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The vitreous humor of the eye is mainly composed of fibrillary collagen and semiflexible hyaluronic acid (HA). To mimic this macromolecular composition of the vitreous, we previously developed an injectable two-component hydrogel composed of a fibrillary gellan and a semiflexible polyelectrolyte, poly[methacrylamide

Presbyopia, the inability to focus at arm's length, and cataracts that cloud vision are associated primarily with changes in the mechanical and optical properties of the lens. The optical properties, particularly the refractive index, of the human lens originate from the cytoplasm of the lens fiber, which contains a highly concentrated solution (∼40%) of globular proteins referred to as α, β, and γ crystallins, of which β is the most abundant. In this study, we focus on the synthesis and characterization of a β-crystallin biomimetic in an effort to understand and develop treatments for presbyopia and cataract. Polyacrylamide was used as a protein analogue. The side chains were endowed with aromatic and acidic functionality. Acrylic acid was incorporated into the copolymer and cross-linked with diamines to form nanoparticles. The composition and cross-linking condition of the biomimetic copolymers were optimized to match the hydrodynamic radius (Rh), refractive index, size, density, and intrinsic and dynamic viscosities with those of βhigh lens crystallins. The refractive indices and densities of the nanoparticles' dispersion at different concentrations matched that of βhigh lens crystallins, and the viscosity of the nanoparticles approached that of βhigh lens crystallins. The biocompatibility findings for primary porcine retinal pigment epithelial (ppRPE) cells and porcine lens epithelial (pLE) cells showed both cell types tolerated up to 30 mg/mL of nanoparticles. These materials have the potential for use as replacements for the crystallins in developing an accommodating intraocular lens nanocomposite hydrogel that closely replicates the natural autofocusing ability of the original.

Our objective is to improve on our previous work developing thiol-containing water-soluble copolyacrylamides that form hydrogels in situ for use as vitreous substitutes. In this study, we evaluate the incorporation of acrylic acid by varying the feed ratio of acrylic acid monomer from 0 to 40 mol% in combination with acrylamide, and bis-acryloylcystamine as the reversible cross-linker. After polymerization, the formed copolymer hydrogels were reduced with dithiothreitol to cleave the disulfide cross-linkers. Purified, lyophilized copolymers were made in a concentration range of 12.5–17.5 mg/mL (polymer in deionized water) and were gelled by oxidation. Chemical, physical, optical, and rheological characterizations along with in vitro biocompatibility studies were performed using thiazolyl blue and Electric Cell–substrate Impedance Sensing. Increasing the percentage of acrylic acid caused the polymer to gel at 12.5 mg/mL as opposed to 20 mg/mL without acrylic acid. Storage modulus values covered the range of natural vitreous (1–108 Pa). Biocompatibility testing in tissue culture with retinal pigment epithelial cells (ARPE-19) showed no toxicity at 10 mg/mL or less when compared to controls, higher concentrations. In contrast to our previously reported copolyacrylamide hydrogels, these hydrogels remain optically clear and gel at lower concentrations and have the potential for use as vitreous substitutes.

The vitreous humor of the eye is a biological hydrogel principally composed of collagen fibers interspersed with hyaluronic acid. Certain pathological conditions necessitate its removal and replacement. Current substitutes, like silicone oils and perfluorocarbons, are not biomimetic and have known complications. In this study, we have developed an in situ forming two-component biomimetic hydrogel with tunable mechanical and osmotic properties. The components are gellan, an analogue of collagen, and poly(methacrylamide-co-methacrylate), an analogue of hyaluronic acid; both endowed with thiol side groups. We used response surface methodology to consider seventeen possible hydrogels to determine how each component affects the optical, mechanical, sol-gel transition temperature and swelling properties. The optical and physical properties of the hydrogels were similar to vitreous. The shear storage moduli ranged from 3 to 358 Pa at 1 Hz and sol-gel transition temperatures from 35.5 to 43 °C. The hydrogel had the ability to remain swollen without degradation for four weeks in vitro. Three hydrogels were tested for biocompatibility on primary porcine retinal pigment epithelial cells, human retinal pigment epithelial cells, and fibroblast (3T3/NIH) cells, by electric cell-substrate impedance sensing system. The two-component hydrogels allowed for the tuning and optimizing of mechanical, swelling, and transition temperature to obtain three biocompatible hydrogels with properties similar to the vitreous. Future studies include testing of the optimized hydrogels in animal models for use as a long-term substitute, whose preliminary results are mentioned. Statement of Significance Although hydrogels are researched as long-term vitreous substitute, none have advanced sufficiently to reach clinical application. Our work focuses on the development of a novel two component in situ forming hydrogel that bio-mimic the natural vitreous. Our thiol-containing copolymers can be injected as an aqueous solution into the vitreous cavity wherein, at physiological temperature, the rigid component will instantaneously form a physical gel imbedding the random coil copolymer. Upon subsequent oxidation, the two components will form disulfide cross-links and a stable reversible hydrogel capable of providing osmotic pressure to reattach the retina. It may be left in the eye permanently or easily removed by injection of a simple reducing agent to cleave the disulfide bonds, rather than surgery. This contribution is significant because it is expected to provide patients with a much better quality of life by improving surgical outcomes, creating much less post-operative burden, and reducing the need for secondary surgeries.

Synthetic hydrogel systems are promising long-term candidates for therapeutic use as vitreous substitutes. To improve the swelling and mechanical properties of existing synthetic substitutes for the natural vitreous, we incorporate charges and fibrous structures. Using dynamic light scattering, rheology, and swelling techniques we show in vitro that synthetic copolymers of methacrylic acid (MAA), methylacrylamide (MAm), and bismethacryloylcystamine (BMAC) cross-linked with gellan match the water content, transport properties, and dynamic response of the natural vitreous better than previously proposed synthetic substitutes.

In many vitreal diseases, the surgeon removes the natural vitreous and replaces it with silicone oils, gases, or balanced salt solutions to fill the eyeball and hold the retina in position. However, these materials are often associated with complications and have properties that differ from natural vitreous. Herein, we report an extension of our previous work on the synthesis of a biomimetic hydrogel that is composed of thiolated gellan as an analogue of type II collagen and poly(methacrylamide-co-methacrylate-co-bis(methacryloyl)cystamine), a polyelectrolyte, as an analogue of hyaluronic acid. This thermosensitive hydrogel can be injected into the eye as a viscous solution at 45 °C. It then forms a physical gel in situ when it reaches body temperature, and later forms disulfide covalent crosslinks. In this article, we evaluated two different formulations of the biomimetic hydrogels for their physical, mechanical, and optical properties, and we determined their biocompatibility with several cell lines. Finally, we report on the progress of the four-month preclinical evaluation of our bio-inspired vitreous substitute in comparison to silicone oil or a balanced salt solution. We assessed the eyes with a slit-lamp examination, intraocular pressure measurements, electroretinography, and optical coherence tomography. Preliminary results are very encouraging for the continuing evaluation of our bio-inspired hydrogel in clinical trials.

The natural vitreous is a biological hydrogel consisting primarily of a collagen and anionic hyaluronate. It is surgically removed in many ocular diseases and replaced with fluids, gases, or silicone oils. We have been interested in developing synthetic hydrogels as vitreous substitutes. In this study, we combined the stiffness and hydrophobicity of polymethacrylamide (PMAM) and the anionic nature of polymethacrylate (PMAA) to make copolymers that would mimic the natural vitreous. We used bis-methacryloyl cystamine (BMAC) to introduce thiol groups for reversible crosslink. The Mn of copolymers ranged from ∼100 k to ∼200 k Da (polydisperisty index of 1.47-2.63) and their composition as determined by titration, 1 H NMR and disulfide test were close to the feed ratio. The reactivities of monomers were as follows: MAM > MAA ∼ BMAC. Copolymers with higher MAA contents gelled faster, swelled more, and had higher storage modulus (1.5 to 100 Pa) comparable to that of the natural vitreous. We evaluated the biocompatibility of copolymers by electric cell-substrate impedance sensing (ECIS) using human retinal pigment epithelial cells, primary porcine retinal pigmented epithelial cells, human microvascular endothelial cells adult dermis, and a fibroblast line 3T3. The biocompatibility decreases as the content of BMAC increases. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 977-988, 2017.

The mammalian eye lens expresses a high concentration of crystallins (α, β and γ-crystallins) to maintain the refractive index essential for lens transparency. Crystallins are long-lived proteins that do not turnover throughout life. The structural destabilization of crystallins by UV exposure, glycation, oxidative stress and mutations in crystallin genes leads to protein aggregation and development of cataracts. Several destabilizing mutations in crystallin genes are linked with human autosomal dominant hereditary cataracts. To investigate the mechanism by which the α-crystallin mutations Cryaa-R49C and Cryab-R120G lead to cataract formation, we determined whether these mutations cause an altered expression of specific transcripts in the lens at an early postnatal age by RNA-seq analysis. Using knock-in mouse models previously generated in our laboratory, in the present work, we identified genes that exhibited altered abundance in the mutant lenses, including decreased transcripts for Clic5, an intracellular water channel in Cryaa-R49C heterozygous mutant lenses, and increased transcripts for Eno1b in Cryab-R120G heterozygous mutant lenses. In addition, RNA-seq analysis revealed increased histones H2B, H2A, and H4 gene expression in Cryaa-R49C mutant lenses, suggesting that the αA-crystallin mutation regulates histone expression via a transcriptional mechanism. Additionally, these studies confirmed the increased expression of histones H2B, H2A, and H4 by proteomic analysis of Cryaa-R49C knock-in and Cryaa;Cryab gene knockout lenses reported previously. Taken together, these findings offer additional insight into the early transcriptional changes caused by Cryaa and Cryab mutations associated with autosomal dominant human cataracts, and indicate that the transcript levels of certain genes are affected by the expression of mutant α-crystallin in vivo.

Author(s): Molnar, Kathleen S; Dunyak, Bryan M; Su, Bonnie; Izrayelit, Yevgeniy; McGlasson-Naumann, Brittney; Hamilton, Paul D; Qian, Mingxing; Covey, Douglas F; Gestwicki, Jason E; Makley, Leah N; Andley, Usha P | Abstract: PurposeWe previously identified an oxysterol, VP1-001 (also known as compound 29), that partially restores the transparency of lenses with cataracts. To understand the mechanism of VP1-001, we tested the ability of its enantiomer, ent-VP1-001, to bind and stabilize αB-crystallin (cryAB) in vitro and to produce a similar therapeutic effect in cryAB(R120G) mutant and aged wild-type mice with cataracts. VP1-001 and ent-VP1-001 have identical physicochemical properties. These experiments are designed to critically evaluate whether stereoselective binding to cryAB is required for activity.MethodsWe compared the binding of VP1-001 and ent-VP1-001 to cryAB using in silico docking, differential scanning fluorimetry (DSF), and microscale thermophoresis (MST). Compounds were delivered by six topical administrations to mouse eyes over 2 weeks, and the effects on cataracts and lens refractive measures in vivo were examined. Additionally, lens epithelial and fiber cell morphologies were assessed via transmission electron microscopy.ResultsDocking studies suggested greater binding of VP1-001 into a deep groove in the cryAB dimer compared with ent-VP1-001. Consistent with this prediction, DSF and MST experiments showed that VP1-001 bound cryAB, whereas ent-VP1-001 did not. Accordingly, topical treatment of lenses with ent-VP1-001 had no effect, whereas VP1-001 produced a statistically significant improvement in lens clarity and favorable changes in lens morphology.ConclusionsThe ability of VP1-001 to bind native cryAB dimers is important for its ability to reverse lens opacity in mouse models of cataracts.

Mice with deletion of genes for small heat shock proteins αA- and αB-crystallin (αA/αB(-/-)) develop cataracts. We used proteomic analysis to identify lens proteins that change in abundance after deletion of these α-crystallin genes. Wild-type (WT) and αA/αB(-/-) knockout (DKO) mice were compared using two-dimensional difference gel electrophoresis and mass spectrometric analysis, and protein identifications were validated by Mascot proteomic software. The abundance of histones H2A, H4, and H2B fragment, and a low molecular weight β1-catenin increased 2-3-fold in postnatal day 2 lenses of DKO lenses compared with WT lenses. Additional major increases were observed in abundance of βB2-crystallin and vimentin in 30-day-old lenses of DKO animals compared with WT animals. Lenses of DKO mice were comprised of nine protein spots containing βB2-crystallin at 10-40-fold higher abundance and three protein spots containing vimentin at ≥2-fold higher abundance than in WT lenses. Gel permeation chromatography identified a unique 328 kDa protein in DKO lenses, containing β-crystallin, demonstrating aggregation of β-crystallin in the absence of α-crystallins. Together, these changes provide biochemical evidence for possible functions of specific cell adhesion proteins, cytoskeletal proteins, and crystallins in lens opacities caused by the absence of the major chaperones, αA- and αB-crystallins.

Background: Sciatic nerve blocks are used to reduce postoperative pain and allow early discharge for patients undergoing foot and ankle surgery. This study aimed to identify the utilization of this procedure in the US and UK and to establish the standard of care with respect to the level of anesthesia that the patient is under and use of ultrasound localization when performing sciatic nerve blocks. Materials and Methods: A survey of current committee members of AOFAS and members of BOFAS. Results: Two hundred sixty-three surgeons were contacted with a response rate of 44%. Eighty-two percent commonly used a sciatic nerve blockade. Sixty-nine percent never or only sometimes used ultrasonography and variable levels of nerve stimulation were used. Forty-two percent where happy to have the block performed under full anesthesia. There were significant differences between British and American practices regarding the level of nerve stimulation and the level of anesthesia used. The most common complication cited was prolonged anesthesia of which the vast majority spontaneously resolved. Performing blocks awake or sedated did not seem to alter number of complications seen. Conclusion: This study represents a current practice review of sciatic nerve blocks performed amongst senior foot and ankle surgeons. Although no absolute consensus has been reached as to the use of ultrasound or whether the patient needs to be awake for the procedure, it is clear that the standard of care does not mandate either of these. The differences between US and UK practice are probably cultural and do not appear to affect the number of complications encountered.

Water-soluble, commercially-available poly(amidoamine) (PAMAM) dendrimers are highly-branched, well-defined, monodisperse macromolecules having an ethylenediamine core and varying surface functional groups. Dendrimers are being employed in an increasing number of biomedical applications. In this study, commercially obtained generation 5 hydroxyl-terminated (G5OH) PAMAM dendrimers were studied as potential proteomimetics for ophthalmic uses. To this end, the surface of G5OH PAMAM dendrimers were hydrophobically modified with varying amounts of dodecyl moieties, (flexible long aliphatic chains), or cholesteryl moieties (rigid lipid found in abundance in biological systems). Dendrimers were characterized by 1H-NMR, DLS, DSC and HPLC. The hydrophobic modification caused aggregation and molecular interactions between dendrimers that is absent in unmodified dendrimers. In vitro tissue culture showed that increasing the amount of dodecyl modification gave a proportional increase in toxicity of the dendrimers, while with increasing cholesteryl modification there was no corresponding increase in toxicity. Storage and loss modulus were measured for selected formulations. The hydrophobic modification caused an increase in loss modulus, while the effect on storage modulus was more complex. Rheological properties of the dendrimer solutions were comparable to those of porcine lens crystallins.

The mechanism by which the eye dynamically changes focal distance (accommodation), and the mechanism by which this ability is lost with age (presbyopia), are still contested. Due to inherent confounding factors in vivo, in vitro measurements have been undertaken using a robotic lens stretcher to examine these mechanisms as well as the efficacy of lens refilling - a proposed treatment for presbyopia. Dynamic forces, anterior and posterior curvatures, and lens thickness are all correlated for young natural and refilled porcine lenses. Comparisons are made to lenses refilled with a homogeneous polymer system. The amplitude of accommodation of the young porcine lens is very small such that it may be a suitable model for presbyopia. The behavior of refilled lenses was highly dependent on the refill volume. The volume could be tuned to maximize accommodative amplitude in the refilled lens.

AlphaA-crystallin is a small heat shock protein that functions as a molecular chaperone and a lens structural protein. The R49C single-point mutation in alphaA-crystallin causes hereditary human cataracts. We have previously investigated the in vivo properties of this mutant in a gene knock-in mouse model. Remarkably, homozygous mice carrying the alphaA-R49C mutant exhibit nearly complete lens opacity concurrent with small lenses and small eyes. Here we have investigated the 90 degrees light scattering, viscosity, refractive index, and bis-ANS fluorescence of lens proteins isolated from the alphaA-R49C mouse lenses and found that the concentration of total water-soluble proteins showed a pronounced decrease in alphaA-R49C homozygous lenses. Light scattering measurements on proteins separated by gel permeation chromatography showed a small amount of high-molecular mass aggregated material in the void volume which still remains soluble in alphaA-R49C homozygous lens homogenates. An increased level of binding of beta- and gamma-crystallin to the alpha-crystallin fraction was observed in alphaA-R49C heterozygous and homozygous lenses but not in wild-type lenses. Quantitative analysis with the hydrophobic fluorescence probe bis-ANS showed a pronounced increase in fluorescence yield upon binding to alpha-crystallin from mutant as compared with the wild-type lenses. These results suggest that the decrease in the solubility of the alphaA-R49C mutant protein was due to an increase in its hydrophobicity and supra-aggregation of alphaA-crystallin that leads to cataract formation. Our study further shows that analysis of mutant proteins from the mouse model is an effective way to understand the mechanism of protein insolubilization in hereditary cataracts.

The soluble proteins present in the ocular lens impart important optical and dynamic mechanical properties on the lens. The short-range order of crystallin proteins grants transparency to a very concentrated protein solution. This unique protein system directly enables proper visual function of the eye. These proteins were investigated in steady and oscillatory shear. Steady shear data were fitted with a modified Herschel-Bulkley yield stress model that allows for a Newtonian plateau at low shear rates. The Cox-Merz rule was used in conjunction with large amplitude oscillatory shear to give insight into the degradation of the fluid structure with increasing strain. The shear thinning viscoelastic behavior of these proteins gives rise to beneficial mechanical properties and results from the same short-range order granting optical transparency.

Before 2003, all NHS trusts charged locally determined prices for the provision of hip and knee arthroplasty services to their primary care trust (PCT). These charges could be fi xed irrespective of the activity provided (Department of Health (DH), 2002). In 2003–2004, the DH set up a national tariff system so that hospitals would charge PCTs per treatment performed. Similar treatments are grouped together under the same tariff and referred to as a Healthcare Resource Group (HRG).

Risk management in the water utility sector is becoming increasingly explicit. However, due to the novelty and complexity of the discipline, utilities are encountering difficulties in defining and institutionalising their risk management processes. In response, the authors have developed a sector specific capability maturity methodology for benchmarking and improving risk management. The research, conducted in consultation with water utility practitioners, has distilled risk management into a coherent, process-based framework. We identified eleven risk management processes, and eight key attributes with characterise the extent to which these processes are defined, controlled and institutionalised. Implementation of the model should enable utilities to more effectively employ their portfolio of risk analysis techniques for optimal, credible and defensible decision making.

Risk management in the water utility sector is fast becoming explicit. Here, we describe application of a capability model to benchmark the risk management maturity of eight water utilities from the UK, Australia and the USA. Our analysis codifies risk management practice and offers practical guidance as to how utilities may more effectively employ their portfolio of risk analysis techniques for optimal, credible, and defensible decision making. For risk analysis, observed good practices include the use of initiation criteria for applying risk assessment techniques; the adoption of formalised procedures to guide their application; and auditing and peer reviews to ensure procedural compliance and provide quality assurance. Additionally, we have identified common weaknesses likely to be representative of the sector as a whole, in particular a need for improved risk knowledge management and education and training in the discipline.

Over the last decade, suppliers of drinking water have recognised the limitations of relying solely on end-product monitoring to ensure safe water quality and have sought to reinforce their approach by adopting preventative strategies where risks are proactively identified, assessed and managed. This is leading to the development of water safety plans; structured ‘route maps’ for managing risks to water supply, from catchment to consumer taps. This paper reviews the Hazard Analysis and Critical Control Point (HACCP) procedure on which many water safety plans are based and considers its appropriateness in the context of drinking water risk management. We examine water safety plans in a broad context, looking at a variety of monitoring, optimisation and risk management initiatives that can be taken to improve drinking water safety. These are cross-compared using a simple framework that facilitates an integrated approach to water safety. Finally, we look at how risk management practices are being integrated across water companies and how this is likely to affect the future development of water safety plans. Keywords: Water safety plans, HACCP, Risk assessment, Cryptosporidium, Water quality

In this study, the possibility of using the internal osmotic pressure of intraocular polymeric hydrogel materials to attach the retina in the repair of a retinal tear or hole was investigated. This is in contrast to the conventional methods of retinal detachment repair (intraocular gas, polydimethylsiloxane, or n-perfluorooctane), which rely on surface tension and have recognized limits. The system selected for implementation of this scheme was based on an acrylamide copolymer that was crosslinked in an aqueous solution to provide a transparent hydrogel which allowed control of the swelling pressure. Synthetic hydrogels, such as those selected here, provide an alternative to materials currently used as vitreous prostheses.

We have developed a new experimental technique for determining the elastic modulus of the ocular lens by using hydrogel phantoms. We successfully prepared disulfide-reversible polyacrylamide hydrogels in the forms of a square column, a sphere, and a lens. The Mn (number average molecular weight) of the reduced copolymer ranged from 696,800 to 870,900 Da. The physical and swelling properties of the gels were independent of shape. The Mc (molecular weight between cross-links) ranged from 1776 to 1887 compared with the theoretical Mc of 1638 Da. The gels exhibited non-linear rubber elasticity, but at low strains the elastic moduli (E) were 4680±150, 5010±280, and 4870±220 Pa for the square column, sphere, and lens, respectively. The shear modulus (G) was 1531±70 Pa with an E/G ratio of approximately 3:1, indicating an incompressible gel at low strains. At high strains (∼15%) the Mooney–Rivlin plot was linear and the magnitude of 2C1 was 1515 Pa, which was comparable to the shear modulus of the gels. Finally, the Tatara mechanical model for large deformation of rubber spheres was successfully applied to extract the elastic modulus of the lens. The modulus of the lens obtained with this technique was consistent with the moduli for the square column and sphere. The new technique will be used to determine the mechanical properties of the ocular lens.

Financial pressures, regulatory reform, and sectoral restructuring are requiring water utilities to move from technically inclined, risk-averse management approaches toward more commercial, business-oriented practices. Risk analysis strategies and techniques traditionally applied to public health protection are now seeing broader application for asset management, assessing competition risks, and potential threats to the security of supplies. Water utility managers have to consider these risks alongside one another, employ a range of techniques, and devise business plans that prioritize resources on the basis of risk. We present a comprehensive review of risk analysis and management strategies for application in the water utility sector at the strategic, program, and operational levels of decision making.

A hydrogel nanocomposite was designed, synthesized, and evaluated for use as an auto-focusing intra-ocular lens. The hydrogel scaffold was composed of a monomer-free, thiol that contained polyacrylamide (5%), which was allowed to gel in the presence of nanoparticles at pH 7.4, 25° C. The nanoparticles consisted of a proteo-mimetic polyacrylamide nanogel (∼42 nm), bovine serum albumin (BSA) (∼6 nm), and hydrophilized silica (∼3 nm). The extent of nanoparticle loading increased with decreasing particle size. The elastic modulus increased with increasing loading of the proteo-mimetic nanogels and BSA, and it decreased with hydrophilized silica. In this investigation, the hydrogel that contained silica was the most promising class of nanocomposite hydrogels with properties comparable to that of a young porcine lens. A nanocomposite that consisted of 10% hydrogel scaffold and 24% hydrophilized silica (elastic [E] modulus of ∼1.0 kPa and refractive index [RI] of 1.42) was injected into a pre-evacuated porcine lens capsular bag. The composite lens was evaluated in a custom-designed four-arm radial stretcher, and its force-time spectrum was characterized by time constants of 60 ± 8.9 and 800 ± 32 ms. These results were comparable to a young porcine lens (E Modulus of 1.2 kPa; Rl of 1.4105; time constants of 48.3 ± 0.58 and 668 ± 24.6 ms, respectively).

Nondegradable polyamides with protein-like properties were designed and prepared as a copolymeric hydrogel by the free-radical polymerization of acrylamide with a disulfide-containing cross-linking agent, N, N-bisacryloylcystamine. Copolyacrylamides containing pendent thiol groups (BSH) were obtained after reduction of the disulfide bonds in the hydrogel, followed by precipitation in methanol. Ellman's analysis and gel-permeation chromatography (GPC) were used to characterize the copolymer. Redissolution of the copolymer in water at very dilute concentrations, followed by air oxidation under physiological conditions (pH 7.5), resulted in nanoparticles (nanogels). The dilute concentration favored intramolecular disulfide crosslinking in the polymer chain, resulting primarily in a single-chain nanogel. Nanogel formation was confirmed and characterized using GPC, dynamic light scattering and atomic force microscopy. The size of the nanogels ranged from 20–200nm.

The provision of wholesome, affordable and safe drinking water that has the trust of customers is the goal of the international water utility sector. Risk management, in terms of protecting the public health from pathogenic and chemical hazards has driven and continues to drive developments within the sector. In common with much of industry, the water sector is formalizing and making explicit approaches to risk management and decision-making that have formerly been implicit. Here, we review the risk management frameworks and risk analysis tools and techniques used within the water sector, considering their application at the strategic, programme and operational levels of decision-making. Our analysis extends the application beyond that of public health to issues of financial risk management, reliability and risk-based maintenance and the application of business risk maturity models.

The characteristics of poly(ethylene glycol) (PEG)–acrylate hydrogel networks were investigated as a function of the ethanol–water solvent composition during free-radical crosslinking copolymerization. Macromonomer (88% ω-methoxy-PEG–acrylate and 10% ω-phenoxy-PEG–acrylate) and crosslinker (2% PEG–diacrylate) concentrations were kept constant. As the copolymerization progressed, the polymer solution in 100% ethanol became increasingly turbid, indicating the development of a heterogeneous network structure. In 100% water, however, the initially turbid polymer solution became increasingly transparent as the crosslinking copolymerization progressed. All the gels were optically clear upon equilibration in water. Kinetic studies, with attenuated total reflectance-infrared, showed a long induction period, along with a lowered reaction rate, in 100% ethanol, and a decrease in conversion with an increase in ethanol content. These results agree with the UV analysis of the sol fractions, which indicated an increase in the amounts of unreacted PEG–acrylates with an increase in the ethanol content. The gels which were formed with a high ethanol concentration exhibited lower Young's modulus and higher swelling ability, suggesting that the network structure was significantly affected by the solvent composition during free-radical crosslinking copolymerization. From the stress–strain and swelling experiments, the Flory–Huggins interaction parameter was evaluated. The creep characteristics of the hydrogels were modeled with two Kelvin elements. © 2002 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 40: 2677–2684, 2002

Growth factors rapidly induce numerous genes that encode regulatory proteins, many of which have been identified by cDNA cloning. In this study, differential hybridization was used to screen a cDNA library constructed from human mammary carcinoma MDA-468 cells that were stimulated with transforming growth factor beta-1 (TGF-beta 1) in the presence of cycloheximide. One of the cDNA clones that was induced by TGF-beta 1 was found to have a nucleotide sequence that predicts a 9,450-Da protein with homology to regulatory DNA-binding proteins. This clone was designated metallopan-stimulin-1 (MPS-1) because it encodes a metalloprotein whose mRNA is expressed in a wide variety of actively proliferating cells and tumor tissues. MPS-1 protein contains one "zinc finger" domain of the C4 type, similar to those present in proteins of the steroid/thyroid hormone receptor superfamily and other DNA-binding proteins. The mRNA for MPS-1 was induced in MDA-468 cells by fetal calf serum, TGF-beta 1, TGF-beta 2, and cAMP analogues. The MPS-1 gene is expressed at relatively high levels in several human carcinoma cell lines, particularly those derived from ectodermal layers, and at higher levels in melanomas (ontogenically of neural origin). In contrast, the MPS-1 mRNA is expressed at low levels in normal WI-38 human lung diploid fibroblasts in culture. We hypothesize that MPS-1 protein may play a role as a potentially important mediator of cellular proliferative responses to various growth factors and other environmental signals.

A series of highly water-soluble organo-silica nanoparticles, ranging from 2 to 10 nm in diameter, were synthesized by the cohydrolysis and copolycondensation reactions. ω-methoxy(polyethyleneoxy)propyltrimethoxysilane (PEG6-9) and hydroxymethyltriethoxysilane (HMTEOS) mixtures were catalyzed by sodium hydroxide in the presence of surfactant benzethonium chloride (BTC) with various ratios of PEG6-9/HMTEOS at room temperature. The synthesized organo-silica nanoparticles possess a core–shell structure with a core of organo-silica resulting from HMTEOS and a monolayer shell of PEG6-9. The chemo-physical characteristics of the particles were studied by gel permeation chromatography (GPC), Fourier transform infrared (FTIR) spectroscopy, 29Si nuclear magnetic resonance (NMR), dynamic light scattering (DLS), transmission electron microscopy (TEM), and thermogravimetric analysis (TGA). The molecular weight and particle size of the particles increased with increasing HMTEOS molar ratios. The richest HMTEOS composition for the water-soluble particles was found to be HMTEOS:PEG6-9 = 80:20, where the particles had a 6 nm diameter core and a 0.8 nm thick shell. We propose that these water-soluble organo-silica nanoparticles will be suitable for biomedical applications.

The human vitreous is a gelatinous substance predominantly composed of water (97-99%). Vitreous substitutes are needed for treatment of retinal detachments by reapproximating the retina to the back of the eye, or during vitrectomies for maintenance of ocular volume. None of the current substitutes can be used long-term due to their short retention time, toxicity, or complications such as glaucoma or cataracts. In addition, all of the current compounds have a specific gravity less than water and so are not appropriate for inferior retinal detachments. The viscoelastic properties of the porcine vitreous were analyzed to develop a model for ideal substitutes. Synthetic polymers that form hydrogels in situ were researched for the development of artificial vitreous. In this study, the physical, mechanical, and optical properties of four self-gelling polyacrylamide copolymeric hydrogels were determined and compared with those of the porcine vitreous. The 2% formulation of high crosslink density, hydrophobically modified hydrogel most closely mimicked the porcine vitreous. The viscoelastic properties of hydrogels capable of being formed in situ were compared to those of the porcine vitreous to determine their efficacy as vitreous substitutes.

We developed a dynamic lens stretching device to quantitatively determine the relationships between force, equatorial displacement, and anterior curvature. A computer-controlled four-arm lens stretcher, equipped with real-time force transducers in each arm, was designed and constructed to obtain transient force measurements during lens stretching. The force-decay spectrum was fitted with a seven-parameter viscoelastic model characterized by three time constants. A corneal topography unit was used to measure the curvature of fresh porcine lenses ex vivo and in vitro in a four-arm lens stretcher at various equatorial displacements. The lens stretcher and corneal topography unit provided detailed information regarding the behavior of the porcine lens in vitro. For all lenses, the central portion of the anterior surface flattened as zonular force increased. Force increased nonlinearly with equatorial displacement. Relaxation time constants for accommodation were 34.5+/-12.2 ms, 310+/-122 ms, and 12,800+/-9490 ms. Time constants for disaccommodation were 34.9+/-4.7 ms, 291+/-79.1 ms, and 3400+/-775 ms, which were not statistically different from those measured for accommodation. The lens stretcher acts as a robotic analog of the ciliary body. This device allows direct, quantitative measurement of the forces and curvature changes relevant to accommodation. However, distortions were present due to the use of only four arms.

Risk analysis in the water utility sector is fast becoming explicit. Here, we describe application of a capability model to benchmark the risk analysis maturity of a sub-sample of eight water utilities from the USA, the UK and Australia. Our analysis codifies risk analysis practice and offers practical guidance as to how utilities may more effectively employ their portfolio of risk analysis techniques for optimal, credible, and defensible decision making.

Polymer solutions can fill any potential irregular cavity using minimally invasive techniques and thus have potential uses in ophthalmology. We prepared acrylamide hydrogels containing disulfide bonds by free radical polymerization in aqueous ethanol. The hydrogels were liquefied using dithiothreitol to yield water-soluble acrylamide copolymers containing pendant thiol (-SH) groups. The weight average molecular weights of the copolymers ranged from 1.43 x 10(5) to 9.22 x 10(5) daltons by GPC. Ellman's analysis and Raman spectroscopy confirmed the presence of -SH. The aqueous solutions of these purified thiol-containing copolymers were oxidized with 3,3'-dithiodipropionic acid or air to reform the hydrogels. The moduli of the reformed hydrogels ranged from 0.27 to 1.1 kPa depending on concentration and thiol content. Rapid endocapsular gelation yielded optically clear gel within the lens capsular bag. This technique now enables us to validate methods to determine the biomechanics of the lens and its role in accommodation.

Background Metallopanstimulin (MPS-1) is a ribosomal protein that is found in elevated amounts in the sera of patients with head and neck squamous cell carcinoma (HNSCC). We used a test, denoted MPS-H, which detects MPS-1 and MPS-1-like proteins, to determine the relationship between MPS-H serum levels and clinical status of patients with, or at risk for, HNSCC. Patients and methods A total of 125 patients were prospectively enrolled from a university head and neck oncology clinic. Participants included only newly diagnosed HNSCC patients. Two control groups, including 25 non-smokers and 64 smokers, were studied for comparison. A total of 821 serum samples collected over a twenty-four month period were analyzed by the MPS-H radioimmunoassay. Results HNSCC, non-smokers, and smokers had average MPS-H values of 41.5 ng/mL, 10.2 ng/mL, and 12.8 ng/mL, respectively (p = 0.0001). Conclusion We conclude that MPS-1 and MPS-1-like proteins are elevated in patients with HNSCC, and that MPS-H appears to be a promising marker of presence of disease and response to treatment in HNSCC patients.

We have used genomics and proteomics based technologies to study tissue and serum protein profiles in patients with breast cancer (BC) in comparison to control healthy subjects. One critical objective of this study was to identify and characterize new tissue and serum biomarkers of BC using differential screening of a recombinant cDNA human BC expression library. A second major objective of this study was to evaluate the clinical utility of Metallolpanstimulin (MPS-1/S27 ribosomal) protein as a biomarker for the early detection and monitoring of BC by immunoassay measurements of serum MPS-1 protein levels and to identify MPS-1 protein in sera of BC patients. A third objective was to present data on cloned BC genes denoted protein subgroup-30 (PS-30), consisting of mitochondria, nuclear, and ribosomal proteins which are increased after growth factor stimulation of BC cells in tissue culture. To study in detail MPS-1 protein expression in BC, the MPS-1 concentrations were determined in the blood of 117 females free of any disease, and in 203 female patients diagnosed with primary BC. The results indicate that increased serum MPS-1 levels can be used for the early detection of BC. Normal subjects have low concentrations of MPS-1 protein in sera. Moreover, changes in MPS-1 protein serum levels can be used for the study of BC progression or regression after various types of therapy. In both the low and high value range, MPS-1 is 10-fold more effective than CA-15-3 in modifying the probability of the target condition -breast cancer. The use of HPLC, Western blot, Immuno-Mass Spectrometry, and protein sequencing confirmed the presence of authentic MPS-1 in sera of patients with BC. Negligible levels of MPS-1 protein were detected in sera from normal subjects. We conclude that (1) the increase in serum MPS-1 can be used for the early detection of BC; and (2) MPS-1 proved to be reliable in the follow-up of patients with advanced BC as demonstrated by the close correlation between MPS-1 protein levels and BC progression or regression after various types of therapy. Furthermore, all proteins denoted group -30 (Mr30,000), consisting of ribosomal, nuclear and mitochondria proteins, were found to be significantly increased in BC tissues in comparison to control tissues, suggesting that these proteins may be useful markers for detection of BC. Finally, several serum reactive proteins such as haptoglobin and C3 complement components provided valuable information on oncogenic activity in BC patients.

An autosomal dominant missense mutation in αB-crystallin (αB-R120G) causes cataracts and desmin-related myopathy, but the underlying mechanisms are unknown. Here, we report the development of an αB-R120G crystallin knock-in mouse model of these disorders. Knock-in αB-R120G mice were generated and analyzed with slit lamp imaging, gel permeation chromatography, immunofluorescence, immunoprecipitation, histology, and muscle strength assays. Wild-type, age-matched mice were used as controls for all studies. Both heterozygous and homozygous mutant mice developed myopathy. Moreover, homozygous mutant mice were significantly weaker than wild-type control littermates at 6 months of age. Cataract severity increased with age and mutant gene dosage. The total mass, precipitation, and interaction with the intermediate filament protein vimentin, as well as light scattering of αB-crystallin, also increased in mutant lenses. In skeletal muscle, αB-R120G co-aggregated with desmin, became detergent insoluble, and was ubiquitinated in heterozygous and homozygous mutant mice. These data suggest that the cataract and myopathy pathologies in αB-R120G knock-in mice share common mechanisms, including increased insolubility of αB-crystallin and co-aggregation of αB-crystallin with intermediate filament proteins. These knock-in αB-R120G mice are a valuable model of the developmental and molecular biological mechanisms that underlie the pathophysiology of human hereditary cataracts and myopathy.

Purpose An in situ forming hydrogel was evaluated as a potential vitreous substitute in rabbits. Methods The hydrogel used a disulfide cross-linker that was then reduced to produce an injectable thiol-containing polymer solution. The disulfide cross-links reformed by air oxidation of the thiols and produced a stable hydrogel once inside the eye. The polymer was clear, autoclavable, and could be stored easily in the presence of nitrogen gas. Capillary rheometry was used to measure the viscoelastic properties of the hydrogels and the porcine vitreous. Fourteen black rabbits underwent a pars plana, 25-gauge, three-port vitrectomy by a single surgeon with injection of a vitreous substitute. Results The refractive indices of the hydrogels were measured by refractometry and were shown to be close to 1.33, and the 2% hydrogel matched the mechanical properties of the natural vitreous humor. The reduced polymeric hydrogel was easily injectable through a small-gauge needle into the vitreous cavity and did not show any fragmentation. The material underwent gelation within the eye, remained optically clear, and appeared well tolerated clinically. Slit lamp examination, dilated fundus examination, and electroretinograms showed no evidence of vitritis, uveitis, or endophthalmitis after 1 week. Histopathologic evaluation did not reveal any overt toxicity or gross morphologic changes in the retina. Conclusions The fact that this process of in situ gelation gives rise to hydrogels that are biocompatible and physically and optically similar to the natural vitreous suggests its suitability as a permanent vitreous substitute. Hydrogel candidates will be further studied to evaluate long-term biocompatibility and degradation in vivo.

We have examined the epidermal growth factor (EGF) dependence of the transcription of different proto-oncogenes, using cultured human mammary carcinoma MDA-468 cells and a nuclear run-on transcription assay. We found that the stimulation of MDA-468 cells with EGF regulates moderately and to different extents the transcription of the EGF-receptor(R) and c-erbB-2 proto-oncogenes. In contrast, the transcription of other proto-oncogenes, including c-myc, c-H-ras, and c-fps, was unchanged. Furthermore, we provide evidence that transforming growth factor beta 1 (TGF-beta 1) selectively and to different degrees modulates the EGF-dependent transcription of EGF-R and c-erbB-2 genes. In this study, we also discovered that T3 (triiodothyronine) exerts synergistic control on the action of EGF alone or in association with TGF-beta 1, on EGF-R and c-erbB-2 gene transcription. Moreover, we established that within 6 h after the addition of EGF, cytoplasmic EGF-R mRNA levels are increased several-fold and that this accumulation is enhanced by the presence of TGF-beta 1 and/or T3. The results described here are consistent with the hypothesis that a complex program of cooperative interactions among EGF-, TGF-beta 1-, and T3-generated signals at the transcriptional level may mediate, at least in part, the combined actions of EGF, TGF-beta 1, and T3 in target cells.

Alterations in c-myc proto-oncogene expression after treatment of human mammary carcinoma MDA-468 cells with epidermal growth factor (EGF) and/or transforming growth factor beta (TGF beta) have been investigated. A stimulation of c-myc messenger RNA was detected within 60 min after treatment with EGF. This induction persisted for at least 24 hr, albeit to a lower extent. The early and late increase in c-myc mRNA levels induced by EGF were inhibited by the presence of TGF beta. TGF beta alone induced little change in c-myc mRNA levels. The effect of TGF beta represents a novel action of this hormone at the level of gene expression.

We have investigated the actions of transforming growth factor (TGF) type alpha on epidermal growth factor (EGF) receptor mRNA expression in MDA-468 human mammary carcinoma cells in serum-free media. We found that exposure of MDA-468 cells to TGF alpha results in elevated levels of EGF receptor mRNA. This increase in mRNA accumulation showed time and dose dependence. Addition of TGF beta 1 enhanced the accumulation of EGF receptor mRNA induced by TGF alpha in a time- and dose-dependent manner. We also found that triiodothyronine at physiological concentrations exerts synergistic control on the action of TGF alpha alone, or in association with TGF beta 1, on EGF receptor mRNA expression. Similarly, retinoic acid treatment also enhanced in a time- and dose-dependent manner the TGF alpha-dependent response of EGF receptor mRNA and acted synergistically with TGF beta 1. The results described here suggest that optimum regulation of EGF receptor gene expression by TGF alpha is a complex process involving synergistic interactions with heterologous growth factors and hormones.