Search

Trypanosoma cruzi, the etiologic agent of American trypanosomiasis, is a vector-borne zoonotic parasite which has been little studied regarding its infection in domestic animals. In this study, we evaluated the occurrence of natural infection by T. cruzi in farm animals using molecular markers and phylogenetic analysis in blood clot samples of 60 sheep (Ovis aires), 22 goats (Capra hircus), and 14 horses (Equus caballus) in eight municipalities located in an infection risk area in the state of Rio Grande do Norte (RN), Northeast Region of Brazil. Trypanosoma spp. infection was identified by amplifying the rRNA 18S SSU gene in 48.9% of the samples. The SH022 sample showed 99.8% similarity with the Y strain of T. cruzi in phylogeny, grouped in the DTU II clade. Blood clots of sheep, goats, and horses detected T. cruzi kDNA in 28.3% (17/60), 22.7% (5/22), and 15.4% (2/14) of the samples, respectively. These animals were distributed in the three studied mesoregions throughout the state of RN. The identification of natural infection in domestic animals contributes to expand the epidemiological transmission scenario in an area where T. brasiliensis is the main vector.

Delivering therapeutics to the posterior segment of the eye is challenging due to various anatomical and physical barriers. While significant improvements have been realized by introducing direct injections to diseased sites, these approaches come with potential side effects that can range from simple inflammation to severe retinal damage. The topical instillation of drugs remains a safer and preferred alternative for patients’ compliance. Here, we report the synthesis of penetratin-complexed, redox-responsive hyaluronic acid-based nanogels for the triggered release and delivery of therapeutics to the posterior part of the eye via topical application. The synthesized nanogels were shown to release their load only when exposed to a reducing environment, similar to the cytoplasm. As a model drug, visual chromophore analog, 9-cis-retinal, was loaded into nanogels and efficiently delivered to the mouse retina’s photoreceptors when applied topically. Electroretinogram measurements showed a partial recovery of photoreceptor function in all treated eyes versus untreated controls. To the best of our knowledge, this report constitutes the first attempt to use a topically applied triggered-release drug delivery system to target the pigmented layer of the retina, in addition to the first attempt to deliver the visual chromophore topically.

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

The existence of polycyclic aromatic hydrocarbons (PAHs) in ambient air is an escalating concern worldwide because of their ability to cause cancer and induce permanent changes in the genetic material. Growing evidence implies that during early life-sensitive stages, the risk of progression of acute and chronic diseases depends on epigenetic changes initiated by the influence of environmental cues. Several reports deciphered the relationship between exposure to environmental chemicals and epigenetics, and have known toxicants that alter the epigenetic states. Amongst PAHs, benzo[a]pyrene (B[a]P) is accepted as a group 1 cancer-causing agent by the International Agency for the Research on Cancer (IARC). B[a]P is a well-studied pro-carcinogen that is metabolically activated by the aryl hydrocarbon receptor (AhR)/cytochrome P450 pathway. Cytochrome P450 plays a pivotal role in the stimulation step, which is essential for DNA adduct formation. Accruing evidence suggests that epigenetic alterations assume a fundamental part in PAH-promoted carcinogenesis. This interaction between PAHs and epigenetic factors results in an altered profile of these marks, globally and locus-specific. Some of the epigenetic changes due to exposure to PAHs lead to increased disease susceptibility and progression. It is well understood that exposure to environmental carcinogens, such as PAH triggers disease pathways through changes in the genome. Several evidence reported due to the epigenome-wide association studies, that early life adverse environmental events may trigger widespread and persistent variations in transcriptional profiling. Moreover, these variations respond to DNA damage and/or a consequence of epigenetic modifications that need further investigation. Growing evidence has associated PAHs with epigenetic variations involving alterations in DNA methylation, histone modification, and micro RNA (miRNA) regulation. Epigenetic alterations to PAH exposure were related to chronic diseases, such as pulmonary disease, cardiovascular disease, endocrine disruptor, nervous system disorder, and cancer. This hormetic response gives a novel perception concerning the toxicity of PAHs and the biological reaction that may be a distinct reliance on exposure. This review sheds light on understanding the latest evidence about how PAHs can alter epigenetic patterns and human health. In conclusion, as several epigenetic change mechanisms remain unclear yet, further analyses derived from PAHs exposure must be performed to find new targets and disease biomarkers. In spite of the current limitations, numerous evidence supports the perception that epigenetics grips substantial potential for advancing our knowledge about the molecular mechanisms of environmental toxicants, also for predicting health-associated risks due to environmental circumstances exposure and individual susceptibility.

More than a third of the world’s population relies on solid fuels for cooking and heating, with major health consequences. Although solid fuel combustion emissions are known to increase the prevalence of illnesses such as chronic obstructive pulmonary disease and lung cancer, however, their effect on the eyes is underexplored. This study assesses the acute toxicity of solid fuel combustion emissions on healthy ocular cells and a cancer cell line. Three healthy ocular cell lines (corneal, lens, and retinal epithelial cells) and a cancer cell line (Chinese hamster ovary cells) were exposed to liquid and gas phase emissions from applewood and coal combustion. Following the exposure, real-time cell attachment behavior was monitored for at least 120 hours with electrical cell impedance spectroscopy. The viability of the cells, amount of apoptotic cells, and generation of reactive oxygen species (ROS) were quantified with MTT, ApoTox-Glo, and ROS-Glo H2O2 assays, respectively. The results showed that coal emissions compromised the viability of ocular cells more than applewood emissions. Interestingly, the cancer cells, although their viability was not compromised, generated 1.7 to 2.7 times more ROS than healthy cells. This acute exposure study provides compelling proof that biomass combustion emissions compromise the viability of ocular cells and increase ROS generation. The increased ROS generation was fatal for ocular cells, but it promoted the growth of cancer cells.

Federal University of Rio Grande do Norte. Graduate Program in Parasitic Biology. Natal, RN, Brazil. Federal University of Rio Grande do Norte. Graduate Program in Pharmaceutical Sciences. Natal, RN, Brazil. Federal University of Rio Grande do Norte. Pharmacy Undergraduate Course. Natal, RN, Brazil. State Secretariat of Public Health of Rio Grande do Norte. Natal, RN, Brazil. Funda??o Oswaldo Cruz. Ren? Rachou Institute. Triatomine Research Group. Belo Horizonte, MG, Brazil. Funda??o Oswaldo Cruz. Ren? Rachou Institute. Triatomine Research Group. Belo Horizonte, MG, Brazil. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Laborat?rio de Geoprocessamento. Ananindeua, PA, Brasil. Federal University of Rio Grande do Norte. Graduate Program in Pharmaceutical Sciences. Natal, RN, Brazil / Federal University of Rio Grande do Norte. Graduate Program in Health Sciences. Natal, RN, Brazil. Federal University of Rio Grande do Norte. Graduate Program in Parasitic Biology. Natal, RN, Brazil / Federal University of Rio Grande do Norte. Graduate Program in Pharmaceutical Sciences. Natal, RN, Brazil. The occurrence of triatomine species, their bloodmeal sources and the discrete typing units (DTUs) of Trypanosoma cruzi isolated from them were determined in different municipalities of the state of Rio Grande do Norte, Brazil. Triatomine captures were carried out in the rural areas of 23 municipalities. The genotyping of T. cruzi isolates was performed using the mitochondrial cytochrome c oxidase subunit 2 (coii) gene, the D7 region of the 24S? rDNA, and the spliced leader intergenic region (SL-IR). Five triatomine species were captured, and the most frequent was Triatoma brasiliensis (84.3%; 916/1086), which was found in 16 of the 23 municipalities surveyed, and infested all types of environment investigated. The TcI DTU was found in all mesoregions surveyed in 51.5% (17/33) of the culture-positive samples. In contrast, TcII (9.1%; 3/33) was detected in the Central mesoregion, while TcIII (27.3%; 9/33) was found in all mesoregions. The geographic distribution and spatial overlap of different DTUs was inferred using the superposition of the radius of occurrence of isolates and using ecological niche distribution modelling. Triatoma brasiliensis was found infected in all mesoregions and with all three T. cruzi DTUs, including mixed infections. With regard to bloodmeal sources, the DNA of rodents was found in triatomines infected with either TcI or TcIII, while that of domestic animals and humans was associated with both single and mixed infections. Our findings demonstrate that different DTUs of T. cruzi are widely dispersed among triatomines in our study area. The association of T. brasiliensis with several different mammalian hosts, as well as overlapping areas with different DTUs, suggests that this triatomine species may have an important role as a vector in both anthropic and sylvatic environments.

This work demonstrates the application of hyaluronan-conjugated nitrogen-doped carbon quantum dots (HA-nCQDs) for bioimaging of tumor cells and illustrates their potential use as carriers in targeted drug delivery. Quantum dots are challenging to deliver with specificity, which hinders their application. To facilitate targeted internalization by cancer cells, hyaluronic acid, a natural ligand of CD44 receptors, was covalently grafted on nCQDs. The HA-nCQD conjugate was synthesized by carbodiimide coupling of the amine moieties on nCQDs and the carboxylic acids on HA chains. Conjugated HA-nCQD retained sufficient fluorescence, although with 30% lower quantum efficiency than the original nCQDs. Confocal microscopy showed enhanced internalization of HA-nCQDs, facilitated by CD44 receptors. To demonstrate the specificity of HA-nCQDs toward human tumor cells, patient-derived breast cancer tissue with high-CD44 expression was implanted in adult mice. The tumors were allowed to grow up to 200–250 mm(3) prior to the injection of HA-nCQDs. With either local or systemic injection, we achieved a high level of tumor specificity judged by a strong signal-to-noise ratio between the tumor and the surrounding tissue in vivo. Overall, the results show that HA-nCQDs can be used for imaging of CD44-specific tumors in preclinical models of human cancer and potentially used as carriers for targeted drug delivery into CD44-rich cells.

INTRODUCTION: Oral infection by Trypanosoma cruzi is currently the most important route of transmission of acute Chagas disease (ACD) in the North region of Brazil, and the reported outbreaks are usually related to ingestion of contaminated food, especially unprocessed açaí pulp. METHODS A retrospective cohort study was performed to analyze the epidemiological profile of individuals with suspected cases of ACD in the municipality of Breves, located in the state of Pará, Brazil. Therefore, notifications of suspected cases of ACD were collected from the Municipal Health Department of Breves from January 2007 to December 2017. RESULTS A total of 265 individuals were registered, and the majority were male (54.7%; 145/265). Age ranged from nine months to 79 years, with a greater number of notifications for individuals aged between 1 and 39 years (71.3%; 189/265). Most of them had a low level of education (74.3%, 197/265), were living in rural and urban areas (58.9%; 156/265 and 37.7%; 100/265, respectively). Infection occurred mainly in the domestic environment (96.2%; 255/265) through oral transmission (98.1%; 260/265). There were a greater number of notifications in November, December and January. CONCLUSIONS These data showed that oral transmission of T. cruzi has become increasingly high in the study region, and health education programs need to be implemented as strategies to ensure good manufacturing practices of unprocessed food.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES Este trabalho avaliou a presença, a distribuição de triatomíneos e unidades distintas de tipagem (DTUs, Discrete Typing Units) do Trypanosoma cruzi isolados de vetores em diferentes mesorregiões do estado do Rio Grande do Norte (RN). As buscas ativas dos triatomíneos foram realizadas em áreas rurais de 21 municípios, 71 comunidades rurais e 345 unidades domiciliares no período de 2015 a 2019. A genotipagem foi realizada pelos marcadores do gene mitocondrial da subunidade 2 do citocromo oxidase (CO II), domínio divergente D71 do gene 24Sα do DNA ribossomal (rDNA) e espaçador intergênico dos genes miniexon (SL-IR).A infestação foi observada em 7,5% (26/345) dos intradomicílios e em 16,2% (56/345) dos peridomicílios pesquisados. A presença de ninfas no peridomicílio foi observada em 94,7% (45/47) das unidades domiciliares infestadas, indicando colonização neste ambiente. Um total de 1.084 triatomíneos foram capturados em ambientes antrópicos e silvestres, sendo o Triatoma brasiliensis (84,5%) a espécie mais encontrada, seguida de Triatoma pseudomaculata (14,9%), Panstrongylus lutzi (0,5%), Rhodnius nasutus (0,1%). O T. brasiliensis foi a única espécie encontrada no intradomicílio, peridomicílio e ambiente silvestre, e também infestou a maior variedade de ecótopos. A infecção natural pelo T. cruzi foi observada em 11% (96/872) triatomíneos, sendo T. brasiliensis a espécie com maior número de exemplares (95,0%). Dos 96 triatomíneos infectados, foi possível isolar o parasito de 33 (34,4%) amostras, todas foram genotipadas. A DTUI: haplótipo A no COII, rDNA 2 e SL-IR com 150pb foi identificada em 51,5% (17/33) dos isolados do parasito no peridomicílio infectando T. brasiliensis nas três mesorregiões estudadas, e T. pseudomaculata, na mesorregião Oeste. A DTU II: haplótipo C no COII, rDNA 1, SLIR com 150pb foi observado em 9,1% (3/33) dos isolados em T. brasiliensis apenas na mesorregião Central tanto no peridomicílio quanto em ambiente silvestre. Enquanto a DTU III: haplótipo B no COII, rDNA 2, SL-IR com 200pb estava em 27,3% (9/33) das amostras e também foi encontrada em todas as mesorregiões, no intradomicílio na mesorregião Oeste, em áreas silvestres no Centro e em peridomicílio no Agreste. A proximidade e até sobreposição de áreas de ocorrência dos genótipos foi observada em várias áreas. Em Caicó, este fato foi evidenciado pela detecção de infecções mistas com TcI e TcII. Estes resultados mostram que o T. brasiliensis continua sendo a espécie com maior importância epidemiológica no semiárido do RN, devido à ampla distribuição geográfica, alto grau de adaptação aos diferentes ambientes, ecótopos, elevado índice de infecção natural pelo T. cruzi e a diversidade de DTUs, e reforçam a necessidade de vigilância entomológica contínua no estado, a fim de impedir o contato desta espécie com os seres humanos e animais domésticos. This work evaluated the presence, distribution of triatomines and discrete typing units (DTUs) of Trypanosoma cruzi isolated from vectors in different mesoregions of the state of Rio Grande do Norte (RN). The captures of triatomines were carried out in rural areas of 21 municipalities, 71 rural communities e 345 domiciles in the period from 2015 to 2019. The genotyping was performed using the markers mitochondrial cytochrome oxidase subunit 2 (COII) gene, the region of the 24Salpha rRNA, spliced leader intergenic region (SL-IR). The infestation was observed in 7.5% (26/345) of households and 16.2% (56/345) of the surveyed households. The presence of nymphs in the home was observed in 94.7% (45/47) of the infested households, indicating colonization in this environment. A total of 1,084 triatomines were captured in anthropic and wild environments, with Triatoma brasiliensis (84.5%) being the most found species, followed by Triatoma pseudomaculata (14.9%), Panstrongylus lutzi (0.5%), Rhodnius nasutus (0.1%). T. brasiliensis was the only species found in the home, peridomicile and wild environment, and also the one that was able to infest the largest variety of infested ecotopes. Natural infection by T. cruzi was observed in 11.0% (96/872) triatomines, with T. brasiliensis being the species with the highest number of specimens (95.0%). Of the 96 infected triatomines, it was possible to isolate the parasite from 33 (34.4%) samples, all of which were genotyped. DTU I: haplotype A in COII, rDNA 2 e SL-IR with 150pb (51.5%; 17/33) was identified in the peridomicile infecting T. brasiliensis in the three studied mesoregions, and T. pseudomaculata, in the western mesoregion. DTU II: haplotype C in COII, rDNA 1, SL-IR with 150pb: (9.1%; 3/33) was found in T. brasiliensis only in the Central mesoregion, both in the home and in the wild. DTU III: haplotype B in COII, rDNA 2, SL-IR with 200pb:(27.3%, 9/33) was also found in all mesoregions, in the home area in the West mesoregion, in wild areas in the Center and the home area in Agreste. The proximity and even overlap of areas of occurrence of the genotypes were observed in several areas. In Caicó, this fact was evidenced by the detection of mixed infections with TcI and TcII. These results show that T. brasiliensis remains the species with the greatest epidemiological importance in the semi-arid region of RN, due to its wide geographical distribution, high degree of adaptation to different environments and ecotopes, high rate of natural infection by T. cruzi and the diversity of DTUs that can house, and reinforce the need for continuous entomological surveillance in the state, in order to prevent contact of this species with humans and domestic animals.

INTRODUCTION: Knowledge of triatomine bloodmeal sources is essential for understanding vector-host interactions in Trypanosoma cruzi transmission cycles. Expensive commercial deoxyribonucleic acid (DNA) extraction kits are widely used for bloodmeal identification. This study assessed the performance of an inexpensive phenol-chloroform DNA extraction protocol for identification of triatomine bloodmeal sources, comparing it with a commercially available kit. METHODS: Both methods were used to obtain DNA from the intestinal contents of Triatoma brasiliensis blood-fed on either Columba sp., Mus musculus, or Gallus gallus. Subsequently, the mitochondrial 12S ribosomal ribonucleic acid (rRNA) gene was amplified by polymerase chain reaction, sequenced, and compared with GenBank data. RESULTS: Twelve (80%) samples extracted with the commercial kit and four (26.7%) with phenol-chloroform were pure (according to the A260/A280 ratio). Samples extracted with phenol-chloroform, except for Columba sp. samples, had higher DNA concentration than those extracted with the commercial kit. Samples extracted using phenol-chloroform and blood-fed on G. gallus had significantly higher DNA concentration than those blood-fed on Columba sp. (p-value

Synthesizing highly efficient red-emissive carbon dots (CDs) is a challenge that still impedes widespread applications of CDs in bioimaging. Herein, we demonstrate a facile, isolation-free synthesis of deep red (600–700 nm) emissive nitrogen-doped CDs (nCDs) based on microwave-assisted pyrolysis of citric acid and ethylenediamine. The duration of pyrolysis, the molar ratio of acid to amine, and the concentration of reactants were optimized using Central Composite Design and Response Surface Methodology to yield deep red fluorescence. We demonstrated their applicability on three different cell lines (retinal epithelial, lens epithelial, and Chinese hamster ovary cells). We measured the viability, the generation of reactive oxygen species, and percentage of apoptotic cells to determine their level of toxicity in cell culture. Confocal images showed that the nCDs fluoresced at different wavelengths depending upon the excitation wavelength and were excitable up to 635 nm. Furthermore, the ex vivo imaging of p...

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.

Vector transmission of Trypanosoma cruzi occurs in several areas of Brazil, including the northeastern region, and domestic animals can serve as reservoirs of the parasite. The aim of this study was to monitor dogs as domestic reservoirs for infection by T. cruzi, and the main triatomine species involved in parasite transmission in rural areas of municipalities in the State of Rio Grande do Norte, in northeastern, Brazil. Blood samples from dogs (n = 40) and manual triatomine capture were performed in domiciliary and peridomiciliary environments in rural areas of the towns of Acari, Caraubas and Marcelino Vieira, between 2013 and 2016. Subsequently, infection of dogs was determined by Polymerase Chain Reaction (PCR), Enzyme-Linked Immunosorbent Assay (ELISA) for the detection of IgM and IgG isotypes and Indirect Immunofluorescence (IIF) reactions for detection of IgG. Triatomine infection was determined by PCR. Forty (16/40) percent of the dogs were seropositive for T. cruzi; 20.0% (8/40) of such reactivity indicated the acute phase, and 20.0% (8/40), the chronic phase. PCR was positive in 42.5% (17/40) of the dogs’ blood samples. Specimens of Triatoma brasiliensis, Triatoma pseudomaculata, Rhodnius nasutus and Panstrongylus lutzi were found to be infected; however only T. brasiliensis nymphs and adults were infected in both environments. Triatomines evaluation showed 82.5% (94/114) of PCR positivity. Taken together, our results confirm that dogs are domestic reservoirs of T. cruzi in northeastern Brazil and T. brasiliensis is the main triatomine species correlated with parasite transmission in domiciliary environments. There is a continuing need to control peridomiciliary populations of triatomines and to implement continuous surveillance strategies for reservoirs with the help from the community.

Gold nanoparticles (Au NPs) have been tested as targeted delivery agents because of their high chemical stability and surface plasmon properties. Here, we investigated the biocompatibility of Au spheres (5-, 10-, 20-, 30-, 50-. and 100-nm), cubes (50-nm), and rods (10×90nm) on a retinal pigment epithelial (ARPE-19) cell line. The lethal dose for killing 50% of the cells (LD50) was evaluated using an MTT (3-[4, 5 dimethyl-thiazoly-2-yl] 2-5 diphenyl tetrazolium bromide) assay. At and above LD50, based on mass concentrations, the confluent cell layer began to detach, as shown by real-time measurements of electric impedance. We found that the biocompatibility of spheres improved with increasing nanoparticle size. The Au rods were less biocompatible than 10-nm spheres. Confocal microscopy showed that cubic (50-nm) and spherical NPs (50- and 100-nm) neither had cytotoxic effects nor entered cells. Lethal doses for internalized spherical NPs, which were toxic, were recalculated based on surface area (LD50,A) concentrations. Indeed, when biocompatibility was expressed as the surface area concentration of NPs, the curve was independent of size. The LD50,A of Au nanospheres was 23cm2/ml. Our findings demonstrate that the sole modulation of the surface area would make it possible to use Au NPs for therapeutic purposes.

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.

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.

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.

For targeted delivery with nanoparticles (NPs) as drug carriers, it is imperative that the NPs are internalized into the targeted cell. Surface properties of NPs influence their interactions with cells. We examined the responses of retinal pigment epithelial cells, NIH 3T3 fibroblast cells, and Chinese hamster ovary cells to gold nanoparticles (Au NPs) in their nascent form as well as coated with end-thiolated hyaluronate (HS-HA). The grafting density of HS-HA on Au NPs was calculated based on total organic carbon measurements and thermal gravimetric analysis. We imaged the intracellular NPs by 3D confocal microscopy. We quantified viability and generation of reactive oxygen species (ROS) of the cells to Au NPs and monitored cell-surface attachment via electrical cell-substrate impedance sensing. The results confirmed that receptors on cell surfaces, for HA, are critical in internalizing HS-HA-Au NPs, and HA may mitigate ROS pathways known to lead to cell death. The 50- and 100-nm HS-HA-Au NPs were able to enter the cells; however, their nascent forms could not. This study shows that the delivery of larger Au NPs is enhanced with HS-HA coating and illustrates the potential of HA-coated NPs as a drug delivery agent for inflamed, proliferating, and cancer cells that express CD44 receptors.

Hyaluronan (HA) is a naturally occurring glycosaminoglycan widely researched for its use as a biomaterial in tissue engineering, drug delivery, angiogenesis, and ophthalmic surgeries. The mechanical properties of this biomaterial can be altered to a required extent by chemically modifying the pendant reactive groups. However, derivatizing these polymers to a predetermined extent has been the Achilles heel for this process. In this study, we have investigated the factors controlling the derivatization of the carboxyl moieties of HA with amine containing thiol, cystamine dihydrochloride (Cys), via carbodiimide crosslinking chemistry. We used fractional factorial design to screen and identify the significant factor(s) affecting the reaction, and response surface methodology (RSM) to develop a model equation for predicting the degree of thiolation of HA. Also, we analyzed the reaction mechanism for potential side reactions. We observed that N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) (mole ratio with repeat unit of HA) is the significant factor controlling the degree of amidation. The quadratic equations developed from RSM predict the formulation for a desired degree of amidation of HA and percentage of potential side product. Hence, derivatizing HA to a predetermined extent with minimal side product can be achieved using the statistical design of experiments.

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.

Sulfonate-containing hydrogels are of particular interest because of their tunable mechanical and swelling properties, as well as their biological effects. Polysulfonate copolymers were synthesized by reacting 2-acrylamido-2-methylpropanesulfonic acid (AMPS), acrylamide (AM), and acrylic acid (AA). We found that the incorporation rate of sulfonate-containing monomer and the molecular weight of the copolymer were significantly enhanced by increasing the ionic strength of the solution. We introduced thiol groups by modifying the pendant carboxylates or copolymerizing along with a disulfide-containing monomer. The thiol-containing copolymers were reacted with a 4-arm acrylamide-terminated poly(ethylene glycol) via a thiol–ene click reaction, which was mediated by a photoinitiator, a redox initiator, or a base-catalyzed Michael-Addition. We were able to tailor the storage modulus (33–1800 Pa) and swelling capacity (1–91 wt %) of the hydrogel by varying the concentration of the copolymers. We determined that the injectable sulfonate-containing hydrogels were biocompatible up to 20 mg/mL, as observed by an electric cell–substrate impedance sensing (ECIS) technique, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay using three different cell lines: human retinal pigment epithelial cells (ARPE-19), fibroblasts (NIH 3T3), and Chinese hamster ovary cells (CHO).

In this chapter, the optimization of the design of in situ-forming hydrogel vitreous substitutes is discussed. First, a review of the structure and function, development and growth, and biomechanical properties of the vitreous humor is given. This review provides a foundation for designing a biomimetic vitreous substitute. Past and current treatments for vitreous substitution are also discussed. Recent research advances utilizing hydrogels as vitreous substitutes and proposed solutions for the design of vitreous substitutes are discussed in detail. Particular emphasis is given to the use of in situ-forming hydrogels for vitreous substitution and exploiting the swelling properties of hydrogels to reattach the retina.

The Veteran’s Health Administration (VHA) is the largest integrated health care system in the United States, forming the arm of the Department of Veterans Affairs (VA) that delivers medical services. From a troubled past, the VHA today is regarded as a model for healthcare transformation. The VA has evaluated and adopted a variety of cutting-edge approaches to foster greater efficiency and effectiveness in healthcare delivery as part of their systems redesign initiative. This paper discusses the integration of two health care analysis platforms: Discrete Event Simulation (DES), and Real Time Locating systems (RTLS) presenting examples of work done at the St. Louis VA Medical Center. Use of RTLS data for generation and validation of DES models is detailed, with prescriptive discussion of methodologies. The authors recommend the careful consideration of these relatively new approaches which show promise in assisting systems redesign initiatives across the health care spectrum.

In 2009, the St. Louis Veterans Affairs Medical Center (VAMC) installed a Real Time Locating system (RTLS) in their eye clinic. The system tracks staff and patient movement through a combination of radio-frequency identification and infra-red technology. This system, in addition to its primary purpose as a throughput monitoring system, was used to gather data for the validation of a Discrete Event Simulation (DES) of the eye clinic. Use of the RTLS gathered data greatly diminished the time required to validate the simulation, as well as the cost of labor needed to observe and record the data points necessary. RTLS systems may be employed by operations researchers and systems engineers to assist in patient flow analysis in capacities beyond the ‘Real Time’ aspects of the locating system.

Like many others, the St. Louis Veterans Administration Medical Center (VAMC) Pharmacy help desk receives far more calls than can be processed by current staffing levels. The objective of the study is to improve pharmaceutical services provided by the call center, by using queueing theory and discrete event dynamic simulation to analyze incoming telephone traffic to the help desk. Queueing and simulation models using both archival and hand-gathered data over a 1-year period were created, compared, and presented in order to determine the minimum quantities of staff needed to reach the desired service threshold. The simulation model was validated in comparison with real-world data. Results suggest that telephone traffic congestion in this setting may be alleviated by increasing the number of staff responsible for telephone services from 2 to 6 throughout the week, with an additional one serving on Monday. Both queueing and simulative models can be used to improve overwhelm pharmacy call centers, by determining the theoretical minimal staff needed to reach a service threshold.

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.