Technical Brief: A novel strategy for enrichment of trabecular meshwork protease proteome

We present a novel and simple enrichment strategy to capture trabecular meshwork (TM) protease proteome. The method relies on fractionation of TM tissue into cytosolic and nuclear extracts and subsequent affinity enrichment of proteases on peptide inhibitors. A large repertoire of available protease substrate analog peptides enables an improved capture of TM protease proteome compared to SDS–PAGE fractionation alone. Peptide analog inhibitors of protease substrates are immobilized on a protein A or G column using 254 nm intense ultraviolet (UV) light. The TM cytosolic protein extract incubated on the column is eluted with salt or a buffer with a low hydrogen ion concentration. The resultant protein solution is precipitated with acetone, fractionated on SDS–PAGE, in situ trypsin digested, and subjected to mass spectrometry. This relatively simple protocol enables improved capture of cytosolic proteases. We identified 20 previously reported TM proteins from a single donor tissue using affinity enrichment. The majority of identified proteins were either intracellular proteases or known protease inhibitors. Both serine and cysteine proteases were captured using this strategy with improved coverage compared to our previous identification without affinity enrichment

Comparative lipid profiling dataset of the inflammation-induced optic nerve regeneration.

In adult mammals, retinal ganglion cells (RGCs) fail to regenerate following damage. As a result, RGCs die after acute injury and in progressive degenerative diseases such as glaucoma; this can lead to permanent vision loss and, eventually, blindness. Lipids are crucial for the development and maintenance of cell membranes, myelin sheaths, and cellular signaling pathways, however, little is known about their role in axon injury and repair. Studies examining changes to the lipidome during optic nerve (ON) regeneration could greatly inform treatment strategies, yet these are largely lacking. Experimental animal models of ON regeneration have facilitated the exploration of the molecular determinants that affect RGC axon regeneration. Here, we analyzed lipid profiles of the ON and retina in an ON crush rat model using liquid chromatography-mass spectrometry. Furthermore, we investigated lipidome changes after ON crush followed by intravitreal treatment with Zymosan, a yeast cell wall derivative known to enhance RGC regeneration. This data is available at the NIH Common Fund's Metabolomics Data Repository and Coordinating Center (supported by NIH grant, U01-DK097430) website, the Metabolomics Workbench, http://www.metabolomicsworkbench.org, where it has been assigned Project ID: PR000661. The data can be accessed directly via it's Project DOI: doi: 10.21,228/M87D53

Hydrogen production by Cyanobacteria

The limited fossil fuel prompts the prospecting of various unconventional energy sources to take over the traditional fossil fuel energy source. In this respect the use of hydrogen gas is an attractive alternate source. Attributed by its numerous advantages including those of environmentally clean, efficiency and renew ability, hydrogen gas is considered to be one of the most desired alternate. Cyanobacteria are highly promising microorganism for hydrogen production. In comparison to the traditional ways of hydrogen production (chemical, photoelectrical), Cyanobacterial hydrogen production is commercially viable. This review highlights the basic biology of cynobacterial hydrogen production, strains involved, large-scale hydrogen production and its future prospects. While integrating the existing knowledge and technology, much future improvement and progress is to be done before hydrogen is accepted as a commercial primary energy source

ETX1 is over-expressed in the glaucomatous trabecular meshwork

To determine whether exon-trapped X chromosome clone 1 (ETX1) is overexpressed in the trabecular meshwork (TM) of glaucomatous human eyes compared to controls. Immunohistochemical, western blot, and enzyme-linked immunosorbent assay analysis were used with human tissues and TM protein extracts. Reverse transcription-PCR was performed on isolated mRNA-derived cDNA preparations. Elevated expression levels of ETX1 were detected in glaucomatous compared to control TM tissue. This corroborates previous detection of ETX1 in glaucomatous TM by proteomic analysis. ETX1 mRNA is present in TM tissue, suggesting ETX1 protein is locally produced within TM cells. This is the first report demonstrating overexpression of ETX1 in glaucomatous TM. ETX1 expression may regulate TM protein interactions involved in cell adhesion, and its aberrant overexpression may be part of the pathophysiological pathway in the development of glaucoma

Aqueous Humor Dynamics: A Review

Glaucoma is a family of optic neuropathies which cause irreversible but potentially preventable vision loss. Vision loss in most forms of glaucoma is related to elevated IOP with subsequent injury to the optic nerve. Secretion of aqueous humor and regulation of its outflow are physiologically important processes for maintaining IOP in the normal range. Thus, understanding the complex mechanisms that regulate aqueous humor circulation is essential for management of glaucoma. The two main structures related to aqueous humor dynamics are the ciliary body and the trabecular meshwork (TM). Three mechanisms are involved in aqueous humor formation: diffusion, ultrafiltration and active secretion. Active secretion is the major contributor to aqueous humor formation. The aqueous humor flow in humans follows a circadian rhythm, being higher in the morning than at night. The aqueous humor leaves the eye by passive flow via two pathways - the trabecular meshwork and the uveoscleral pathway. In humans, 75% of the resistance to aqueous humor outflow is localized within the TM with the juxtacanalicular portion of the TM being the main site of outflow resistance. Glycosaminoglycan deposition in the TM extracellular matrix (ECM) has been suggested to be responsible for increased outflow resistance at this specific site whereas others have suggested deposition of proteins, such as cochlin, obstruct the aqueous humor outflow through the TM. The uveoscleral outflow pathway is relatively independent of the intraocular pressure and the proportion of aqueous humor exiting the eye via the uveoscleral pathway decreases with age

Interaction of cochlin and mechanosensitive channel TREK-1 in trabecular meshwork cells influences the regulation of intraocular pressure.

This work was funded by National Institute of Health Grants R01 EY016112, EY015266, and EY014801 and an unrestricted grant to the University of Miami's Bascom Palmer Eye Institute from Research to Prevent Blindness. Financial support from Fight for Sight is gratefully acknowledged. Funding to XG was provided by Instituto de Salud Carlos III, Spain (FIS PI14/00141 and RETIC RD12/0034/0003) and Generalitat de Catalunya (2014SGR1165). In the eye, intraocular pressure (IOP) is tightly regulated and its persistent increase leads to ocular hypertension and glaucoma. We have previously shown that trabecular meshwork (TM) cells might detect aqueous humor fluid shear stress via interaction of the extracellular matrix (ECM) protein cochlin with the cell surface bound and stretch-activated channel TREK-1. We provide evidence here that interaction between both proteins are involved in IOP regulation. Silencing of TREK-1 in mice prevents the previously demonstrated cochlin-overexpression mediated increase in IOP. Biochemical and electrophysiological experiments demonstrate that high shear stress-induced multimeric cochlin produces a qualitatively different interaction with TREK-1 compared to monomeric cochlin. Physiological concentrations of multimeric but not monomeric cochlin reduce TREK-1 current. Results presented here indicate that the interaction of TREK-1 and cochlin play an important role for maintaining IOP homeostasis

Potential use of sugar binding proteins in reactors for regeneration of CO(2 )fixation acceptor D-Ribulose-1,5-bisphosphate

Sugar binding proteins and binders of intermediate sugar metabolites derived from microbes are increasingly being used as reagents in new and expanding areas of biotechnology. The fixation of carbon dioxide at emission source has recently emerged as a technology with potentially significant implications for environmental biotechnology. Carbon dioxide is fixed onto a five carbon sugar D-ribulose-1,5-bisphosphate. We present a review of enzymatic and non-enzymatic binding proteins, for 3-phosphoglycerate (3PGA), 3-phosphoglyceraldehyde (3PGAL), dihydroxyacetone phosphate (DHAP), xylulose-5-phosphate (X5P) and ribulose-1,5-bisphosphate (RuBP) which could be potentially used in reactors regenerating RuBP from 3PGA. A series of reactors combined in a linear fashion has been previously shown to convert 3-PGA, (the product of fixed CO(2 )on RuBP as starting material) into RuBP (Bhattacharya et al., 2004; Bhattacharya, 2001). This was the basis for designing reactors harboring enzyme complexes/mixtures instead of linear combination of single-enzyme reactors for conversion of 3PGA into RuBP. Specific sugars in such enzyme-complex harboring reactors requires removal at key steps and fed to different reactors necessitating reversible sugar binders. In this review we present an account of existing microbial sugar binding proteins and their potential utility in these operations

New ZZ Ceti stars from the LAMOST survey

The spectroscopic sky survey carried out by the Large Sky Area Multi-Object Fiber Spectroscopic Telescope (LAMOST) provides the largest stellar spectra library in the world until now. A large number of new DA white dwarfs had been identified based on the LAMOST spectra. The effective temperature ($T_{\rm eff}$) and surface gravity ($\log g$) of most DA white dwarfs were determined and published in the catalogs, e.g. Zhao et al. (2013), Rebassa-Mansergas et al. (2015), Gentile Fusillo et al. (2015) and Guo et al. (2015). We selected ZZ Ceti candidates from the published catalogs by considering whether their $T_{\rm eff}$ are situated in the ZZ Ceti instability strip. The follow-up time-series photometric observations for the candidates were performed in 2015 and 2016. Four stars: LAMOST J004628.31+343319.90, LAMOST J062159.49+252335.9, LAMOST J010302.46+433756.2 and LAMOST J013033.90+273757.9 are finally confirmed to be new ZZ Ceti stars. They show dominant peaks with amplitudes rising above the 99.9% confidence level in the amplitude spectra. As LAMOST J004628.31+343319.90 has an estimated mass of $\sim$ 0.40 $M_{\odot}$ and LAMOST J013033.90+273757.9 has a mass of $\sim$ 0.45 $M_{\odot}$ derived from their $\log g$ values, these two stars are inferred to be potential helium-core white dwarfs.Comment: 15 pages, 8 figures, 3 tables, accepted for publication in Ap