Compulsory Patent Licensing: Is It a Viable Solution in the United States

As technology continues to advance at a rapid pace, so do the number of patents that cover every aspect of making, using, and selling these innovations. In 1996, to compound the rapid change of technology, the U.S. Supreme Court affirmed that business methods are also patentable. Hence in the current environment, scores of patents, assigned to many different parties, may cover a single electronic device or software--making it increasingly impossible to manufacture an electronic device without receiving a cease and desist letter or other notice from a patentee demanding a large royalty or threatening an injunction. Companies, particularly those in the high technology sector, have been asserting for some time now that they are under constant threat of lawsuits that threaten to shut them down. As a result, numerous radical changes to the U.S. Patent Act and patent practice before the U.S. Patent & Trademark Office have been proposed. Certain proposed changes, however, are meeting with resistance because of a reliance on long term patent protection and exclusivity of patent rights by different industries. Notwithstanding, certain foreign governments have already enacted provisions making it possible to obtain a compulsory patent license in the event that a patentee is not practicing his invention or is simply refusing to license the rights to his invention for a reasonable royalty fee

Nonessential role of beta3 and beta5 integrin subunits for efficient clearance of cellular debris after light-induced photoreceptor degeneration

PURPOSE: During light-induced photoreceptor degeneration, large amounts of cellular debris are formed that must be cleared from the subretinal space. The integrins alphavbeta5 and alphavbeta3 are involved in the normal physiological process of phagocytosis in the retina. This study was conducted to investigate the question of whether the lack of beta5 and/or beta3 integrin subunits might influence the course of retinal degeneration and/or clearance of photoreceptor debris induced by acute exposure to light. METHODS: Wild-type, beta5(-/-) and beta3(-/-) single-knockout, and beta3(-/-)/beta5(-/-) Ccl2(-/-)/beta5(-/-) double-knockout mice were exposed to 13,000 lux of white light for 2 hours to induce severe photoreceptor degeneration. Real-time PCR and Western blot analysis were used to analyze gene and protein expression, light- and electron microscopy to judge retinal morphology, and immunofluorescence to study retinal distribution of proteins. RESULTS: Individual or combined deletion of beta3 and beta5 integrin subunits did not affect the pattern of photoreceptor cell loss or the clearance of photoreceptor debris in mice compared with that in wild-type mice. Invading macrophages may contribute to efficient phagocytosis. However, ablation of the MCP-1 gene did not prevent macrophage recruitment. Several chemokines in addition to MCP-1 were induced after light-induced damage that may have compensated for the deletion of MCP-1. CONCLUSIONS: Acute clearance of a large amount of cellular debris from the subretinal space involves invading macrophages and does not depend on beta3 and beta5 integrins

Retinal degeneration modulates intracellular localization of CDC42 in photoreceptors

Purpose: Rho GTPases such as RAS-related C3 botulinum substrate 1 (RAC1) and cell division cycle 42 homolog (S. cerevisiae; CDC42) have been linked to cellular processes including movement, development, and apoptosis. Recently, RAC1 has been shown to be a pro-apoptotic factor in the retina during light-induced photoreceptor degeneration. Here, we analyzed the role of CDC42 in the degenerating retina. Methods: Photoreceptor degeneration was studied in a mouse model for autosomal dominant retinitis pigmentosa (VPP) with or without a rod-specific knockdown of Cdc42, as well as in wild-type and Cdc42 knockdown mice after light exposure. Gene and protein expression were analyzed by real-time PCR, western blotting, and immunofluorescence. Retinal morphology and function were assessed by light microscopy and electroretinography, respectively. Results: CDC42 accumulated in the perinuclear region of terminal deoxynucleotidyl transferase dUTP nick end labeling–negative photoreceptors during retinal degeneration induced by excessive light exposure and in the rd1, rd10, and VPP mouse models of retinitis pigmentosa. The knockdown of Cdc42 did not affect retinal morphology or function in the adult mice and did not influence photoreceptor apoptosis or molecular signaling during induced and inherited retinal degeneration. Conclusions: Retinal degeneration induces the accumulation of CDC42 in the perinuclear region of photoreceptors. In contrast to RAC1, however, lack of CDC42 does not affect the progression of degeneration. CDC42 is also dispensable for normal morphology and function of adult rod photoreceptor cells

Stanniocalcin2, but Not Stanniocalcin1, Responds to Hypoxia in a HIF1-Dependent Manner in the Retina

The quest for neuroprotective factors that can prevent or slow down the progression of retinal degeneration is still ongoing. Acute hypoxic stress has been shown to provide transient protection against subsequent damage in the retina. Stanniocalcins – STC1 and STC2 – are secreted glycoproteins that are hypoxia-regulated and were shown to be cytoprotective in various in vitro studies. Hence, we investigated the expression of stanniocalcins in the normal, degenerating and hypoxic retina. We show that the expression of Stc1 and Stc2 in the retina was detectable as early as postnatal day 10 and persisted during aging. Retinal expression of Stc2, but not Stc1, was induced in mice in an in vivo model of acute hypoxia and a genetic model of chronic hypoxia. Furthermore, we show that HIF1, not HIF2, is responsible for regulating Stc2 in cells with the molecular response to hypoxia activated due to the absence of von Hippel Lindau protein. Surprisingly, Stc2 was not normally expressed in photoreceptors but in the inner retina, as shown by laser capture microdissection and immunofluorescence data. The expression of both Stc1 and Stc2 remained unchanged in the degenerative retina with an almost complete loss of photoreceptors, confirming their expression in the inner retina. However, the absence of either Stc1 or Stc2 had no effect on retinal architecture, as was evident from retinal morphology of the respective knockout mice. Taken together our data provides evidence for the differential regulation of STC1 and STC2 in the retina and the prospect of investigating STC2 as a retinal neuroprotective factor

Cone Genesis Tracing by the Chrnb4-EGFP Mouse Line: Evidences of Cellular Material Fusion after Cone Precursor Transplantation.

The cone function is essential to mediate high visual acuity, color vision, and daylight vision. Inherited cone dystrophies and age-related macular degeneration affect a substantial percentage of the world population. To identify and isolate the most competent cells for transplantation and integration into the retina, cone tracing during development would be an important added value. To that aim, the Chrnb4-EGFP mouse line was characterized throughout retinogenesis. It revealed a sub-population of early retinal progenitors expressing the reporter gene that is progressively restricted to mature cones during retina development. The presence of the native CHRNB4 protein was confirmed in EGFP-positive cells, and it presents a similar pattern in the human retina. Sub-retinal transplantations of distinct subpopulations of Chrnb4-EGFP-expressing cells revealed the embryonic day 15.5 high-EGFP population the most efficient cells to interact with host retinas to provoke the appearance of EGFP-positive cones in the photoreceptor layer. Importantly, transplantations into the DsRed retinas revealed material exchanges between donor and host retinas, as >80% of transplanted EGFP-positive cones also were DsRed positive. Whether this cell material fusion is of significant therapeutic advantage requires further thorough investigations. The Chrnb4-EGFP mouse line definitely opens new research perspectives in cone genesis and retina repair

Retinal Layer Separation (ReLayS) method enables the molecular analysis of photoreceptor segments and cell bodies, as well as the inner retina

Understanding the physiology of the retina, and especially of the highly polarized photoreceptors, is essential not only to broaden our knowledge of the processes required for normal vision, but also to develop effective therapies to prevent or slow retinal degenerative diseases. However, the molecular analysis of photoreceptors is a challenge due to the heterogeneity of the retinal tissue and the lack of easy and reliable methods for cell separation. Here we present the ReLayS method—a simple technique for the separation of photoreceptor segments (PS) containing both inner and outer segments, outer nuclear layer (ONL), and inner retina (InR) that contains the remaining retinal layers. The layer-specific material isolated from a mouse half-retina with the ReLayS method was sufficient for protein isolation and Western blotting or RNA isolation and real-time PCR studies. The separation of PS, ONL, and InR was successfully validated by Western blotting and real-time PCR using proteins and genes with known expression profiles within the retina. Furthermore, the separation of the PS from the ONL enabled the detection of light-driven translocation of transducin from the PS to the soma. ReLayS is a simple and useful method to address protein and possibly metabolites distribution in photoreceptor compartments in various situations including development, ageing, and degenerative diseases

Computing zero deficiency realizations of kinetic systems

In the literature, there exist strong results on the qualitative dynamical properties of chemical reaction networks (also called kinetic systems) governed by the mass action law and having zero deficiency. However, it is known that different network structures with different deficiencies may correspond to the same kinetic differential equations. In this paper, an optimization-based approach is presented for the computation of deficiency zero reaction network structures that are linearly conjugate to a given kinetic dynamics. Through establishing an equivalent condition for zero deficiency, the problem is traced back to the solution of an appropriately constructed mixed integer linear programming problem. Furthermore, it is shown that weakly reversible deficiency zero realizations can be determined in polynomial time using standard linear programming. Two examples are given for the illustration of the proposed methods. © 2015 Elsevier B.V. All rights reserved

Unique proteome signature of post-chemotherapy ovarian cancer ascites-derived tumor cells

Eighty % of ovarian cancer patients diagnosed at an advanced-stage have complete remission afterinitial surgery and chemotherapy. However, most patients die within <5 years due to episodesof recurrences resulting from the growth of residual chemoresistant cells. In an effort to identifymechanisms associated with chemoresistance and recurrence, we compared the expression of proteinsin ascites-derived tumor cells isolated from advanced-stage ovarian cancer patients obtained atdiagnosis (chemonaive, CN) and after chemotherapy treatments (chemoresistant/at recurrence, CR)by using in-depth, high-resolution label-free quantitative proteomic profiling. A total of 2,999 proteinswere identified. Using a stringent selection criterion to define only significantly differentially expressedproteins, we report identification of 353 proteins. There were significant differences in proteinsencoding for immune surveillance, DNA repair mechanisms, cytoskeleton rearrangement, cell-celladhesion, cell cycle pathways, cellular transport, and proteins involved with glycine/proline/argininesynthesis in tumor cells isolated from CR relative to CN patients. Pathway analyses revealed enrichmentof metabolic pathways, DNA repair mechanisms and energy metabolism pathways in CR tumor cells.In conclusion, this is the first proteomics study to comprehensively analyze ascites-derived tumor cellsfrom CN and CR ovarian cancer patients

Deficits in mitochondrial TCA cycle and OXPHOS precede rod photoreceptor degeneration during chronic HIF activation

Background: Major retinal degenerative diseases, including age-related macular degeneration, diabetic retinopathy and retinal detachment, are associated with a local decrease in oxygen availability causing the formation of hypoxic areas affecting the photoreceptor (PR) cells. Here, we addressed the underlying pathological mechanisms of PR degeneration by focusing on energy metabolism during chronic activation of hypoxia-inducible factors (HIFs) in rod PR. Methods: We used two-photon laser scanning microscopy (TPLSM) of genetically encoded biosensors delivered by adeno-associated viruses (AAV) to determine lactate and glucose dynamics in PR and inner retinal cells. Retinal layer-specific proteomics, in situ enzymatic assays and immunofluorescence studies were used to analyse mitochondrial metabolism in rod PRs during chronic HIF activation. Results: PRs exhibited remarkably higher glycolytic flux through the hexokinases than neurons of the inner retina. Chronic HIF activation in rods did not cause overt change in glucose dynamics but an increase in lactate production nonetheless. Furthermore, dysregulation of the oxidative phosphorylation pathway (OXPHOS) and tricarboxylic acid (TCA) cycle in rods with an activated hypoxic response decelerated cellular anabolism causing shortening of rod photoreceptor outer segments (OS) before onset of cell degeneration. Interestingly, rods with deficient OXPHOS but an intact TCA cycle did not exhibit these early signs of anabolic dysregulation and showed a slower course of degeneration. Conclusion: Together, these data indicate an exceeding high glycolytic flux in rods and highlight the importance of mitochondrial metabolism and especially of the TCA cycle for PR survival in conditions of increased HIF activity

Retinal regions shape human and murine Müller cell proteome profile and functionality

The human macula is a highly specialized retinal region with pit‐like morphology and rich in cones. How Müller cells, the principal glial cell type in the retina, are adapted to this environment is still poorly understood. We compared proteomic data from cone‐ and rod‐rich retinae from human and mice and identified different expression profiles of cone‐ and rod‐associated Müller cells that converged on pathways representing extracellular matrix and cell adhesion. In particular, epiplakin (EPPK1), which is thought to play a role in intermediate filament organization, was highly expressed in macular Müller cells. Furthermore, EPPK1 knockout in a human Müller cell‐derived cell line led to a decrease in traction forces as well as to changes in cell size, shape, and filopodia characteristics. We here identified EPPK1 as a central molecular player in the region‐specific architecture of the human retina, which likely enables specific functions under the immense mechanical loads in vivo