Unlocking Access to Broad Molecular Profiling: Benefits, Barriers, and Policy Solutions.

OBJECTIVES: "Personalized healthcare" is generating new approaches to disease management by considering inter-individual variability in genes, environment, and lifestyle. Technologies such as comprehensive genomic profiling (CGP) are drivers of this shift. Here, we address the significant hurdles to the equitable implementation of CGP into routine clinical practice. METHODS: This article draws on published evidence on the value of genomic profiling, as well as interviews with nine academic and clinical experts from six different countries to validate findings and test policy proposals for reforms. RESULTS: The potential benefits of CGP extend beyond direct patient outcomes, to healthcare systems with societal and economic impacts. Among key barriers impeding integration into routine clinical practice are the lack of infrastructure to ensure reliable clinical testing and the limited understanding of genomics among healthcare personnel. In addition, the absence of health economic evidence supporting broader use of CGP is creating concerns for payers regarding the systemic benefits and affordability of this technology. CONCLUSION: Policy proposals that aim to improve equitable patient access to CGP will need to consider new funding models, health technology assessment processes that capture both patient and systemic benefits, and appropriate regulatory standards to determine the quality of genomic profiling tests.

Transcriptional regulation of the purine de novo synthesis gene Prat in Drosophila melanogaster.

The first step of the purine de novo synthesis pathway is catalyzed by amidophosphoribosyltransferase (E.C.2.4.2.14) which is encoded by two Prat genes in D. melanogaster, Prat and Prat2. Prat is a retrogene duplication of Prat2, where each gene has a distinct expression pattern. Prat transcription is restricted to proliferating tissues such as imaginal discs and the female germ line. Three conserved putative DNA replication-related element binding factor (DREF) sites lie upstream of the Prat coding region. These elements are upstream of many genes important in cell proliferation. We have found that DREF binds directly upstream of Prat and that the DRE sites associated with its activity are necessary for Prat expression; furthermore, we have determined that a second cis-acting element is present upstream of the Prat gene. Finally, the genes Distal-less, Mi-2 and dMyc, which influence Dref activity, do not appear to affect Prat transcription.

Biosynthetic corneal implants for replacement of pathologic corneal tissue: performance in a controlled rabbit alkali burn model.

PURPOSE: To evaluate the performance of structurally reinforced, stabilized recombinant human collagen-phosphorylcholine (RHCIII-MPC) hydrogels as corneal substitutes in a rabbit model of severe corneal damage. METHODS: One eye each of 12 rabbits received a deep corneal alkali wound. Four corneas were implanted with RHCIII-MPC hydrogels. The other eight control corneas were implanted with either allografts or a simple cross-linked RHCIII hydrogel. In all cases, 6.25 mm diameter, 350 µm thick buttons were implanted by anterior lamellar keratoplasty to replace damaged corneal tissue. Implants were followed for nine months by clinical examination and in vivo confocal microscopy, after which implanted corneas were removed and processed for histopathological and ultrastructural examination. RESULTS: Alkali exposure induced extensive central corneal scarring, ocular surface irregularity, and neovascularization in one case. All implants showed complete epithelial coverage by four weeks postoperative, but with accompanying suture-induced vascularization in 6 out of 12 cases. A stable, stratified epithelium with hemidesmosomal adhesion complexes regenerated over all implants, and subbasal nerve regeneration was observed in allograft and RHCIII-MPC implants. Initially acellular biosynthetic implants were populated with host-derived keratocytes as stromal haze subsided and stromal collagen was remodeled. Notably, RHCIII-MPC implants exhibited resistance to vascular ingrowth while supporting endogenous cell and nerve repopulation. CONCLUSIONS: Biosynthetic implants based on RHC promoted cell and nerve repopulation in alkali burned rabbit eyes. In RHCIII-MPC implants, evidence of an enhanced resistance to neovascularization was additionally noted.

Femtosecond-UVA-riboflavin (FUR) cross-linking approach to penetrating keratoplasty and anterior lamellar keratoplasty.

PURPOSE: To introduce femtosecond laser wound design combined with riboflavin/ultraviolet light-A (UVA) collagen cross-linking at the wound for penetrating (PKP) and anterior lamellar keratoplasty (ALK). Primary outcomes were intraocular pressure (IOP in mmHg) at burst point for the PKP group, and tensile strength (kPa) until dehiscence for the ALK group. METHODS: Human corneoscleral rims (N = 20) were mounted on artificial anterior chambers. PKP specimens underwent FUR, femtosecond laser-cut without cross-linking, or conventional corneal transplantation. PKP maximum burst IOP with progressive suture removal was assessed by a digital manometer, in triplicate and by three observers. ALK involved whole human globes (N = 10) divided into three groups using a 200-micron, 8 mm diameter donor lenticule, with or without cross-linking. Cross-linked specimens were exposed to UVA light (3 mW/cm(2) irradiance, 3.4 J, 370 nm wavelength) for 30 min with 0.1% riboflavin (20% Dextran) applied every 2-min. ALK tensile strength was determined using a digital tensiometer. RESULTS: In PKP, burst IOP was 31.32 mmHg greater for corneas that underwent the UVA-riboflavin treatment than for those that did not (p < 0.05). There was no significant relationship (p = 0.719) established between cut design (femtosecond versus conventional). On multivariate analysis, there was a mean of 15.82 mmHg higher sustainable pressure for each stabilization suture present (p < 0.0001). In ALK, specimens comprised of human donor and human recipient tissue combined with UVA-riboflavin therapy experienced the greatest level of adhesion strength (954.7 ± 290.4 kPa) as shown by the force required to separate the tissues, and compared to non-cross-linked specimens. Electron microscopy of ALK specimens showed non-fused and fused longitudinal cross-linked collagen fibers as well as bridges, densities, attachment plaques and primitive plasmalemmal densities. CONCLUSIONS: Cross-linking effects of the FUR technique enable a stronger graft-recipient adhesion compared to conventional penetrating and anterior lamellar keratoplasty. Electron microscopy enabled visualization of cross-linked interface and potential bonding. The FUR approach may further lead to sutureless transplantation techniques in the future. SETTING/VENUE: ImagePlus Laser Eye Centre, Winnipeg, and University of Ottawa Eye Institute, Ottawa, Canada.

Collagen and glycopolymer based hydrogel for potential corneal application.

6-Methacryloyl-alpha-D-galactopyranose (MG) was synthesized, and characterized by Fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR) spectrometry, and single-crystal X-ray diffraction. A series of interpenetrating polymer network (IPN) hydrogels was fabricated by simultaneously photocuring MG crosslinked by poly(ethylene glycol) diacrylate and chemically crosslinking type I collagen with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide. The successful incorporation of the glycopolymer, polymer MG, into collagen hydrogel was confirmed by FTIR and solid-state (13)C NMR. The optical characteristics of the IPN hydrogels are comparable to those of human corneas. The tensile strength and modulus of the hydrogels are enhanced by incorporation of polymer MG in comparison to that of the control collagen hydrogel. Biodegradation results indicated that polymer MG enhanced the stability of the composite hydrogels against collagenase. In vitro results demonstrated that the IPN hydrogel supported the adhesion and proliferation of human corneal epithelial cells and outperformed human cornea in blocking bacteria adhesion. Taken together, the IPN hydrogel might be a promising material for use in corneal lamellar keratoplasty.

Modifiers of Prat, a de novo purine synthesis gene, in Drosophila melanogaster.

Drosophila melanogaster was used to identify genes with a potential role in genetic regulation of purine biosynthesis. In this study we examine two dominant genetic modifiers of the essential gene Prat, which encodes amidophosphoribosyltransferase (EC 2.4.2.14). We found that Mod(Prat:bw)3-1 enhances Prat expression only in female heads, whereas Mod(Prat:bw)3-5 suppresses Prat in all stages and tissues examined for both sexes. For Mod-3-5, gene expression microarrays were used to identify other genes that are affected by the modifier. Three mapping approaches were used to localize these modifiers. Deficiency and meiotic mapping showed that the complex lethal complementation group previously associated with Mod-3-1 and Mod-3-5 is actually due to shared second-site lethal mutations. Using male recombination mapping, Mod-3-1 was localized to a 21 kilobase region containing nine genes, and Mod-3-5 was localized to a 53 kilobase region containing eight genes.