Diffusion Of Fluorescein Research Articles

Lactobacilli cell-free supernatants: Potential green and natural enhancers for nose-to-brain delivery of small hydrophilic molecules

Concerns about environmental health are driving pharmaceutical industries towards more eco-friendly and biocompatible products. Lactobacilli cell-free supernatants (CFS) are mixtures of soluble factors derived from the microbial growth of beneficial bacteria with the potential to serve as natural and sustainable excipients in emerging formulations. This work aims to verify the usefulness of the CFS obtained from human L. crispatus BC5, L. gasseri BC9 and BC12 as enhancers in the nose-to-brain absorption of small hydrophilic molecules. CFS influence on cell viability was investigated together with their ability to increase the diffusion of sodium fluorescein across in vitro models of the olfactory epithelium. The enhancing mechanism was studied through differential scanning calorimetry analysis and immunostaining assay of zonula occludens-1. The use of 25 % (v/v) of all lactobacilli CFS on porcine olfactory epithelial primary cells was not associated with any cytotoxic effect, but only the CFS obtained from BC5 was pointed out as a permeation enhancer across both the biomimetic membrane PermeaPad®, the porcine olfactory tissue, and the primary cell model. The enhancing mechanism seems to rely on the CFS perturbation effect on lipid membranes that, in the case of the cell-based model, probably also results in an alteration of tight junctions’ activity.

Diffusion of small molecule drugs is affected by surface interactions and crowder proteins

SummaryCrowded environments are known to affect the diffusion of macromolecules, but their effects on the diffusion of small molecules are largely uncharacterized. We investigate how three protein crowders, bovine serum albumin (BSA), hen egg-white lysozyme, and myoglobin, influence the diffusion rates and interactions of four small molecules: fluorescein, and three drugs, doxorubicin, glycogen synthase kinase-3 inhibitor SB216763, and quinacrine. Using Line-FRAP measurements, Brownian dynamics simulations, and molecular docking, we find that the diffusion rates of the small molecules are highly affected by self-aggregation, interactions with the proteins, and surface adsorption. The diffusion of fluorescein is decreased because of its interactions with the protein crowders and their surface adsorption. Protein crowders increase the diffusion rates of doxorubicin and SB216763 by reducing surface interactions and self-aggregation, respectively. Quinacrine diffusion was not affected by protein crowders. The mechanistic insights gained here may assist in optimization of compounds for higher mobility in complex macromolecular environments.

Molecular Diffusion of Ions in Nanoscale Confinement.

We measured the diffusion of an anion, fluorescein, confined to a nanoscale (10-100 nm) aqueous film between two glass walls. The two glass walls were very slightly angled to form a crack. The diffusion of fluorescein was strongly influenced by the presence of an inert electrolyte, NaCl, in the film prior to the diffusion of charged fluorescein into the crack. The time to reach an equilibrium distribution of fluorescein was 10 times longer without the inert electrolyte than when the electrolyte was present. In applications where rapid diffusion of ions is important, it would therefore be advisable to not prewet a confined space with pure water. We attribute this phenomenon to the effect of the electrical potential of the confining walls. Unscreened surface potential in a thin film severely hinders the diffusion of the fluorescein ion. As salt diffuses into the thin film, the electrostatic double layer shrinks in thickness and further diffusion of ions is less hindered. On the other hand, diffusion of ions into the film is only weakly affected by the Debye length of the solution, provided that the surface potential inside a thin film is initially screened by even a low concentration of electrolyte inside the film. The time evolution of the concentration profile for different Debye lengths matches a diffusion model developed with the finite difference method (FDM).

The protective effects of butorphanol tartrate against homocysteine-induced blood-brain barrier dysfunction

ABSTRACT A high concentration of homocysteine (Hcy) has been recently reported to be closely associated with the development of stroke, which is related to the Hcy-induced blood-brain barrier (BBB) dysfunction. Butorphanol tartrate is a promising analgesic agent that targets the opiate receptor and shows promising protective effects on ischemia/reperfusion injury. The present research proposes to investigate the protective effect of butorphanol tartrate on Hcy-induced BBB disruption to explore the potential application of butorphanol tartrate in treating Hcy-induced stroke. Hcy was utilized to establish both an in vivo animal model and in vitro human brain vascular endothelial cells (HBVECs) injury model. We found that the increased diffusion of sodium fluorescein and Evan’s blue, declined expression of Claudin-5, and increased production of interleukin- 6 (IL-6) and tumor necrosis factor-α (TNF-α) were observed in Hcy-treated mice, which were all significantly reversed by butorphanol tartrate. In Hcy-stimulated HBVECs, increased endothelial permeability and reduced expression levels of Claudin-5 and Krüppel-like factor 5 (KLF5) were observed, all of which were dramatically rescued by 2 and 5 µM butorphanol tartrate. Lastly, the protective function of butorphanol tartrate in Hcy-stimulated HBVECs was dramatically abolished by the knockdown of KLF5. Collectively, butorphanol tartrate showed protective effects on Hcy-induced BBB disruption by upregulating the KLF5/Claudin-5 axis.

Depth- and direction-dependent changes in solute transport following cross-linking with riboflavin and UVA light in ex vivo porcine cornea

Diffusion is an important mechanism of transport for nutrients and drugs throughout the avascular corneal stroma. The purpose of this study was to investigate the depth- and direction-dependent changes in stromal transport properties and their relationship to changes in collagen structure following ultraviolet A (UVA)-riboflavin induced corneal collagen cross-linking (CXL). After cross-linking in ex vivo porcine eyes, fluorescence recovery after photobleaching (FRAP) was performed to measure fluorescein diffusion in the nasal-temporal (NT) and anterior-posterior (AP) directions at corneal depths of 100, 200, and 300 μm. Second harmonic generation (SHG) imaging was also performed at these three corneal depths to quantify fiber alignment. For additional confirmation, an electrical conductivity method was employed to quantify ion permeability in the AP direction in corneal buttons and immunohistochemistry (IHC) was used to image collagen structure. Cross-linked corneas were compared to a control treatment that received the riboflavin solution without UVA light (SHAM). The results of FRAP revealed that fluorescein diffusivity decreased from 23.39 ± 11.60 μm2/s in the SHAM group to 19.87 ± 10.10 μm2/s in the CXL group. This change was dependent on depth and direction: the decrease was more pronounced in the 100 μm depth (P = 0.0005) and AP direction (P = 0.001) when compared to the effect in deeper locations and in the NT direction, respectively. Conductivity experiments confirmed a decrease in solute transport in the AP direction (P < 0.0001). FRAP also detected diffusional anisotropy in the porcine cornea: the fluorescein diffusivity in the NT direction was higher than the diffusivity in the AP direction. This anisotropy was increased following CXL treatment. Both SHG and IHC revealed a qualitative decrease in collagen crimping following CXL. Analysis of SHG images revealed an increase in coherency in the anterior 200 μm of CXL treated corneas when compared to SHAM treated corneas (P < 0.01). In conclusion, CXL results in a decrease in stromal solute transport, and this decrease is concentrated in the most anterior region and AP direction. Solute transport in the porcine cornea is anisotropic, and an increase in anisotropy with CXL may be explained by a decrease in collagen crimping.

Stellar Neuroretinitis as Presenting Sign of Behcet’s Disease

A 40-year-old One-eyed patient (right eye) acuity history of post-traumatic burst in his left eye at the age of 6 years. Consult for a decrease of vision on his right eye that has progressed for 3 weeks. Ophthalmic examination found visual acuity: 2/10 P10 in the right eye, the left eye presents enophthalmos with an opacified cornea; the anterior segment was normal in the right eye, fundus examination shows hyalitis graded 1+, papillary edema, peripapillary hemorrhages and stellar macular exudates, cottony nodules, copper arteries with vascular sheathing of the superior temporal branch and areas of retinal ischemia (Figure 1). The inflammatory and serological assessment is negative. Fluorescein retinal angiography shows papillary diffusion of fluorescein and vasculitis (Figure 2). We find serous retinal detachment on OCT (Figure 3). The general examination found bipolar aphthosis. The diagnosis of Behçet’s disease is retained. Treatment with immunosuppressants allowed the regression of papillary edema with persistence of some macular exudates and great improvingin visual acuity.

Effects of salinity on the onset of elastic turbulence in swirling flow and curvilinear microchannels

Elastic turbulence, which is sensitive to geometry and polymer rheology, has shown great potential for improving the performance of mixing, heat transfer, and even oil recovery. Recent studies showed the importance of the rheological properties of polymer solutions on the onset of elastic turbulence. However, variations of rheological properties based on polymer sensitivities such as salinity and its corresponding effects on the elastic turbulence have not been revealed. This work investigated systematically the effects of salinity on the onset of elastic turbulence in both swirling flow and curvilinear microchannels. The variations of statistical properties, such as probability distribution functions (PDFs) and power spectral density of injected power (PSD), were analyzed for characterization. The onset conditions of elastic turbulence are postponed by high salinity, which is consistent with the mixing performance in a curvilinear microchannel. A salinity independent power-law exponent at a value of −4.3 is observed in a fully developed elastic regime for all polymer solutions. Particularly, the diffusion of fluorescein at a low flow rate in the microchannel is possible due to the existence of a steady secondary flow before the onset of elastic instability.

Diffusion through the ex vivo vitreal body – Bovine, porcine, and ovine models are poor surrogates for the human vitreous

The human vitreous humour is a complex gel structure whose composition and physical properties can vary considerably from person to person and also change with age. To date, the viscoelastic properties of the human vitreous gel has not been thoroughly investigated and despite many years of intensive research, an ideal vitreous substitute remains a challenge. Understanding the physical structure and properties of the vitreous is of fundamental and therapeutic interest, providing a clear insight into diffusion and transport of administered ophthalmic drug molecules into the vitreous. A number of mammalian surrogates, mainly bovine, porcine and ovine vitreous humours have been used in the literature as a means of studying ophthalmic drug transport and diffusion. In this study, the mechanical, physical and rheological properties of ovine, porcine, and bovine surrogates were investigated and compared to human vitreous. In addition, a bespoke Franz cell construct was used to compare the diffusion of a model drug (fluorescein) through vitreous samples. Despite the similarity in rheological properties between bovine, porcine and human vitreous samples, diffusion of fluorescein through the different vitreous samples revealed great differences in values of steady-state flux and diffusion coefficient. In addition, a first-generation vitreous mimic, composed of 4.5 mg/mL hyaluronic acid with complex viscosity of 0.3 ± 0.01 Pa has been evaluated and was demonstrated to be a better mimic of the human vitreous than the mammalian samples investigated.

Digital Manufacturing of Selective Porous Barriers in Microchannels Using Multi-Material Stereolithography.

We have developed a sequential stereolithographic co-printing process using two different resins for fabricating porous barriers in microfluidic devices. We 3D-printed microfluidic channels with a resin made of poly(ethylene glycol) diacrylate (MW = 258) (PEG-DA-258), a UV photoinitiator, and a UV sensitizer. The porous barriers were created within the microchannels in a different resin made of either PEG-DA (MW = 575) (PEG-DA-575) or 40% (w/w in water) PEG-DA (MW = 700) (40% PEG-DA-700). We showed selective hydrogen ion diffusion across a 3D-printed PEG-DA-575 porous barrier in a cross-channel diffusion chip by observing color changes in phenol red, a pH indicator. We also demonstrated the diffusion of fluorescein across a 3D-printed 40% PEG-DA-700 porous barrier in a symmetric-channel diffusion chip by measuring fluorescence intensity changes across the porous barrier. Creating microfluidic chips with integrated porous barriers using a semi-automated 3D printing process shortens the design and processing time, avoids assembly and bonding complications, and reduces manufacturing costs compared to micromolding processes. We believe that our digital manufacturing method for fabricating selective porous barriers provides an inexpensive, simple, convenient and reproducible route to molecule delivery in the fields of molecular filtration and cell-based microdevices.

Direct enrichment of pathogens from physiological samples of high conductivity and viscosity using H-filter and positive dielectrophoresis.

The full potential of microfluidic techniques as rapid and accurate methods for the detection of disease-causing agents and foodborne pathogens is critically limited by the complex sample preparation process, which commonly comprises the enrichment of bacterial cells to detectable levels. In this manuscript, we describe a microfluidic device which integrates H-filter desalination with positive dielectrophoresis (pDEP) for direct enrichment of bacterial cells from physiological samples of high conductivity and viscosity, such as cow's milk and whole human blood. The device contained a winding channel in which electrolytes in the samples continuously diffused into deionized (DI) water (desalination), while the bacterial cells remained in the samples. The length of the main channel was optimized by numerical simulation and experimentally evaluated by the diffusion of fluorescein into DI water. The effects of another three factors on H-filter desalination were also investigated, including (a) the flow rate ratio between the sample and DI water, (b) sample viscosity, and (c) non-Newtonian fluids. After H-filter desalination, the samples were withdrawn into the dielectrophoresis chamber in which the bacterial cells were captured by pDEP. The feasibility of the device was demonstrated by the direct capture of the bacterial cells in 1× PBS buffer, cow's milk, and whole human blood after H-filter desalination, with the capture efficiencies of 70.7%, 90.0%, and 80.2%, respectively. We believe that this simple method can be easily integrated into portable microfluidic diagnosis devices for rapid and accurate detection of disease-causing agents and foodborne pathogens.

Welcome to clinical ophthalmology.

Purpose: Cortical cataracts commonly occur in people with presbyopia and may be due to age-related loss of accommodation. Wedge-shaped or spoke-like opacities develop underneath insertion points of lens zonules, suggesting a possible link between lens zonular tension and cortical cataracts. Previous studies have suggested an association between accommodative stress and formation of cortical cataracts. We tested the hypothesis that increasing the tension of lens zonules by paralyzing ciliary muscles with cycloplegic drugs may promote cortical cataractogenesis. Methods: Diabetic rats were treated with cycloplegic eye drops (atropine or cyclopentolate) to paralyze the ciliary muscles and produce lens zonular tension. Lens opacification, diffusion of fluorescein into the lenses, and protein leakage into aqueous humor, were assessed at various time points. For human comparison, visual accommodative amplitude data from patients with cortical cataracts as well as agematched normal controls were collected and analyzed. Results: Atropine and cyclopentolate-treated diabetic rats developed lens opacities earlier and with greater severity than diabetic control rats. Fluorescein diffusion into the lens, and leakage of proteins in the aqueous in atropine-treated diabetic eyes was significantly higher than in diabetic controls, and undetectable in nondiabetic controls. Clinical data showed significantly lower visual accommodative amplitude in cortical cataract patients than in normal controls. Conclusion: Chronic use of cycloplegic drugs that paralyze ciliary muscles and increase lens zonular tension can accelerate lens leakage and opacification, contributing to formation of cortical cataracts. Clinical and experimental data support the hypothesis that increased lens zonular tension, which can be associated with presbyopia and clinical pupillary dilation, may promote cortical cataractogenesis.

Floating Droplet Array: An Ultrahigh-Throughput Device for Droplet Trapping, Real-time Analysisand Recovery

We describe the design, fabrication and use of a dual-layered microfluidic device for ultrahigh-throughput droplet trapping, analysis, and recovery using droplet buoyancy. To demonstrate the utility of this device for digital quantification of analytes, we quantify the number of droplets, which contain a β-galactosidase-conjugated bead among more than 100,000 immobilized droplets. In addition, we demonstrate that this device can be used for droplet clustering and real-time analysis by clustering several droplets together into microwells and monitoring diffusion of fluorescein, a product of the enzymatic reaction of β-galactosidase and its fluorogenic substrate FDG, between droplets.

In Vivo Measurement of Site-Specific Peritoneal Solute Transport Using a Fiber-Optic-based Fluorescence Photobleaching Technique

Fluorescence recovery after photobleaching (FRAP) is a well-established method commonly used to measure the diffusion of fluorescent solutes and biomolecules in living cells or tissues. Here a fiber-optic-based FRAP (f-FRAP) system was developed, and validated using macromolecules in water and agarose gels of different concentrations. We applied f-FRAP to measure the site-specific diffusion of fluorescein (NaFluo) in peritoneal membranes (PMs) on the liver, cecum, and kidney of a living rat during peritoneal dialysis. Diffusion of fluorescein in PM varied in a time-dependent manner according to the type of organ (<TEX>$D_{PM\;on\;Liver}/D_{NaFluo}=0.199{\pm}0.085$</TEX>, <TEX>$D_{PM\;on\;Cecum}/D_{NaFluo}=0.292{\pm}0.151$</TEX>, <TEX>$D_{PM\;on\;Kidney}/D_{NaFluo}=0.218{\pm}0.110$</TEX>). The proposed method allows direct quantitative measurement of the three-dimensional diffusion in local PM in vivo, which was previously inaccessible by peritoneal function test methods such as peritoneal equilibration test (PET) and standardized PM assessment (SPA). f-FRAP is promising for local and dynamic assessments of peritoneal pathophysiology and the mass transport properties of PMs, presumed to be affected by variation of tissue structures over different organs and functional changes of the PM with years of peritoneal dialysis.

Advanced glycation end-product accumulation reduces vitreous permeability.

To evaluate the effect of nonenzymatic cross-linking (glycation) upon the permeability of the vitreous to small- and large-solute diffusion. Vitreous from freshly excised porcine eyes was treated for 30 minutes with control or 0.01%, 0.1%, or 1% methylglyoxal (MG) solution. The efficacy of the glycation regimen was verified by measuring nonenzymatic cross-link density by fluorescence in the vitreous samples. Resistance to collagenase digestion as well as N(ε)-(carboxyethyl) lysine (CEL) content were also measured. The permeability coefficient for fluorescein and fluorescein isothiocyanate (FITC)-IgG diffusion through 3 mL of the vitreous samples was determined by using a custom permeability tester. Vitreous cross-linking with MG treatment was confirmed by increased fluorescence, increased CEL concentration, and increased resistance to collagenase digestion. Vitreous glycation resulted in a statistically significant decrease in the permeability coefficient for fluorescein diffusion when either 0.1% or 1% MG solution was used (5.36 ± 5.24 × 10(-5) cm s(-1), P = 0.04; and 4.03 ± 2.1 × 10(-5) cm s(-1), P = 0.001; respectively, compared with control, 9.77 ± 5.45 × 10(-5) cm s(-1)). The permeability coefficient for diffusion of FITC-IgG between control (9.9 ± 6.37 × 10(-5) cm s(-1)) and treatment groups was statistically significant at all MG concentrations (0.01% MG: 3.95 ± 3.44 × 10(-5) cm s(-1), P = 0.003; 0.1% MG: 4.27 ± 1.32 × 10(-5) cm s(-1), P = 0.004; and 0.1% MG: 3.72 ± 2.49 × 10(-5) cm s(-1), P = 0.001). Advanced glycation end-product (AGE) accumulation reduces vitreous permeability when glycation is performed in ex vivo porcine vitreous. The permeability change was more pronounced for the larger solute, suggesting a lower threshold for AGE-induced permeability changes to impact the movement of proteins through the vitreous when compared with smaller molecules.

Dynamics and function of the tear film in relation to the blink cycle.

Great strides have recently been made in quantitative measurements of tear film thickness and thinning, mathematical modeling thereof and linking these to sensory perception. This paper summarizes recent progress in these areas and reports on new results. The complete blink cycle is used as a framework that attempts to unify the results that are currently available. Understanding of tear film dynamics is aided by combining information from different imaging methods, including fluorescence, retroillumination and a new high-speed stroboscopic imaging system developed for studying the tear film during the blink cycle. During the downstroke of the blink, lipid is compressed as a thick layer just under the upper lid which is often released as a narrow thick band of lipid at the beginning of the upstroke. "Rippling" of the tear film/air interface due to motion of the tear film over the corneal surface, somewhat like the flow of water in a shallow stream over a rocky streambed, was observed during lid motion and treated theoretically here. New mathematical predictions of tear film osmolarity over the exposed ocular surface and in tear breakup are presented; the latter is closely linked to new in vivo observations. Models include the effects of evaporation, osmotic flow through the cornea and conjunctiva, quenching of fluorescence, tangential flow of aqueous tears and diffusion of tear solutes and fluorescein. These and other combinations of experiment and theory increase our understanding of the fluid dynamics of the tear film and its potential impact on the ocular surface.

Brazilian green propolis modulates inflammation, angiogenesis and fibrogenesis in intraperitoneal implant in mice.

BackgroundChronic inflammatory processes in the peritoneal cavity develop as a result of ischemia, foreign body reaction, and trauma. Brazilian green propolis, a beeswax product, has been shown to exhibit multiple actions on inflammation and tissue repair. Our aim was to investigate the effects of this natural product on the inflammatory, angiogenic, and fibrogenic components of the peritoneal fibroproliferative tissue induced by a synthetic matrix.MethodsChronic inflammation was induced by placing polyether-polyurethane sponge discs in the abdominal cavity of anesthetized Swiss mice. Oral administration of propolis (500/mg/kg/day) by gavage started 24 hours after injury for four days. The effect of propolis on peritoneal permeability was evaluated through fluorescein diffusion rate 4 days post implantation. The effects of propolis on the inflammatory (myeloperoxidase and n-acetyl-β-D-glucosaminidase activities and TNF-α levels), angiogenic (hemoglobin content-Hb), and fibrogenic (TGF-β1 and collagen deposition) components of the fibrovascular tissue in the implants were determined 5 days after the injury.ResultsPropolis was able to decrease intraperitoneal permeability. The time taken for fluorescence to peak in the systemic circulation was 20 ± 1 min in the treated group in contrast with 15 ± 1 min in the control group. In addition, the treatment was shown to down-regulate angiogenesis (Hb content) and fibrosis by decreasing TGF-β1 levels and collagen deposition in fibroproliferative tissue induced by the synthetic implants. Conversely, the treatment up-regulated inflammatory enzyme activities, TNF-α levels and gene expression of NOS2 and IFN-γ (23 and 7 fold, respectively), and of FIZZ1 and YM1 (8 and 2 fold) when compared with the untreated group.ConclusionsThese observations show for the first time the effects of propolis modulating intraperitoneal inflammatory angiogenesis in mice and disclose important action mechanisms of the compound (downregulation of angiogenic components and activation of murine macrophage pathways).

Disruption of the Blood–Aqueous Barrier and Lens Abnormalities in Mice Lacking Lysyl Oxidase-Like 1 (LOXL1)

Exfoliation syndrome (ES) is commonly associated with glaucoma, premature cataracts, and other ocular and systemic pathologies. LOXL1 gene variants are significantly associated with ES; however, the role of the protein in ES development remains unclear. The purpose of this study was to characterize the ocular phenotype in Loxl1(-/-) (null) mice. Loxl1 null mice and strain-matched controls (C57BL) were evaluated by clinical and histologic analyses. Anterior segment histology showed a pronounced vesiculation of the anterior lens in the null mice. The lesions were subcapsular and in direct apposition with the posterior iris surface. Fluorescein angiography showed increased diffusion of fluorescein into the anterior chamber of the null mice compared with age-matched controls (P = 0.003, two-tailed, unequal variance t-test), suggesting compromise of the blood-aqueous barrier. Intraocular pressure measurements were within the normal range (16.5 ± 2.0 mm Hg) in null mice up to 1 year of age. Immunohistochemistry showed decreased elastin in the iris and ciliary body in the null mouse compared with controls. Elimination of LOXL1 in mice impairs the blood-aqueous humor barrier in the ocular anterior segment and causes lens abnormalities consistent with cataract formation, but does not result in deposition of macromolecular material or glaucoma. These results show that mice lacking LOXL1 have some ES features but that complete disease manifestation requires other factors that could be genetic and/or environmental.

Using fluorescein and bromide tracers to investigate the role of baseflow in a small suburban watershed in Iowa, USA

Fluorescein and bromide tracers were used to study baseflow mechanisms of a small suburban watershed in northeast Iowa, USA. The tracers were applied to ten injection holes ranging from 1.3 to 3.0 ft in depth in two phases. Separately, two PVC wells (15 and 16 ft deep) were used to investigate tracer movement in a deeper flow system. Over 30 days of phase 1, none of the tracers was detected in the creek water. In phase 2, fluorescein was irregularly detected in the creek at two sites, whereas bromide was detected at one site only. Meanwhile, soil analysis detected measurable diffusion of bromide and fluorescein at four sites. At each of these sites, the tracer was found to be diffusing toward the creek. None of the tracers applied to the deeper PVC wells showed any movement toward the creek over 1 month of continuous sampling. Isotopic composition of water samples varied spatially as well as temporally going from the deep well (δ18O = −8.89‰) to the injection holes (average δ18O = −8.42‰), to the creek (average δ18O = −7.86‰), and further to the rain samples (average δ18O = −4.68‰). The analytical error margin is ±0.09‰. Samples from the injection holes were generally heavier than the deep well sample and lighter than the creek samples, indicating that there was no significant connection between the surface and the subsurface systems. Furthermore, the sporadic appearance of bromide and fluorescein both spatially and temporally points to the fact that baseflow does not constitute a significant part of the area's stream discharge. Copyright © 2014 John Wiley & Sons, Ltd.

Cell-Free Protein Synthesis in Miniaturized Array Devices and Effects of Device Orientation

Cell-free protein synthesis (CFPS) has been used as an alternative to cell-based recombinant technology for protein production in academic and industrial labs. The continuous-exchange format generally has higher expression yield by constantly supplying a nutrient solution and removing inhibitory by-products through a porous membrane. Because of the concern of possible membrane clogging by large molecules in the CFPS solution, we investigated the effects of membrane orientation on protein synthesis. We fabricated a miniaturized array device called Vertical-I with its membrane oriented vertically in reference to the table surface and found that the protein synthesis yield in the Vertical-I device was 144% higher than the Horizontal Device reported previously. The reaction time was also faster; β-glucuronidase reached the synthesis yield plateau after 2 h in the Vertical-I device versus 4 h in the Horizontal Device. Possible clogging of membrane pores was confirmed by fluorescein diffusion measurement. Using these results, we designed a device called Vertical-II that would fit into a 96 well plate holder for compatibility with commercial reagent dispensers and microplate readers. The experimentally optimized device increased protein expression 406% over the Horizontal Device and consumes 5 times fewer reagents than a commercial device, showing the potential for high-throughput protein synthesis.

Cross-Linking with Ultraviolet-A and Riboflavin Reduces Corneal Permeability

To investigate the effect of cross-linking treatment on corneal permeability in a live animal model. Rabbit eyes were selected at random to be left unoperated or to undergo epithelial debridement with or without treatment consisting of cross-linking (CXL) with riboflavin and ultraviolet-A. Nine eyes received a total dose of 3.6 J/cm² and after epithelial healing the corneas were placed in a two-chamber system for quantification of the diffusion of fluorescein compared with controls. Thirty eyes received a total dose of 5.4 J/cm² and, after epithelial healing, in vivo corneal permeability was quantified as the pupillary response over a 30-minute period to a dose of topical pilocarpine compared with controls. In the ex vivo assay, the mean permeability coefficient in the CXL group (2.42 × 10⁻⁷) was reduced when compared with the unoperated controls (3.73 × 10⁻⁷; P = 0.007) and to the eyes that received epithelial debridement alone (3.74 × 10⁻⁷; P = 0.01). In the in vivo permeability assay, the change in pupillary diameter at 30 minutes after pilocarpine administration was smaller in the CXL group (-1.9 mm), compared with the epithelial debridement group (-2.6 mm; P < 0.001) and with the unoperated controls (-2.7 mm; P = 0.003). Corneal cross-linking with ultraviolet-A and riboflavin results in a statistically significant reduction in corneal permeability. These findings suggest that dosing of topical medications may need to be increased in eyes with a history of CXL to achieve expected therapeutic effects, and they may have implications for the long-term health of the cornea.