Indoxyl sulfate induces retinal microvascular injury via COX-2/PGE2 activation in diabetic retinopathy.

BACKGROUND: Diabetic retinopathy (DR), the principal cause of acquired blindness among the working-age population, is the most frequent microvascular complication of diabetes. Although metabolic disorders are hypothesized to play a role in its pathogenesis, the underlying mechanism remains largely elusive. METHODS: To elucidate the mechanism, we initially compared metabolite profiles of vitreous fluid between 23 patients with DR and 12 non-diabetic controls using liquid chromatography/tandem mass spectrometry, identifying the distinct metabolite indoxyl sulfate (IS). Subsequently, streptozotocin (STZ)-induced diabetic and IS-injected rat models were established to examine the effects of IS on retinal microvasculature. RNA sequencing was conducted to identify potential regulatory mechanisms in IS-treated human retinal endothelial cells (HREC). Finally, target gene knockdown in HREC and treatment of IS-injected rats with inhibitors (targeting IS production or downstream regulators) were employed to elucidate the detailed mechanisms and identify therapeutic targets for DR. RESULTS: Metabolomics identified 172 significantly altered metabolites in the vitreous humor of diabetics, including the dysregulated tryptophan metabolite indoxyl sulfate (IS). IS was observed to breach the blood-retinal barrier and accumulate in the intraocular fluid of diabetic rats. Both in vivo and in vitro experiments indicated that elevated levels of IS induced endothelial apoptosis and disrupted cell junctions. RNA sequencing pinpointed prostaglandin E2 (PGE2) synthetase-cyclooxygenase 2 (COX-2) as a potential target of IS. Validation experiments demonstrated that IS enhanced COX-2 expression, which subsequently increased PGE2 secretion by promoting transcription factor EGR1 binding to COX-2 DNA following entry into cells via organic anion transporting polypeptides (OATP2B1). Furthermore, inhibition of COX-2 in vivo or silencing EGR1/OATP2B1 in HREC mitigated IS-induced microcapillary damage and the activation of COX-2/PGE2. CONCLUSION: Our study demonstrated that indoxyl sulfate (IS), a uremic toxin originating from the gut microbiota product indole, increased significantly and contributed to retinal microvascular damage in diabetic retinopathy (DR). Mechanistically, IS impaired retinal microvascular integrity by inducing the expression of COX-2 and the production of PGE2. Consequently, targeting the gut microbiota or the PGE2 pathway may offer effective therapeutic strategies for the treatment of DR.

Chemotaxis and Motility of Spongospora subterranea Zoospores in Response to Potato Root Exudate Constituents and pH.

Spongospora subterranea f. sp. subterranea is an important pathogen of potato responsible for major losses in most potato growing regions of the world. Infection is initiated by biflagellated motile zoospores released from long-lived resting spores. Zoospore chemotaxis to the host plant root is widely believed to be stimulated by host root exudate compounds, although direct evidence is lacking. This study refined the traditional chemotaxis capillary assay, with which we provided the first empirical evidence of S. subterranea zoospore chemotaxis. Individual potato root exudate metabolites were either taxis neutral, inhibitory, or attractant to the zoospores. L-Glutamine was the strongest chemoattractant, while spermine was the most inhibitory. Zoospore motility and chemotaxis were constrained by strongly acidic or alkaline solutions of pH < 5.3 and >8.5, respectively. Beyond pH, ionic constituents of the test solution affected zoospore motility as Sorensen's phosphate buffer stalled zoospore motility, but HEPES buffer at the same concentration and pH had little or no negative motility effect. Zoospore motility, as characterized by several parameters, influenced chemotaxis. Among the parameters measured, total distance traveled was the best predictor of zoospore chemotaxis. The characterization of environmental and ecological effects on zoospore motility and chemotaxis highlights useful targets for S. subterranea disease control through manipulation of zoospore taxis or selection of host resistance traits.

"One suction and one extrusion" mode-based wash-free platform for determination of breast cancer cell-derived exosomes.

A simple and wash-free POCT platform based on microcapillary was developed, using breast cancer cell-derived exosomes as a model. This method adopted the "one suction and one extrusion" mode. The hybridized complex of epithelial cell adhesion molecule (EpCAM) aptamer and complementary DNA-horseradish peroxidase conjugate (CDNA-HRP) was pre-modified on the microcapillary's inner surface. "One suction" meant inhaling the sample into the functionalized microcapillary. The exosomes could specifically bind with the EpCAM aptamer on the microcapillary's inner wall, and then the CDNA-HRP complex was released. "One extrusion" referred to squeezing the shedding CDNA-HRP into the 3,3',5,5'-tetramethylbenzidine (TMB)/H2O2 solution, and then the enzyme-catalyzed reaction would occur to make the solution yellow using sulfuric acid as the terminator. Therefore, exosome detection could be realized. The limit of detection was 2.69 × 104 particles mL-1 and the signal value had excellent linearity in the concentration range from 2.75 × 104 to 2.75 × 108 particles⋅mL-1 exosomes. In addition, the wash-free POCT platform also displayed a favorable reproducibility (RSD = 2.9%) in exosome detection. This method could effectively differentiate breast cancer patients from healthy donors. This work provided an easy-to-operate method for detecting cancer-derived exosomes without complex cleaning steps, which is expected to be applied to breast cancer screening.

Analysis and comparison of tear protein profiles in dogs using different tear collection methods.

BACKGROUND: Tear proteomic analysis has become an important tool in medical and veterinary research. The tear collection method could influence the tear protein profile. This study aims to evaluate the protein profiles of dog tears collected using microcapillary tubes (MT), Schirmer tear strips (ST), and ophthalmic sponges (OS). METHODS: The tear samples were collected using MT, ST, and OS. Tear protein profiles were analyzed using two-dimensional electrophoresis (2-DE) and the different protein spots' expression was compared. Fourteen protein spots were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: Tear protein concentrations ranged from 2.80 to 4.03 µg/µL, with no statistically significant differences among collection methods. Protein expression in each collection method differed in terms of both the number and intensity of the spots. There were 249, 327, and 330 protein spots found from tears collected with MT, ST, and OS, respectively. The proteins albumin, haptoglobin, and lactoferrin identified from OS were found to have higher spot intensities than other methods of collection. The use of MT demonstrated the downregulation of nine proteins. CONCLUSIONS: The recent study supported that tear protein analysis is affected by different tear collection methods. Although ST is commonly used for tear collection, it provides insufficient information to study particular tear proteins.

Microcapillary LAMP for rapid and sensitive detection of pathogen in bovine semen.

A microcapillary-based loop-mediated isothermal amplification (µcLAMP) has been described for specific detection of infectious reproductive pathogens in semen samples of cattle without sophisticated instrumentation. Brucella abortus, Leptospira interrogans serovar Pomona and bovine herpesvirus 1 (BoHV-1) cultures were mixed in bovine semen samples. The µcLAMP assay is portable, user-friendly, cost-effective, and suitable to be performed as a POC diagnostic test. We have demonstrated high sensitivity and specificity of µcLAMP for detection of Brucella, Leptospira, and BoHV-1 in bovine semen samples comparable to PCR and qPCR assays. Thus, µcLAMP would be a promising field-based test for monitoring various infectious pathogens in biological samples.HighlightsDetect infectious organism in bovines semenReduction in carryover contamination is an important attribute, which may reduce the false-positive reaction.µcLAMP is a miniaturized form, which could be performed with a minimum volume of reagents.The µcLAMP assay is portable, user-friendly, and suitable to be performed as a POC diagnostic test.

Spectral Fingerprint Investigation in the near Infra-Red to Distinguish Harmful Ethylene Glycol from Isopropanol in a Microchannel.

Ethylene glycol (EG) and isopropanol (ISO) are among the major toxic alcohols that pose a risk to human health. However, it is important to distinguish them, since EG is more prone to cause renal failure, and can thus be more dangerous when ingested than ISO. Analysis of alcohols such as isopropanol and ethylene glycol generally can be performed with a complex chromatographic method. Here, we present an optical method based on absorption spectroscopy, performed remotely on EG-ISO mixtures filling a microchannel. Mixtures of ethylene glycol in isopropanol at different volume concentrations were analyzed in a contactless manner in a rectangular-section glass micro-capillary provided with integrated reflectors. Fiber-coupled broadband light in the wavelength range 1.3-1.7 µm crossed the microchannel multiple times before being directed towards an optical spectrum analyzer. The induced zig-zag path increased the fluid-light interaction length and enhanced the effect of optical absorption. A sophisticated theoretical model was developed and the results of our simulations were in very good agreement with the results of the experimental spectral measurements. Moreover, from the acquired data, we retrieved a responsivity parameter, defined as power ratio at two wavelengths, that is linearly related to the EG concentration in the alcoholic mixtures.

Nanomechanical Molecular Mass Sensing Using Suspended Microchannel Resonators.

In this work we study the different phenomena taking place when a hydrostatic pressure is applied in the inner fluid of a suspended microchannel resonator. Additionally to pressure-induced stiffness terms, we have theoretically predicted and experimentally demonstrated that the pressure also induces mass effects which depend on both the applied pressure and the fluid properties. We have used these phenomena to characterize the frequency response of the device as a function of the fluid compressibility and molecular masses of different fluids ranging from liquids to gases. The proposed device in this work can measure the mass density of an unknown liquid sample with a resolution of 0.7 µg/mL and perform gas mixtures characterization by measuring its average molecular mass with a resolution of 0.01 atomic mass units.

Coherent Optical Transduction of Suspended Microcapillary Resonators for Multi-Parameter Sensing Applications.

Characterization of micro and nanoparticle mass has become increasingly relevant in a wide range of fields, from materials science to drug development. The real-time analysis of complex mixtures in liquids demands very high mass sensitivity and high throughput. One of the most promising approaches for real-time measurements in liquid, with an excellent mass sensitivity, is the use of suspended microchannel resonators, where a carrier liquid containing the analytes flows through a nanomechanical resonator while tracking its resonance frequency shift. To this end, an extremely sensitive mechanical displacement technique is necessary. Here, we have developed an optomechanical transduction technique to enhance the mechanical displacement sensitivity of optically transparent hollow nanomechanical resonators. The capillaries have been fabricated by using a thermal stretching technique, which allows to accurately control the final dimensions of the device. We have experimentally demonstrated the light coupling into the fused silica capillary walls and how the evanescent light coming out from the silica interferes with the surrounding electromagnetic field distribution, a standing wave sustained by the incident laser and the reflected power from the substrate, modulating the reflectivity. The enhancement of the displacement sensitivity due to this interferometric modulation (two orders of magnitude better than compared with previous accomplishments) has been theoretically predicted and experimentally demonstrated.

Detection of catalase activity with aldehyde-doped liquid crystals confined in microcapillaries.

In this study, a simple, rapid, and label-free sensor was developed for detecting the enzymatic activity of catalase (CAT) with liquid crystals (LCs) confined in microcapillaries. Inside a microcapillary functionalized with n-octyltrichlorosilane, aldehyde-doped LCs anchored radially so that a pattern of straight lines was observed under a polarized optical microscope (POM). However, once hydrogen peroxide (HP) oxidized the aldehyde into carboxylic acid, which has surface activity, the orientation of the LCs at the interface changed, resulting in a distinct pattern change, from straight to crossed. In this system, the enzymatic activity of CAT could be detected as it inhibits the oxidation by decomposing HP; as a result, the pattern changed back to the straight one. From the orientational and optical shift, the enzymatic activity of CAT was detected up to a concentration of 0.8 fM under mild experimental conditions and 8 aM at pH 9.0. This result suggests the need for further study of microcapillary systems to develop simple and sensitive sensors for biochemical interactions.

Tear ferning test in healthy dogs.

PURPOSE: To evaluate and compare three tear sampling methods using two grading scales for administering the tear ferning test (TFT) to healthy dogs. METHODS: In total, 90 dogs (180 eyes) were subjected to tear sampling using millimetered strips, reused after the Schirmer tear test (STT) (Schirmer group, SG). Then, the dogs were subdivided into three groups according to sampling approach: micropipette (MPG), microcapillary (MCG), and Schirmer sample 2 (S2G). The collected tears were dried on a clean microscope glass slide at room temperature and humidity. The ferning patterns were observed under a polarized light microscope and classified according to the Rolando and Masmali grading scales. RESULTS: Although all three methods were feasible, the STT was easier to perform in clinical settings. Type I and Grade 1 were the most commonly observed (64.17% and 61.7%, respectively) regardless of collection method. There was no significant difference between the STT median values and the TFT classifications. CONCLUSIONS: The TFT is appropriate for dogs and can be performed using the three suggested sampling methods, with a higher frequency of Type I and Grade 1. Thus, it is possible to use both grading scales in the classification of tear ferning in dogs.

Micro-Capillary Coatings Based on Spiropyran Polymeric Brushes for Metal Ion Binding, Detection, and Release in Continuous Flow.

Micro-capillaries, capable of light-regulated binding and qualitative detection of divalent metal ions in continuous flow, have been realised through functionalisation with spiropyran photochromic brush-type coatings. Upon irradiation with UV light, the coating switches from the passive non-binding spiropyran form to the active merocyanine form, which binds different divalent metal ions (Zn2+, Co2+, Cu2+, Ni2+, Cd2+), as they pass through the micro-capillary. Furthermore, the merocyanine visible absorbance spectrum changes upon metal ion binding, enabling the ion uptake to be detected optically. Irradiation with white light causes reversion of the merocyanine to the passive spiropyran form, with simultaneous release of the bound metal ion from the micro-capillary coating.

Photo inactivation of virus particles in microfluidic capillary systems.

It has long been established that UVC light is a very effective method for inactivating pathogens in a fluid, yet the application of UVC irradiation to modern biotechnological processes is limited by the intrinsic short penetration distance of UVC light in optically dense protein solutions. This experimental and numerical study establishes that irradiating a fluid flowing continuously in a microfluidic capillary system, in which the diameter of the capillary is tuned to the depth of penetration of UVC light, uniquely treats the whole volume of the fluid to UVC light, resulting in fast and effective inactivation of pathogens, with particular focus to virus particles. This was demonstrated by inactivating human herpes simplex virus type-1 (HSV-1, a large enveloped virus) on a dense 10% fetal calf serum solution in a range of fluoropolymer capillary systems, including a 0.75 mm and 1.50 mm internal diameter capillaries and a high-throughput MicroCapillary Film with mean hydraulic diameter of 206 µm. Up to 99.96% of HSV-1 virus particles were effectively inactivated with a mean exposure time of up to 10 s, with undetectable collateral damage to solution proteins. The kinetics of virus inactivation matched well the results from a new mathematical model that considers the parabolic flow profile in the capillaries, and showed the methodology is fully predictable and scalable and avoids both the side effect of UVC light to proteins and the dilution of the fluid in current tubular UVC inactivation systems. This is expected to speed up the industrial adoption of non-invasive UVC virus inactivation in clinical biotechnology and biomanufacturing of therapeutic molecules. Biotechnol. Bioeng. 2016;113: 1481-1492. © 2015 Wiley Periodicals, Inc.

Cryopreservation of human spermatozoa with minimal non-permeable cryoprotectant.

Cryopreservation of human spermatozoa is a commonly used technique in assisted reproduction, however freezing low concentrations of sperm while maintaining adequate post-thaw motility remains a challenge. In an effort to optimize post-thaw motility yields, low volumes of human sperm were frozen in polyimide-coated fused silica micro-capillaries using 0.065 M, 0.125 M, 0.25 M, or 0.5 M trehalose as the only cryoprotectant. Micro-capillaries were either initially incubated in liquid nitrogen vapor before plunging into liquid nitrogen, or directly plunged into liquid nitrogen. Post thaw sperm counts and motility were estimated. Spermatozoa that were initially incubated in liquid nitrogen vapor had greater post thaw motility than those plunged immediately into liquid nitrogen independent of trehalose concentration. The protective effect of 0.125 M d-glucose, 3-O-methyl-d-glucopyranose, trehalose, sucrose, raffinose, or stachyose were evaluated individually. Trehalose and sucrose were the most effective cryoprotectants, recovering 69.0% and 68.9% of initial sperm motility, respectively.

Low-Coherence Reflectometry for Refractive Index Measurements of Cells in Micro-Capillaries.

The refractive index of cells provides insights into their composition, organization and function. Moreover, a good knowledge of the cell refractive index would allow an improvement of optical cytometric and diagnostic systems. Although interferometric techniques undoubtedly represent a good solution for quantifying optical path variation, obtaining the refractive index of a population of cells non-invasively remains challenging because of the variability in the geometrical thickness of the sample. In this paper, we demonstrate the use of infrared low-coherence reflectometry for non-invasively quantifying the average refractive index of cell populations gently confined in rectangular glass micro-capillaries. A suspension of human red blood cells in plasma is tested as a reference. As a use example, we apply this technique to estimate the average refractive index of cell populations belonging to epithelial and hematological families.

Modeling of Magnetite Nanoparticles Behavior under Conditions of Microcirculation and Analysis of In Vivo Toxicity.

The behavior of magnetite nanoparticles was studied in the cell chip microcapillaries. No aggregation of magnetite nanoparticles under conditions of long-term circulation was noted. Biodistribution and toxicity of magnetite nanoparticles (14 nm) and aminated magnetite after their intragastric administration to mice were studied in vivo. According to mass spectrometry and microscopy data, accumulation of nanoparticles occurred mainly in the liver cells.

Sample holder for axial rotation of specimens in 3D microscopy.

In common light microscopy, observation of samples is only possible from one perspective. However, especially for larger three-dimensional specimens observation from different views is desirable. Therefore, we are presenting a sample holder permitting rotation of the specimen around an axis perpendicular to the light path of the microscope. Thus, images can be put into a defined multidimensional context, enabling reliable three-dimensional reconstructions. The device can be easily adapted to a great variety of common light microscopes and is suitable for various applications in science, education and industry, where the observation of three-dimensional specimens is essential. Fluorescence z-projection images of copepods and ixodidae ticks at different rotation angles obtained by confocal laser scanning microscopy and light sheet fluorescence microscopy are reported as representative results.

Improved low-CPA vitrification of mouse oocytes using quartz microcapillary.

Cryopreservation by low-cryoprotectant (CPA) vitrification has the potential to combine all the advantages of the conventional high-CPA vitrification and slow-freezing approaches while avoiding their drawbacks. However, current low-CPA vitrification protocol for cryopreservation of oocytes requires a lengthy and multi-step procedure for unloading CPAs. In this study, we report a much-simplified procedure of using quartz microcapillary (QMC) for low-CPA vitrification of mouse oocytes with only one step for unloading CPAs. The immediate viability of oocytes after the improved low-CPA vitrification was determined to be more than 90%. Moreover, no significant difference was observed in terms of embryonic development from the two-cell to blastocyst stages between the fresh and vitrified oocytes after in vitro fertilization (IVF). This improved low-CPA vitrification technology has the potential for efficient cryopreservation of oocytes to preserve the fertility of mammals including humans for assisted reproductive medicine, maintenance of animal resource and endangered species, and livestock management.

Visualization of Microcapillary Tips Using Waveguided Light.

The microcapillary, a glass tube with a nano/micrometer scale aperture, is used for manipulating small objects across diverse disciplines. A primary concern in using the microcapillary involves tip breakage upon contact. Here, we report a method for visualizing the microcapillary tip, enabling precise and instant determination of its contact with other objects. Illumination directed to the back aperture of the microcapillary induces waveguiding through the glass wall, enabling the visualization of the tip through scattering. We demonstrate that the tip scattering is sensitive to contact with an adjacent object owing to the near-field interaction of the waveguided light, providing a clear distinction between the contact and noncontact states. The key advantage of our method encompasses its minimal influence, irrespective of conductivity, and applicability to nanoscale systems. The versatility of our method is shown by the application to a wide range of tip diameters, various substrate and in-filling materials.

Brain Sample Preparation After Performing in Utero Electroporation to Proliferating and Differentiated Cells in the Developing Mouse Neocortex.

In utero electroporation (IUE) enables labeling and manipulating specific types of cells by introducing DNA plasmids with desired promoters. After the surgery, mouse brains are fixed at any stage and analyzed after staining using specific antibodies. Here, we describe the flow of the IUE experiment from the preparation to microscopic observations.

Differential proteomics profile of microcapillary networks in response to sound pattern-driven local cell density enhancement.

Spatial cell organization and biofabrication of microcapillary networks in vitro has a great potential in tissue engineering and regenerative medicine. This study explores the impact of local cell density enhancement achieved through an innovative sound-based patterning on microcapillary networks formation and their proteomic profile. Human umbilical vein endothelial cells (HUVEC) and human pericytes from placenta (hPC-PL) were mixed in a fibrin suspension. The mild effect of sound-induced hydrodynamic forces condensed cells into architected geometries showing good fidelity to the numerical simulation of the physical process. Local cell density increased significantly within the patterned areas and the capillary-like structures formed following the cell density gradient. Over five days, these patterns were well-maintained, resulting in concentric circles and honeycomb-like structures. Proteomic analysis of the pre-condensed cells cultured for 5 days, revealed over 900 differentially expressed proteins when cells were preassembled through mild-hydrodynamic forces. Gene ontology (GO) enrichment analysis identified cellular components, molecular functions, and biological processes that were up- and down-regulated, providing insights regarding molecular processes influenced by the local density enhancement. Furthermore, we employed Ingenuity Pathway Analysis (IPA) to identify altered pathways and predict upstream regulators. Notably, VEGF-A emerged as one of the most prominent upstream regulators. Accordingly, this study initiates the unraveling of the changes in microcapillary networks at both molecular and proteins level induced by cell condensation obtained through sound patterning. The findings provide valuable insights for further investigation into sound patterning as a biofabrication technique for creating more complex microcapillary networks and advancing in vitro models.