Study of Hydrolysis Kinetics & Lactone-Acid- Salt Equilibria of γ-D-Mannonoiactone

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Large-scale interrogation of retinal cell functions by 1-photon light-sheet microscopy

Visual processing in the retina depends on the collective activity of large ensembles of neurons organized in different layers. Current techniques for measuring activity of layer-specific neural ensembles rely on expensive pulsed infrared lasers to drive 2-photon activation of calcium-dependent fluorescent reporters. We present a 1-photon light-sheet imaging system that can measure the activity in hundreds of neurons in the ex vivo retina over a large field of view while presenting visual stimuli. This allows for a reliable functional classification of different retinal cell types. We also demonstrate that the system has sufficient resolution to image calcium entry at individual synaptic release sites across the axon terminals of dozens of simultaneously imaged bipolar cells. The simple design, large field of view, and fast image acquisition make this a powerful system for high-throughput and high-resolution measurements of retinal processing at a fraction of the cost of alternative approaches

SC65 directly interacts with prolyl 3-hydroxylase 3 (P3H3).

<p>a) Lysates of 714 mouse embryonic fibroblasts stably expressing SC65-Flag or EV control were used for IP experiments utilizing a Flag antibody (upper panel) or a P3H3 antibody (lower panel). 10% of total inputs and immuno-precipitates were separated on a 10% SDS-PAGE gel, blotted and probed with antibodies against FLAG and P3H3. The reciprocal interaction of SC65-Flag with P3H3 is confirmed in both experiments. b) Western blot of primary calvarial osteoblast and skin fibroblast lysates from WT and <i>Sc65KO</i> 3 day-old mice (N = 2) showing significantly decreased levels of P3H3 protein in <i>Sc65KO</i> samples. Densitometric quantification of P3H3 protein normalized to β-actin from the western blot shown above (#p<0.01; *p<0.05; error bars represent SD). All experiments were performed at least 3 times.</p

Schematic representation of a fibrillar collagen molecule in the ER.

<p>The long uninterrupted triple-helical domain is shown here already folded and without N- and C-propeptides. Depicted in different colors are some of the prolyl 3-hydroxylase enzymes (P3Hs), lysyl hydroxylase enzymes (LHs) and cyclophilin B (purple) with some of their known substrate residues. Our previous work identified the prolyl 3-hydroxylation complex that modifies P986; our current work suggests the existence of a new complex, possibly including CYPB, responsible for the hydroxylation of K87 and K930. Evidence indicates that CRTAP and SC65 act as unique orchestrators of essential molecular complexes for collagen post-translational modification. (LH2 is likely to also act within a protein complex but direct interactions have not yet been published).</p

Loss of Sc65 results in dermal tears, abnormal collagen fibrils and skin fragility.

<p>a) H&E stained sections of WT and <i>Sc65KO</i> skin. Note the decreased density of collagen, the frayed dermis indicated by arrows and the reduced thickness of the muscle layer in the <i>Sc65-null</i> samples. b) Serial skin sections were stained with Sirius red. <i>Sc65-null</i> skin exhibits fewer large collagen fibers (red staining) and greater number of smaller collagen fibers stained in green compared to WT counterparts. c) Electron micrographs of 7 month-old mouse skin biopsy from WT and <i>Sc65KO</i> mice. Collagen fibrils, shown in cross-section, from <i>Sc65-null</i> skin tended to be smaller and have a decreased range of fibril diameter compared to WT fibrils. Loss of Sc65 also resulted in the presence of collagen fibrils with irregular profile and several large “cauliflower-like” fibrils (red arrow) which indicate abnormal fibrillogenesis (scale bar represents 500nm). d) Distribution of collagen fibril diameter in WT and <i>Sc65KO</i> mouse skin as measured from electron microscopy images. Measurements were collected from three different mice/genotype and >200 fibril/mouse. e) Skin EMs from <i>Sc65KO</i> mice also exhibited significantly more empty space among collagen fibrils compared to WT mice indicating a less densely packed collagen (*p = 0.01). Five electron micrograph images of non-overlapping areas were quantified from each mouse. f-h) Skin samples from WT and <i>Sc65KO</i> mice were subjected to a biomechanical skin loading test to measure tensile strength. Skin that lacks SC65 expression ruptured at a significantly lower peak load compared to WT skin indicating significant skin fragility (*p<0.01).</p

<i>Sc65KO</i> mouse generation and confirmation of bone loss phenotype.

<p>a) Strategy for the creation of the <i>Sc65-null</i> allele. The schematic diagram shows the <i>Sc65</i> wild-type, targeted, floxed and excised allele. <i>Sc65</i> coding regions are in light blue while non-coding regions are in dark blue. Also note the proximity to the <i>Fkbp10</i> gene which is transcribed in the opposite orientation. b) PCR genotyping of <i>Sc65KO</i> mice (upper panel) and Western blot confirmation of SC65 protein (arrow) loss in multiple <i>Sc65KO</i> tissues from 3 day-old mice compared to WT controls (lower panel—Cal = calvaria, Kid = Kidney). c) Immunohistochemistry detection of SC65 in adult femur section from a WT mouse showing specific intracellular staining in bone forming cells (osteoblasts) aligned on the surface of a bone trabecula. <i>Sc65</i> expression is lost in a similar section from a <i>Sc65KO</i> mouse. Scale bars = 100μM (10x) or 20μM (63x). d) MicroCT analysis of long bones from 6 month-old WT and <i>Sc65KO</i> male mice (n = 9). Both femurs and tibias from <i>Sc65KO</i> mice exhibited decreased trabecular bone volume/tissue volume (BV/TV), connectivity density (Conn.D) and cortical thickness (Ct.Th) compared to WT controls (*p<0.05).</p

Characterization of a new SC65/LH1/P3H3 complex in the ER.

<p>a) Lysates of 714 mouse embryonic fibroblasts that were transiently transfected with an HA-tagged LH1 expression construct were immuno-precipitated with a HA antibody (upper panel) or a P3H3 antibody (lower panel). 10% of total inputs and immuno-precipitates were separated on a 8% SDS-PAGE gel, blotted and probed with antibodies against HA and P3H3. Negative controls included non-transfected 714 cells incubated with the HA antibody (for non-specific binding of HA antibody, left lanes) and LH1-HA transfected cells incubated with no antibody (for non-specific proteins binding to beads, middle lanes). In both experiments, LH1-HA and P3H3 were found to interact (right lanes). b) Lysates of 714 mouse embryonic fibroblasts stably expressing SC65-Flag or EV control and transiently transfected with a HA-tagged CYPB were used for IP utilizing an HA antibody. 10% of total input and immuno-precipitates were separated on a 12% SDS-PAGE gel, blotted and probed with antibodies against Flag and HA. The blot detecting SC65-Flag following IP with the HA antibody is shown over-exposed. c) Western blot of primary calvarial osteoblast and skin fibroblast lysates from WT and <i>Sc65KO</i> 3 day-old mice (N = 2) showing similar content of CYPB protein in <i>Sc65KO</i> and WT samples. All experiments were performed at least 3 times.</p