microhemorrhages were more numerous in mice lacking Cx43 or its carboxyl terminus. In perivascular domains of cerebral vessels, we found that density of Cx43 GJs was higher in the truncation mutant, while GJ size was smaller. Density of perivascular particles formed by AQP4 and its extended isoform AQP4ex was inversely related to the presence of full length Cx43, whereas the ratio of sizes of the particles of the AQP4ex isoform to total AQP4 was directly related to the presence of full length Cx43. Confocal analysis showed that Cx43 and Cx30 were substantially colocalized in astrocyte domains near vasculature of truncation mutant mice. These results showing altered distribution of some astrocyte nexus components (AQP4 and Cx30) in Cx43 null mice and in a truncation mutant, together with leakier cerebral vasculature, support the hypothesis that localization and mobility of gap junction proteins and their binding partners influences organization of astrocyte endfeet which in turn impacts BBB integrity of the NVU.

Enzymatically Crosslinked In Situ Synthesized Silk/Gelatin/Calcium Phosphate Hydrogels for Drug Delivery

Our research focuses on combining the valuable properties of silk fibroin (SF) and calcium phosphate (CaP). SF is a natural protein with an easily modifiable structure; CaP is a mineral found in the human body. Most of the new age biocomposites lack interaction between organic/inorganic phase, thus SF/CaP composite could not only mimic the natural bone, but could also be used to make drug delivery systems as well, which can ensure both healing and regeneration. CaP was synthesized in situ in SF at different pH values, and then crosslinked with gelatin (G), horseradish peroxide (HRP), and hydrogen peroxide (H2O2). In addition, dexamethasone phosphate (DEX) was incorporated in the hydrogel and drug delivery kinetics was studied. Hydrogel made at pH 10.0 was found to have the highest gel fraction 110.24%, swelling degree 956.32%, and sustained drug delivery for 72 h. The highest cell viability was observed for the hydrogel, which contained brushite (pH 6)—512.43%.

Cx43 carboxyl terminal domain determines AQP4 and Cx30 endfoot organization and blood brain barrier permeability

The neurovascular unit (NVU) consists of cells intrinsic to the vessel wall, the endothelial cells and pericytes, and astrocyte endfeet that surround the vessel but are separated from it by basement membrane. Endothelial cells are primarily responsible for creating and maintaining blood-brain-barrier (BBB) tightness, but astrocytes contribute to the barrier through paracrine signaling to the endothelial cells and by forming the glia limitans. Gap junctions (GJs) between astrocyte endfeet are composed of connexin 43 (Cx43) and Cx30, which form plaques between cells. GJ plaques formed of Cx43 do not diffuse laterally in the plasma membrane and thus potentially provide stable organizational features to the endfoot domain, whereas GJ plaques formed of other connexins and of Cx43 lacking a large portion of its cytoplasmic carboxyl terminus are quite mobile. In order to examine the organizational features that immobile GJs impose on the endfoot, we have used super-resolution confocal microscopy to map number and sizes of GJ plaques and aquaporin (AQP)-4 channel clusters in the perivascular endfeet of mice in which astrocyte GJs (Cx30, Cx43) were deleted or the carboxyl terminus of Cx43 was truncated. To determine if blood-brain-barrier integrity was compromised in these transgenic mice, we conducted perfusion studies under elevated hydrostatic pressure using horseradish peroxide as a molecular probe enabling detection of micro-hemorrhages in brain sections. These studies revealed that microhemorrhages were more numerous in mice lacking Cx43 or its carboxyl terminus. In perivascular domains of cerebral vessels, we found that density of Cx43 GJs was higher in the truncation mutant, while GJ size was smaller. Density of perivascular particles formed by AQP4 and its extended isoform AQP4ex was inversely related to the presence of full length Cx43, whereas the ratio of sizes of the particles of the AQP4ex isoform to total AQP4 was directly related to the presence of full length Cx43. Confocal analysis showed that Cx43 and Cx30 were substantially colocalized in astrocyte domains near vasculature of truncation mutant mice. These results showing altered distribution of some astrocyte nexus components (AQP4 and Cx30) in Cx43 null mice and in a truncation mutant, together with leakier cerebral vasculature, support the hypothesis that localization and mobility of gap junction proteins and their binding partners influences organization of astrocyte endfeet which in turn impacts BBB integrity of the NVU.

Functionalization of Bulk SiO2 Surface with Biomolecules for Sensing Applications: Structural and Functional Characterizations

Biomolecule immobilization on bulk silicon dioxide (SiO2) is an important aspect in the field of Si-based interfaces for biosensing. The approach used for surface preparation should guarantee not only the stable anchoring of biomolecules but also their structural integrity and biological functioning. In this paper, we review our findings on the SiO2 functionalization process to immobilize a variety of biomolecules, including glucose oxidase, horseradish peroxide, metallothionein, and DNA molecules. Morphological and chemical characterization of SiO2 surfaces after biomolecule immobilization using techniques already employed in the microelectronic industry are presented and discussed. Optical and spectrophotometric analysis revealed the preservation of biomolecules’ activity once they are anchored on the biointerface.

Protein-bound polyphenols create “ghost” band artifacts during chemiluminescence-based antigen detection

Antigen detection during Western blotting commonly utilizes a horseradish peroxidase-coupled secondary antibody and enhanced chemiluminescent substrate. We utilized this technique to examine the impact of green tea-derived polyphenols on the binding of egg white protein-specific IgE antibodies from allergic human plasma to their cognate antigens. Our experiments unexpectedly showed that green tea-derived polyphenols, when stably complexed with egg white proteins, caused “ghost” band formation in the presence of horseradish peroxide. This study suggests that caution should be taken when evaluating polyphenol-bound proteins by enhanced chemiluminescence Western blotting using horseradish peroxidase and demonstrates that protein-bound polyphenols can be a source of “ghost” band artifacts on Western blots.

Enzymatic Synthesis of Polyaniline/Graphite Oxide Nanocomposites

ABSTRACTIn this work, we prepared graphite oxide (GO)/polyaniline nanocomposites by enzymatic polymerization of aniline in an aqueous dispersion of graphite oxide. Two GO dispersions with sheets having an average lateral size of 12.50 μm and 247 nm were used. The enzymatic polymerization was carried out in aqueous acidic medium using toluenesulfonic acid (TSA) as doping agent, horseradish peroxide (HRP) as catalyst, and hydrogen peroxide as oxidizer. The polymerization reaction was studied using 1.0, 2.5 and 5 wt % of GO and nGO dispersions. No changes were observed in the catalytic activity of the peroxidase during the enzymatic synthesis due to the additionof GO sheets. Scanning electron microscopy images show that PANI colloids were attached on GO sheets. The PANI-GO colloids were characterized by ultraviolet–visible spectroscopy and Fourier transformed infrared spectroscopy, whereas their colloidal stability was evaluated at different pHs. The UV-vis spectroscopy study revealed that GO affects the electronic conjugation of PANI modifying its absorption spectrum.