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The Emory cataract (Em) mouse mutant has long been proposed as an animal model for age-related or senile cataract in humans—a leading cause of visual impairment. However, the genetic defect(s) underlying the autosomal dominant Em phenotype remains elusive. Here, we confirmed development of the cataract phenotype in commercially available Em/J mice [but not ancestral Carworth Farms White (CFW) mice] at 6–8 months of age and undertook whole-exome sequencing of candidate genes for Em. Analysis of coding and splice-site variants did not identify any disease-causing/associated mutations in over 450 genes known to underlie inherited and age-related forms of cataract and other lens disorders in humans and mice, including genes for lens crystallins, membrane/cytoskeleton proteins, DNA/RNA-binding proteins, and those associated with syndromic/systemic forms of cataract. However, we identified three cataract/lens-associated genes each with one novel homozygous variant including predicted missense substitutions in Prx (p.R167C) and Adamts10 (p.P761L) and a disruptive in-frame deletion variant (predicted missense) in Abhd12 (p.L30_A32delinsS) that were absent in CFW and over 35 other mouse strains. In silico analysis predicted that the missense substitutions in Prx and Adamts10 were borderline neutral/damaging and neutral, respectively, at the protein function level, whereas, that in Abhd12 was functionally damaging. Both the human counterparts of Adamts10 and Abhd12 are clinically associated with syndromic forms of cataract known as Weil-Marchesani syndrome 1 and polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and cataract syndrome, respectively. Overall, while we cannot exclude Prx and Adamts10, our data suggest that Abhd12 is a promising candidate gene for cataract in the Em/J mouse.

Charged multivesicular-body protein 4b (CHMP4B) is a core sub-unit of the endosomal sorting complex required for transport-III (ESCRT-III) machinery that serves myriad remodeling and scission processes of biological membranes. Mutation of the human CHMP4B gene underlies rare forms of early-onset lens opacities, or cataracts, and CHMP4B is required for lens growth and differentiation in mice. Here, we determine the sub-cellular distribution of CHMP4B in the lens and uncover a novel association with gap-junction alpha-3 protein (GJA3) or connexin-46 (Cx46) and GJA8 or Cx50. Immunofluorescence confocal microscopy revealed that CHMP4B localized to cell membranes of elongated fiber cells in the outer cortex of the lens - where large gap-junction plaques begin to form - particularly on the broad faces of these flattened hexagon-like cells in cross-section. Dual immunofluorescence imaging showed that CHMP4B co-localized with gap-junction plaques containing Cx46 and/or Cx50. When combined with the in situ proximity ligation assay, immunofluorescence confocal imaging indicated that CHMP4B lay in close physical proximity to Cx46 and Cx50. In Cx46-knockout (Cx46-KO) lenses, CHMP4B-membrane distribution was similar to that of wild-type, whereas, in Cx50-KO lenses CHMP4B localization to fiber cell membranes was lost. Immunoprecipitation and immunoblotting analyses revealed that CHMP4B formed complexes with Cx46 and Cx50 in vitro. Collectively, our data suggest that CHMP4B forms plasma-membrane complexes, either directly and/or indirectly, with gap-junction proteins Cx46 and Cx50 that are often associated with ‘ball-and-socket’ double-membrane junctions during lens fiber cell differentiation.

The linearity of the plastics economy is wasteful and polluting. To encourage recycling and decrease diversion to landfill, new legislation within the EU and UK will tax single-use plastic products made with less than 30% recycled plastic. At present, quantitative determination of recycled content is elusive and existing methods are inconsistent. We present a fluorescence-based analytical technique to determine recycled content in plastic and (single use) packaging. Bathochromic shifts resulting from aggregation of the fluorescent brightener 4,4'-bis(2-benzoxazolyl) stilbene (BBS) in three commodity plastics [high-density polyethylene, polypropylene, and poly(ethylene terephthalate)] at loadings ≤0.5 wt % were used to systematically quantify simulated recycled contents as low as 10 wt %. Linear correlations were found between recycled content and three fluorescence-based properties: emission, lifetime, and resulting color. We demonstrate how this multi-branched verification system is completely independent of sample dimensions and processing conditions, has a negligible effect on polymer properties, and is inexpensive and highly compatible with existing recycling infrastructure.

The synthesis of particles with discrete phases using traditional emulsion polymerisation is a well-established process. Phase-separated particles have a wide range of applications, such as in coatings, drug delivery, impact modification and as supports in catalysis. However, as a dry powder is often desired for the end application, post-polymerisation, energy intensive drying steps are usually required for the removal of water. Alternatively, dispersion polymerisation utilising supercritical carbon dioxide (scCO2) as a reaction medium allows for the production of dry, free-flowing powders upon release of the CO2. Here, we present the innovative use of scCO2 to provide a novel and environmentally acceptable route for creating phase-separated particles. Particles containing a high Tg poly(methyl methacrylate) (PMMA) phase, combined with a low Tg polymer phase of either poly(benzyl acrylate) (PBzA) or poly(butyl acrylate) (PBA), were investigated. Both monomers were added to the reaction after the formation of PMMA seed particles. Benzyl acrylate (BzA) was chosen as a model low Tg monomer, with well-defined and detectable functionality when mixed with PMMA. Butyl acrylate (BA) was also used as an alternative, more industrially relevant monomer. The loading of the low Tg monomer was varied and full characterisation of the particles produced was performed to elucidate their internal morphologies and compositions.

Charged multivesicular body protein 4B (CHMP4B) functions as a core component of the endosome sorting complex required for transport-III (ESCRT-III) machinery that facilitates diverse membrane remodeling and scission processes in eukaryotes. Mutations in the human CHMP4B gene underlie rare, inherited forms of early-onset lens opacities or cataract. Here we have characterized the lens phenotypes of mutant (knock-in) mice harboring a human cataract-associated mutation (p.D129V) in CHMP4B (Chmp4b-mutant) and conditional knockdown mice deficient in lens CHMP4B (Chmp4b-CKD). In situ hybridization localized Chmp4b transcripts to lens epithelial cells and elongating fiber cells at the lens equator. Heterozygous Chmp4b-mutant (D/V) mice were viable and fertile with lenses grossly similar to those of wild-type. However, homozygous Chmp4b-mutant (V/V) mice died by embryonic day 15.5 (E15.5) with grossly abnormal eye and brain histology. Chmp4b-CKD mice displayed variable degrees of lens dysmorphology including lens ablation. Immuno-localization of aquaporin-0 (AQP0) revealed lens fiber cell degeneration in homozygous Chmp4b-mutant (V/V) mouse embryos and in embryonic and postnatal Chmp4b-CKD mice. DNA fragmentation (TUNEL) analysis revealed global cell death in homozygous Chmp4b-mutant (V/V) embryos, whereas, cell death was confined to the lens of Chmp4b-CKD mice. Immuno-localization of the monocyte/macrophage marker macrosialin (CD68) suggested that severe lens degeneration in Chmp4b-CKD mice resulted in an ocular immune cell response. Collectively, these mouse data suggest that (1) heterozygous, germ-line mutations in Chmp4b may not manifest as cataract, (2) homozygous, germ-line mutations in Chmp4b are embryonic lethal, and (3) conditional loss of Chmp4b results in arrest of lens growth and differentiation.

Reversible addition–fragmentation chain transfer (RAFT) mediated dispersion polymerisation in supercritical carbon dioxide (scCO2) is an efficient and green method for synthesising block copolymer microparticles with internal nanostructures. Here we report for the first time the synthesis of phase separated poly(methyl methacrylate-block-styrene-block-4-vinylpyridine) (PMMA-b-PS-b-P4VP) triblock terpolymer microparticles using a simple two-pot sequential synthesis procedure in scCO2, with high monomer conversions and no purification steps. The microparticles, produced directly and without further processing, show a complex internal nanostructure, appearing as a “lamellar with spheres” [L + S(II)] type morphology. The P4VP block is then exploited as a structure-directing agent for the fabrication of TiO2 microparticles. Through a simple and scalable sol–gel and calcination process we produce hollow TiO2 microparticles with a mesoporous outer shell. When directly compared to porous TiO2 particles fabricated using an equivalent PMMA-b-P4VP diblock copolymer, increased surface area and enhanced photocatalytic efficiencies are observed.

© 2020 Wiley-VCH GmbH The size, shape, and underlying chemistries of drug delivery particles are key parameters which govern their ultimate performance in vivo. Responsive particles are desirable for triggered drug delivery, achievable through architecture change and biodegradation to control in vivo fate. Here, polymeric materials are synthesized with linear, hyperbranched, star, and micellar-like architectures based on 2-hydroxypropyl methacrylamide (HPMA), and the effects of 3D architecture and redox-responsive biodegradation on biological transport are investigated. Variations in “stealth” behavior between the materials are quantified in vitro and in vivo, whereby reduction-responsive hyperbranched polymers most successfully avoid accumulation within the liver, and none of the materials target the spleen or lungs. Functionalization of selected architectures with doxorubicin (DOX) demonstrates enhanced efficacy over the free drug in 2D and 3D in vitro models, and enhanced efficacy in vivo in a highly aggressive orthotopic breast cancer model when dosed over schedules accounting for the biodistribution of the carriers. These data show it is possible to direct materials of the same chemistries into different cellular and physiological regions via modulation of their 3D architectures, and thus the work overall provides valuable new insight into how nanoparticle architecture and programmed degradation can be tailored to elicit specific biological responses for drug delivery.

Transient-receptor-potential cation channel, subfamily M, member 3 (TRPM3) serves as a polymodal calcium sensor in diverse mammalian cell-types. Mutation of the human TRPM3 gene (TRPM3) has been linked with inherited forms of early-onset cataract with or without other eye abnormalities. Here we have characterized the ocular phenotypes of germline ‘knock-in’ mice that harbor a human cataract-associated isoleucine-to-methionine mutation (p.I65M) in TRPM3 (Trpm3-mutant) compared with germline ‘knock-out’ mice that functionally lack TRPM3 (Trpm3-null). Despite strong expression of Trpm3 in lens epithelial cells, neither heterozygous (Trpm3(+/−)) nor homozygous (Trpm3(−/−)) Trpm3-null mice developed cataract; however, the latter exhibited a mild impairment of lens growth. By contrast, homozygous Trpm3-M/M mutants developed severe, progressive, anterior pyramid-like cataract with microphthalmia, whereas, heterozygous Trpm3-I/M and hemizygous Trpm3-M/- mutants developed anterior pyramidal cataract with delayed onset and progression - consistent with a semi-dominant lens phenotype. Histochemical staining revealed abnormal accumulation of calcium phosphate-like deposits and collagen fibrils in Trpm3-mutant lenses and immunoblotting detected increased αII-spectrin cleavage products consistent with calpain hyper-activation. Immunofluorescent confocal microscopy of Trpm3-M/M mutant lenses revealed fiber cell membrane degeneration that was accompanied by accumulation of alpha-smooth muscle actin positive (α-SMA+ve) myofibroblast-like cells and macrosialin positive (CD68+ve) macrophage-like cells. Collectively, our mouse model data support an ocular disease association for TRPM3 in humans and suggest that (1) Trpm3 deficiency impaired lens growth but not lens transparency and (2) Trpm3 dysfunction resulted in progressive lens degeneration and calcification coupled with pro-fibrotic (α-SMA+ve) and immune (CD68+ve) cell responses.

Reversible addition-fragmentation chain transfer (RAFT) dispersion polymerisation in supercritical carbon dioxide is an effective process for creating block copolymer microparticles with internal nanostructures. Here we report an alternative synthesis route involving completely independent steps by exploiting the livingness of RAFT-terminated PMMA microparticles and their unique ability to be redispersed in scCO(2). This not only enables a series of block copolymers to be created from a single RAFT dispersion synthesised PMMA homopolymer batch, thus improving reproducibility, but also adds flexibility by allowing the time and concentration requirements for each stage to be decoupled. The internal morphology development and evolution for a series of poly(methyl methacrylate-block-styrene) (PMMA-b-PS) block copolymer microparticles synthesised via this route was monitored via in situ small-angle X-ray scattering (SAXS) using an autoclave with diamond windows. Together with offline kinetics experiments and postmortem transmission electron microscopy imaging, this study provides remarkably detailed insights into block copolymer self-organisation phenomena in scCO(2). Specifically, the time period over which this block copolymer undergoes phase separation before progressing from an arrangement of spheres to lamellae via the hexagonal cylinder phase is elucidated, and the data are used to plot a detailed empirical phase diagram for this block copolymer system in scCO(2).

We report a novel and facile approach to “fix” the internal nanostructure of block copolymer (BCP) microparticles via in situ crosslinking copolymerisation in dispersion in supercritical CO2 (scCO2). By delaying the addition of the crosslinker and a portion of the second monomer, polymerisation induced microphase separation (PIMS) within the microparticles is well preserved, while the growing chains of the precursor poly(methyl methacrylate)-block-poly(4-vinyl pyridine) (PMMA-b-P4VP) or poly(methyl methacrylate)-block-poly(benzyl methacrylate) (PMMA-b-PBzMA) microparticles are crosslinked. The unique structure of the as-synthesised crosslinked microparticles was fully characterised using transmission electron microscopy (TEM), scanning electron microscopy (SEM) and atomic force microscopy (AFM). Moreover, the swelling and solubility behaviour of the crosslinked PMMA-b-P4VP microparticles was investigated. Notably, the porosity generated by swelling in ethanol can be well controlled by the quantity of crosslinker incorporated. Macropores > 100 nm–∼20 nm, sub-10 nm mesopores, and non-porous microparticles were all achieved by varying the crosslinker incorporation from 0, 0.5, 1, to 4 wt%, respectively. In situ AFM nano-mapping of the crosslinked P4VP domains in 80% humidity revealed that microparticles with a high degree of crosslinking (8 wt% divinylbenzene) are highly resistant to swelling in humidity, by contrast to their non-crosslinked counterparts. This versatile approach further expands the available repertoire for fabricating porous BCP microparticles with tunable physico-chemical properties, morphologies and pore sizes, greatly broadening their application potential to more diverse fields.

Devices featuring electrophoretic displays (EPD) have become extremely popular in recent years because of their low power consumption, high readability and thin display designs, but a product with a full colour gamut comparable with liquid crystal displays (LCDs) has not yet been commercialised. In this article, we demonstrate that staggering the addition of methyl methacrylate (MMA) monomer and low quantities of a coloured dye crosslinker is an effective route to producing well-defined and covalently-linked, strongly coloured PMMA microparticles in one-pot, via dispersion polymerisation in supercritical carbon dioxide (scCO2). This novel methodology is synthetically simple, readily scalable and has the added cachet of being cost effective because the functional molecules can be confined on the microparticle surface such that even at low concentrations, the resulting materials are brightly coloured. We then demonstrate the applicability of this approach to another functional comonomer/crosslinker system in 2-dimethylaminoethyl methacrylate (DMAEMA)/ethyleneglycol dimethacrylate (EGDMA), in this case allowing hierarchically structured ‘pomegranate-like’ microparticles with polarisable charge to be produced over a range of DMAEMA loadings as high as 44 wt%. Finally, the performance of these materials in out-of-plane EPD test cells is compared against analogues synthesised in dodecane. These tests revealed that the coloured microparticles fabricated in scCO2 performed as well as or better than their dodecane synthesised counterparts, consistently producing the cleanest white state and achieving effective colour switching over ten cycles.

Metal oxide microparticles with well-defined internal mesostructures are promising materials for a variety of different applications, but practical routes to such materials that allow the constitue...

We find that the topology of phase diagrams for block copolymer/ionic liquid (IL) blends and the scaling of the phase-separated domains are significantly influenced by the cation structure of the IL. We have varied the cation structure of bis(trifluoromethane sulfonyl)imide ILs and investigated the influence of these changes on the thermodynamics of blends with polystyrene-b-poly(methyl methacrylate). Effective χ parameters (χeff) were determined and used to plot phase diagrams, where significant differences were observed, suggesting that the balance of Coulombic and van der Waals interactions was influencing the topology of the phase diagrams. The relationship between IL structure and scaling of domain spacing was also investigated. These important findings shed additional light on the thermodynamic-driven properties of IL-containing block copolymers and have practical implications for how they can be used in nanotemplating or block copolymer lithography applications.

There is currently a strong drive within the cosmetics industry to replace petroleum based solvents and polymers with renewable alternatives. Copolymers of 2-ethylhexyl acrylate (2EHA) and maleic anhydride (MA) are interesting model candidates to be used in make-up and skincare applications as cross-linkable film forming materials, but are currently synthesised in organic solvents and are derived from petroleum. Herein, we have studied and optimised the copolymerisation of these two monomers in supercritical carbon dioxide (scCO2). The kinetics of the reaction and resulting products were compared with analogues synthesised in a 30/70 solvent mixture of ethyl acetate/isododecane (EtOAc/IDD), which mimics current industry practices. Initially, the incompatibility of these two monomers in the absence of a solvent lead to mixed homo- and copolymer products during reactor pressurisation. This was overcome by devising a new synthesis approach, in which MA is first dissolved in scCO2 at high temperature, followed by the addition of 2EHA and a radical initiator in situ via a HPLC pump. Copolymers synthesised using this new methodology were added to simple cosmetic formulations and evaluated for their resistance to olive oil, where they performed comparably with those synthesised in EtOAc/IDD. The renewable content of the reaction was then increased further, by substituting the monomer 2EHA with the terpene-derived alternatives isobornyl acrylate and α-pinene acrylate. This study is an important step towards materials that can achieve outstanding levels of performance in a variety of cosmetic applications, and are also more in phase with green chemistry criteria.

This work presents a robust method to achieve the synthesis of low molecular weight polyesters 26 via ring-opening polymerization (ROP) initiated by 2-hydroxyethyl-methacrylate (HEMA) 27 when using triazabicyclodecene (TBD) as catalyst. The effect that the HEMA:TBD ratio has 28 upon the final reaction rate and final polymer molecular architecture is discussed. The optimum 29 HEMA:TBD ratio and reaction conditions required to minimize competing transesterification 30 reactions were determined, in order to synthesize successfully the target ROP macromonomer 31 species containing only a single 2-methacryloyloxyethyl end-group. Additionally, to confirm 32 the terminal end-group fidelity of the product macromonomers and confirm TBD utility for 33 block copolymer manufacture, a small series of di-block polyesters were synthesized using 34 TBD and shown to exhibit good control over the final polymer structure whilst negating the 35 side transesterification reactions, irrespective of the monomers used.

Metal oxide microparticles with well-defined internal mesostructures are promising materials for a variety of different applications, but practical routes to such materials that allow the constituent structural length scales to be precisely tuned have thus far been difficult to realize. Herein, we describe a novel platform methodology that utilizes self-assembled block copolymer (BCP) microparticles synthesized by dispersion polymerization in supercritical CO

Understanding the phase behavior of block copolymer/ionic liquid mixtures is an important step toward their implementation in commercial devices. Here we report a high throughput and systematic small-angle X-ray scattering study of the lyotropic phase behavior of a series of polystyrene-b-poly(methyl methacrylate) (PS-b-PMMA) block copolymers in the ionic liquid, 1-ethyl-3-methylimidazolium bis(trifluoromethane sulfonyl)imide (EMIM Tf2N). The ionic liquid induces disorder–order transitions for a number of low molecular weight systems, and the onset points of these transitions are used to calculate the dependence of the effective Flory–Huggins interaction parameter (χeff) on the ionic liquid concentration. This enabled construction of an experimental phase diagram, which reveals that after taking volumetric swelling into account, at higher ionic liquid concentrations, the experimental phase boundaries shift significantly when compared to theoretical calculations for block copolymer melts. It is also demons...

Mesoporous titania microspheres (MTMs) have been employed in many applications, including (photo)catalysis as well as energy conversion and storage. Their morphology offers a hierarchical structural design motif that lends itself to being incorporated into established large-scale fabrication processes. Despite the fact that device performance hinges on the precise morphological characteristics of these materials, control over the detailed mesopore structure and the tunability of the pore size remains a challenge. Especially the accessibility of a wide range of mesopore sizes by the same synthesis method is desirable, as this would allow for a comparative study of the relationship between structural features and performance. Here, we report a method that combines sol-gel chemistry with polymer micro- and macrophase separation to synthesize porous titania spheres with diameters in the micrometer range. The as-prepared MTMs exhibit well-defined, accessible porosities with mesopore sizes adjustable by the choice of the polymers. When applied as an anode material in lithium ion batteries (LIBs), the MTMs demonstrate excellent performance. The influence of the pore size and an in situ carbon coating on charge transport and storage is examined, providing important insights for the optimization of structured titania anodes in LIBs. Our synthesis strategy presents a facile one-pot approach that can be applied to different structure-directing agents and inorganic materials, thus further extending its scope of application.

Major intrinsic protein or aquaporin-0 (MIP/AQP0) functions as a water channel and a cell-junction molecule in the vertebrate eye lens. Loss of MIP function in the lens leads to degraded optical quality and cataract formation by pathogenic mechanisms that are unclear. Here we have used microarray-hybridization analysis to detect lens transcriptome changes during cataract formation in mice that are functionally null for MIP (Mip−/−). In newborn Mip−/− lenses (P1) 11 genes were up-regulated and 18 were down-regulated (>2-fold, p=6-fold) in the Mip−/− lens at P1 included those coding for a mitochondrial translocase (Timmdc1), a matrix metallopeptidase (Mmp2), a Rho GTPase-interacting protein (Ubxn11) and a transcription factor (Twist2). Apart from Mip, the most down-regulated genes (>4-fold) in the Mip−/− lens at P1 included those coding for a proteasome sub-unit (Psmd8), a ribonuclease (Pop4), and a heat-shock protein (Hspb1). Lens fiber cell degeneration in the Mip−/− lens was associated with increased numbers of TUNEL-positive cell nuclei and dramatically elevated levels of calpain-mediated proteolysis of αII-spectrin. However red-ox status, measured by glutathione and free-radical levels, was similar to that of wild-type. These data suggest that while relatively few genes (~1.5% of the transcriptome) were differentially regulated >2-fold in the Mip−/− lens, calpain hyper-activation acts as a terminal pathogenic event during lens fiber cell death and cataract formation.

Breast cancer-related lymphoedema of the arm (BCRL) results from impaired lymph drainage after axillary surgery. Little is known about lymphatic changes in the arm between surgery and oedema onset. We measured forearm muscle and subcutis lymph drainage in 36 women at 7 and 30 months after surgery by quantitative lymphoscintigraphy. None had BCRL initially but 19% had BCRL by 30 months. At 7 months muscle and subcutis drainage in both arms of BCRL-destined women exceeded that of non-BCRL women (P < 0.01). Muscle lymph drainage always exceeded subcutis drainage (P < 0.0001). Muscle lymph drainage in the ipsilateral arm was unimpaired relative to the contralateral arm. BCRL therefore developed in women with higher peripheral lymph flows. The major lymphatic load was generated by muscle; there was no pre-BCRL lymphatic impairment in the muscle of the ipsilateral arm. We propose that some women have a defined, constitutive predisposition to secondary lymphoedema. Specifically, women with higher filtration rates, and therefore higher lymph flows through the axilla that are closer to the maximum sustainable, are at greater risk of BCRL following axillary trauma, even following removal of 1-2 nodes.

Primary open-angle glaucoma (POAG) is a clinically important and genetically heterogeneous cause of progressive vision loss as a result of retinal ganglion cell death. Here we have utilized trio-based, whole-exome sequencing to identify the genetic defect underlying an autosomal dominant form of adult-onset POAG segregating in an African-American family. Exome sequencing identified a novel missense variant (c.418C>T, p.Arg140Trp) in exon-5 of the gene coding for epidermal growth factor (EGF) containing fibulin-like extracellular matrix protein 1 (EFEMP1) that co-segregated with disease in the family. Linkage and haplotype analyses with microsatellite markers indicated that the disease interval overlapped a known POAG locus (GLC1H) on chromosome 2p. The p.Arg140Trp substitution was predicted in silico to have damaging effects on protein function and transient expression studies in cultured cells revealed that the Trp140-mutant protein exhibited increased intracellular accumulation compared with wild-type EFEMP1. In situ hybridization of the mouse eye with oligonucleotide probes detected the highest levels of EFEMP1 transcripts in the ciliary body, cornea, inner nuclear layer of the retina, and the optic nerve head. The recent finding that a common variant near EFEMP1 was associated with optic nerve-head morphology supports the possibility that the EFEMP1 variant identified in this POAG family may be pathogenic.

Background Inherited cataract is a clinically important and genetically heterogeneous cause of visual impairment. Typically, it presents at an early age with or without other ocular/systemic signs and lacks clear phenotype-genotype correlation rendering both clinical classification and molecular diagnosis challenging. Here we have utilized trio-based whole exome sequencing to discover mutations in candidate genes underlying autosomal dominant cataract segregating in three nuclear families. Results In family A, we identified a recurrent heterozygous mutation in exon-2 of the gene encoding γD-crystallin (CRYGD; c.70C > A, p.Pro24Thr) that co-segregated with ‘coralliform’ lens opacities. Families B and C were found to harbor different novel variants in exon-2 of the gene coding for gap-junction protein α8 (GJA8; c.20T > C, p.Leu7Pro and c.293A > C, p.His98Pro). Each novel variant co-segregated with disease and was predicted in silico to have damaging effects on protein function. Conclusions Exome sequencing facilitates concurrent mutation-profiling of the burgeoning list of candidate genes for inherited cataract, and the results can provide enhanced clinical diagnosis and genetic counseling for affected families. Electronic supplementary material The online version of this article (doi:10.1186/s40246-014-0019-6) contains supplementary material, which is available to authorized users.

Inherited forms of cataract are a clinically important and genetically heterogeneous cause of visual impairment that usually present at an early age with or without systemic and/or other ocular abnormalities. Here we have identified a new locus for inherited cataract and high-tension glaucoma with variable anterior segment defects, and characterized an underlying mutation in the gene coding for transient receptor potential cation channel, subfamily M, member-3 (TRPM3, melastatin-2). Genome-wide linkage analysis mapped the ocular disease locus to the pericentric region of human chromosome 9. Whole exome and custom-target next-generation sequencing detected a heterozygous A-to-G transition in exon-3 of TRPM3 that co-segregated with disease. As a consequence of alternative splicing this missense mutation was predicted to result in the substitution of isoleucine-to-methionine at codon 65 (c.195A>G; p.I65 M) of TRPM3 transcript variant 9, and at codon 8 (c.24A>G; p.I8 M) of a novel TRPM3 transcript variant expressed in human lens. In both transcript variants the I-to-M substitution was predicted in silico to exert damaging effects on protein function. Furthermore, transient expression studies of a recombinant TRPM3-GFP reporter product predicted that the I-to-M substitution introduced an alternative translation start-site located 89 codons upstream from the native initiator methionine found in eight other TRPM3 transcript variants (1-8). Collectively, these studies have provided the first evidence that TRPM3 is associated with inherited ocular disease in humans, and further provide support for the important role of this cation channel in normal eye development.

Directed self-assembly (DSA) is a promising technique for extending conventional lithographic techniques by being able to print features with critical dimensions under 10 nm. The most widely studied block copolymer system is polystyreneblock- polymethyl methacrylate (PS-b-PMMA). The system is well understood in terms of its synthesis, properties and performance in DSA. However, PS-b-PMMA also has a number of limitations that impact on its performance and hence scope of application. The primary limitation is the low Flory-Huggins polymer-polymer interaction parameter (Χ), which limits the size of features that can be printed by DSA. Another issue with block copolymers in general is that specific molecular weights need to be synthesized to achieve desired morphologies and feature sizes. We are exploring blending ionic liquid additiveswithPS-b-PMMAto increase the Χ parameter. This allows smaller feature sizes to be accessed by PS-b-PMMA. Depending on the amount of additive it is also possible to tune the domain size and the morphology of the systems. These findings may expand the scope of PS-b-PMMA for DSA.

Lens opacities, or cataract(s), may be inherited as a classic Mendelian disorder usually with early-onset or, more commonly, acquired with age as a multi-factorial or complex trait. Many genetic forms of cataract have been described in mice and other animal models. Considerable progress has been made in mapping and identifying the genes and mutations responsible for inherited forms of cataract, and genetic determinants of age-related cataract are beginning to be discovered. To provide a convenient and accurate summary of current information focused on the increasing genetic complexity of Mendelian and age-related cataract we have created an online chromosome map and reference database for cataract in humans and mice (Cat-Map).

Infantile nystagmus is a clinically and genetically heterogeneous eye movement disorder. Here we map and identify the genetic mutation underlying X-linked idiopathic infantile nystagmus (XL-IIN) segregating in two Caucasian-American families.Eye movements were recorded using binocular infrared digital video-oculography. Genomic DNA was prepared from blood or buccal-cells, and linkage analysis was performed using short tandem repeat (STR) and single nucleotide polymorphism (SNP) markers. Pedigree and haplotype data were managed using Cyrillic, and LOD scores calculated using MLINK. Mutation profiling of PCR-amplified exons was performed by dye-terminator cycle-sequencing and analyzed by automated capillary electrophoresis.Video-oculography of affected males recorded conjugate, horizontal, pendular nystagmus with increasing-velocity waveforms in primary gaze converting to jerk nystagmus in eccentric gaze. Linkage analysis detected significantly positive two-point LOD scores (Z) at markers DXS8078 (Z=4.82, recombination fraction [theta]=0) and DXS1047 (Z=3.87, theta=0). Haplotyping indicated that the IIN locus mapped to the physical interval DXS8057-(11.59 Mb)-rs6528335 on Xq25-q26. Sequencing of positional-candidate genes detected a c.425TG transversion in exon-6 of the gene for FERM domain containing-7 (FRMD7) that cosegregated with affected and carrier status. In addition, the same change was found to cosegregate with IIN in a genetically unrelated family but was not detected in 192 control individuals.The c.425TG change is predicted to result in the missense substitution of the phylogenetically conserved leucine at codon 142 for an arginine (p.L142R), and supports a causative role for FRMD7 mutations in the pathogenesis of XL-IIN.

This study examines the association between nodal positivity and risk of developing breast cancer-related lymphedema (BCRL) in patients who underwent axillary lymph node dissection (ALND).The pathophysiology of BCRL is poorly understood. It has been assumed that one of the factors predisposing to the development of BCRL is nodal positivity, although retrospective series have produced contradictory findings. As these studies have included treatment regimens known to cause BCRL, such as axillary radiotherapy, any relationship between nodal positivity and the development of BCRL remains speculative.A total of 212 patients who had undergone ALND for invasive breast cancer had arm volume measurements preoperatively, and at intervals postoperatively. No patient received axillary radiotherapy. Arm volumes were obtained by measuring serial arm circumferences every 4 cm up the arm and then calculated by using the formula for the volume of a truncated cone. Robust regression techniques were used to analyze the effects of node positivity, age, preoperative body mass index, and wound infection on arm volume excess.In all, 64 of 212 (30%) patients were node positive. Contrary to previous assumptions, positive node status was significantly inversely associated with arm volume after adjusting for tumor size, time since operation, and allowing for correlated observations within subjects. Furthermore, the number of positive nodes also correlated inversely with arm volume.These results are counterintuitive to the conventional understanding of the pathophysiology of BCRL. A possible explanation is that patients who develop disease in axillary lymph nodes and subsequently undergo ALND have more time and ability to develop lymphatic collaterals, which may provide adequate lymphatic drainage following surgery, thereby reducing the risk of developing BCRL.

Cataracts are a clinically diverse and genetically heterogeneous disorder of the crystalline lens and a leading cause of visual impairment. Here we report linkage of autosomal dominant "progressive childhood posterior subcapsular" cataracts segregating in a white family to short tandem repeat (STR) markers D20S847 (LOD score [Z] 5.50 at recombination fraction [theta] 0.0) and D20S195 (Z=3.65 at theta =0.0) on 20q, and identify a refined disease interval (rs2057262-(3.8 Mb)-rs1291139) by use of single-nucleotide polymorphism (SNP) markers. Mutation profiling of positional-candidate genes detected a heterozygous transversion (c.386A--T) in exon 3 of the gene for chromatin modifying protein-4B (CHMP4B) that was predicted to result in the nonconservative substitution of a valine residue for a phylogenetically conserved aspartic acid residue at codon 129 (p.D129V). In addition, we have detected a heterozygous transition (c.481G--A) in exon 3 of CHMP4B cosegregating with autosomal dominant posterior polar cataracts in a Japanese family that was predicted to result in the missense substitution of lysine for a conserved glutamic acid residue at codon 161 (p.E161K). Transfection studies of cultured cells revealed that a truncated form of recombinant D129V-CHMP4B had a different subcellular distribution than wild type and an increased capacity to inhibit release of virus-like particles from the cell surface, consistent with deleterious gain-of-function effects. These data provide the first evidence that CHMP4B, which encodes a key component of the endosome sorting complex required for the transport-III (ESCRT-III) system of mammalian cells, plays a vital role in the maintenance of lens transparency.

Autosomal dominant cataracts are a clinically and genetically heterogeneous eye-lens disorder that usually present in childhood with symptoms of impaired vision. The purpose of this study was to map and identify the mutation underlying autosomal dominant nuclear punctate cataracts segregating in a six generation Caucasian pedigree.Genomic DNA was prepared from blood leucocytes, genotyping was performed using microsatellite markers, and LOD scores were calculated using the LINKAGE programs. Mutation detection was performed using direct sequencing and restriction fragment length analysis.Significant evidence of linkage was obtained at marker D13S175 (LOD score [Z]=4.11, recombination fraction [theta]=0.0) and haplotyping indicated that the disease gene lay in the about 2 Mb physical interval between D13S1316 and D13S1236, which contained the gene for gap-junction protein a3 (GJA3) or connexin46. Sequencing of GJA3 detected a C-T transition in exon 2 that resulted in the gain of an Alu 1 restriction site and was predicted to cause a conservative substitution of proline to leucine at codon 59 (P59L). Restriction analysis confirmed that the novel Alu 1 site co-segregated with cataracts in the family but was not detected in a control panel of 170 normal unrelated individuals.The present study has identified a fifth mutation in GJA3, rendering this connexin gene one of the most common non-crystallin genes associated with autosomal dominant cataracts in humans.

Directed self-assembly (DSA) is a promising approach for extending conventional lithographic techniques by being able to print features with critical dimensions under 10 nm. The most widely studied block copolymer system is polystyrene-block-poly(methyl methacrylate) (PS-b-PMMA). This system is well understood in terms of its synthesis, properties, and performance in DSA. However, PS-b-PMMA also has a number of limitations that impact on its performance and hence scope of application. The primary limitation is the low Flory-Huggins polymer-polymer interaction parameter (χ), which limits the size of features that can be printed. Another issue with block copolymers in general is that specific molecular weights need to be synthesized to achieve desired morphologies and feature sizes. Here we explore blending ionic liquid (IL) additives with PS-b-PMMA to increase the χ parameter. ILs have a number of useful properties that include negligible vapor pressure, tunable solvent strength, thermal stability, and chemical stability. The blends of PS-b-PMMA with an IL selective for the PMMA block allowed the resolution of the block copolymer to be improved. Depending on the amount of additive, it is also possible to tune the domain size and the morphology of the systems. These findings may expand the scope of PS-b-PMMA for DSA. © 2014 Society of Photo-Optical Instrumentation Engineers

The Eph receptors constitute the largest subfamily of receptor tyrosine kinases.1 The extracellular domains of Eph receptors interact with membrane-bound ligands, ephrins, located on the surfaces of adjacent cells. Unlike other receptor tyrosine kinases, Eph receptors function primarily in the regulation of cell shape, cell-substrate adhesion, and migration, though in certain contexts Eph receptors can also influence proliferation.2 Eph receptors take their name from the erythropoietin-producing hepatocellular carcinoma cell line from which the first member of the family, EPHA1, was cloned.3 Eph receptors are divided into A and B subclasses.4 In mammals, nine Epha (Epha1–8 and Epha10) and five Ephb (Ephb1–4 and Ephb6) receptors are known. Ephrin ligands are also classified into two groups: ephrin-a ligands (Efna1–5 in mammals) are GPI-linked proteins, whereas ephrin-b ligands (Efnb1–3) are transmembrane proteins. Typically, EphA receptors bind to ephrin-A ligands and EphB receptors bind to ephrin-B ligands, although limited cross-talk between class members has been reported.5 Eph-ephrin binding is unusual in that it can stimulate bidirectional signaling: forward signaling into the receptor-expressing cell and reverse signaling into the ligand-expressing cell.2 As a group, Eph receptors are expressed most prominently during development, when they play critical roles in such processes as limb formation and nervous system patterning. Most Eph receptors are expressed at low levels in adult tissues. A notable exception to this is Epha2 (originally called Eck, for epithelial cell kinase), which was cloned from HeLa cells6 and was shown to be enriched in adult tissues with high epithelial content such as lung, digestive tract, and skin.7 The biology of Epha2 has been studied intensively because the receptor is overexpressed in many human cancers.8–10 Recent studies have identified Epha2 as a surprisingly abundant plasma membrane component in cells of the ocular lens.11 The precise role of Epha2 in the lens is unknown, but mutations in EPHA2 have been shown to underlie inherited forms of cataract in humans.12–14 Moreover, common variants in EPHA2 have been associated with the much more prevalent age-related form of cataract.12,15,16 To gain insights into the role of Epha2 in the lens, we analyzed the lens phenotype of Epha2-null mice. Our data suggest that Epha2 is not required for lens transparency but does play a significant role in cell guidance and refractive organization of the lens.

Major intrinsic protein (MIP) is a functional water-channel (AQP0) that also plays a key role in establishing lens fiber cell architecture. Genetic variants of MIP have been associated with inherited and age-related forms of cataract; however, the underlying pathogenic mechanisms are unclear. Here we have used lens transcriptome profiling by microarray-hybridization and qPCR to identify pathogenic changes during cataract development in Mip-mutant (Lop/+) mice. In postnatal Lop/+ lenses (P7) 99 genes were up-regulated and 75 were down-regulated (>2-fold, p=4-fold) in the Lop/+ lens included Chac1 > Ddit3 > Atf3 > Trib3 > Xbp1 and the most down-regulated genes (>5-fold) included two anti-oxidant genes, Hspb1 and Hmox1. Lop/+ lenses were further characterized by abundant TUNEL-positive nuclei within central degenerating fiber cells, glutathione depletion, free-radical overproduction, and calpain hyper-activation. These data suggest that Lop/+ lenses undergo proteotoxic ER-stress induced cell-death resulting from prolonged activation of the Eif2ak3/Perk-Atf4-Ddit3-Chac1 branch of the UPR coupled with severe oxidative-stress.

We report a facile and versatile strategy for the bespoke fabrication of macro- and mesoporous block copolymer microparticles. A clean synthetic route, RAFT dispersion polymerisation in supercritical carbon dioxide (scCO2), is used to generate block copolymer microparticles. Selective swelling/deswelling is then applied to induce controlled morphology transitions and to trap the resulting porous state. The pore sizes are controllable over a large size range (~20 – 200 nm) by varying the length of the swellable block. Through a systematic approach we then demonstrate that the shape of the pores can also be tailored from isolated spheres through to interconnected/bicontinuous channels by varying the ratio of the two blocks. This process is shown to be applicable to a range of poly(methyl methacrylate) (PMMA)based block copolymer systems, including PMMA-b-poly(4-vinyl pyridine), PMMAb-poly(dimethyl acrylamide) (DMA) and PMMA-bpoly(dimethylaminoethylmethacrylate) (DMAEMA). In each case, the second minority block (e.g. P4VP, etc.) was selectively swollen with an alcohol to induce an order-toorder morphology transition and then quenched rapidly by the non-solvent hexane. This not only takes place on the order of hours, but is also freely scalable for the production of grams of material and beyond in a single step following polymerisation.