Fluorescent Albumin Research Articles

Co-Delivery of Docetaxel and p44/42 MAPK siRNA Using PSMA Antibody-Conjugated BSA-PEI Layer-by-Layer Nanoparticles for Prostate Cancer Target Therapy.

How to overcome the low accumulation of chemotherapeutic agent in tumor tissue and exhibit multitherapeutics remains an ongoing challenge for cancer treatment. Here, a simple method is demonstrated that used to prepare prostate-specific membrane antigen antibody (PSMAab )-conjugated fluorescent bovine serum albumin (BSA)-branched polyethylenimine layer-by-layer nanoparticles (BSA-PEILBL NPs) for co-delivery of docetaxel (DTX) and p44/42 mitogen-activated protein kinase (MAPK) small interfering RNA (p44/42 MAPK siRNA) as synergistic and selective inhibition of cancer cell proliferation platform. The results show the levels of α-tubulin and p44/42 MAPK in CWR22R cells are significantly reduced after treatment with PSMAab -conjugated DTX/BSA-PEILBL /siRNA NPs. Consequently, the 50% cellular growth inhibition (IC50 ) values of the NPs loaded with both DTX and p44/42 MAPK siRNA are ≈2.1-fold less than those for the NPs only loaded with DTX. The median survival significantly prolongs from 18 d to upward 45 d compared to mice that receive same dose (12 mg kg-1 ) of free DTX. The results suggest this synergistic delivery system may be a promising clinical treatment in prostate cancer.

Uptake of leptin and albumin via separate pathways in proximal tubule cells

The adipokine leptin and oncotic protein albumin are endocytosed in the proximal tubule via the scavenger receptor megalin. Leptin reduces megalin expression and activates cell signalling pathways that upregulate fibrotic protein expression. The aim of this study was to investigate if leptin uptake in proximal tubule cells was via the albumin-megalin endocytic complex. In immortalised proximal tubule Opossum kidney cells (OK) fluorescent leptin and albumin co-localised following 5min exposure, however there was no co-localisation at 10, 20 and 30min exposure. In OK cells, acute exposure to leptin for 2h did not alter NHE3, ClC-5, NHERF1 and NHERF2 mRNA. However, acute leptin exposure increased NHERF2 protein expression in proximal tubule cells. In OK cells, immunoprecipitation experimentation indicated leptin did not bind to ClC-5. Leptin uptake in OK cells was enhanced by bafilomycin and ammonium chloride treatment, demonstrating that uptake was not dependent on lysosomal pH. Thus, it is likely that two pools of megalin exist in proximal tubule cells to facilitate separate uptake of leptin and albumin by endocytosis.

Synthesis of tumor-targeted folate conjugated fluorescent magnetic albumin nanoparticles for enhanced intracellular dual-modal imaging into human brain tumor cells

Superparamagnetic iron oxide nanoparticles (SPIO NPs), utilized as carriers are attractive materials widely applied in biomedical fields, but target-specific SPIO NPs with lower toxicity and excellent biocompatibility are still lacking for intracellular visualization in human brain tumor diagnosis and therapy. Herein, bovine serum albumin (BSA) coated superparamagnetic iron oxide, i.e. γ-Fe2O3 nanoparticles (BSA-SPIO NPs), are synthesized. Tumor-specific ligand folic acid (FA) is then conjugated onto BSA-SPIO NPs to fabricate tumor-targeted NPs, FA-BSA-SPIO NPs as a contrast agent for MRI imaging. The FA-BSA-SPIO NPs are also labeled with fluorescein isothiocyanate (FITC) for intracellular visualization after cellular uptake and internalization by glioma U251 cells. The biological effects of the FA-BSA-SPIO NPs are investigated in human brain tumor U251 cells in detail. These results show that the prepared FA-BSA-SPIO NPs display undetectable cytotoxicity, excellent biocompatibility, and potent cellular uptake. Moreover, the study shows that the made FA-BSA-SPIO NPs are effectively internalized for MRI imaging and intracellular visualization after FITC labeling in the targeted U251 cells. Therefore, the present study demonstrates that the fabricated FITC-FA-BSA-SPIO NPs hold promising perspectives by providing a dual-modal imaging as non-toxic and target-specific vehicles in human brain tumor treatment in future.

Self-Assembled Fluorescent Bovine Serum Albumin Nanoprobes for Ratiometric pH Measurement inside Living Cells.

In this work, we demonstrated a new ratiometric method for the quantitative analysis of pH inside living cells. The structure of the nanosensor comprises a biofriendly fluorescent bovine serum albumin (BSA) matrix, acting as a pH probe, and pH-insensitive reference dye Alexa 594 enabling ratiometric quantitative pH measurement. The fluorescent BSA matrix was synthesized by cross-linking of the denatured BSA proteins in ethanol with glutaraldehyde. The size of the as-synthesized BSA nanoparticles can be readily manipulated from 30 to 90 nm, which exhibit decent fluorescence at the peak wavelength of 535 nm with a pH response range of 6-8. The potential of this pH sensor for intracellular pH monitoring was demonstrated inside living HeLa cells, whereby a significant change in fluorescence ratio was observed when the pH of the cell was switched from normal to acidic with anticancer drug treatment. The fast response of the nanosensor makes it a very powerful tool in monitoring the processes occurring within the cytosol.

A systems approach to hemostasis: 4. How hemostatic thrombi limit the loss of plasma-borne molecules from the microvasculature

AbstractPrevious studies have shown that hemostatic thrombi formed in response to penetrating injuries have a core of densely packed, fibrin-associated platelets overlaid by a shell of less-activated, loosely packed platelets. Here we asked, first, how the diverse elements of this structure combine to stem the loss of plasma-borne molecules and, second, whether antiplatelet agents and anticoagulants that perturb thrombus structure affect the re-establishment of a tight vascular seal. The studies combined high-resolution intravital microscopy with a photo-activatable fluorescent albumin marker to simultaneously track thrombus formation and protein transport following injuries to mouse cremaster muscle venules. The results show that protein loss persists after red cell loss has ceased. Blocking platelet deposition with an αIIbβ3 antagonist delays vessel sealing and increases extravascular protein accumulation, as does either inhibiting adenosine 5′-diphosphate (ADP) P2Y12 receptors or reducing integrin-dependent signaling and retraction. In contrast, sealing was unaffected by introducing hirudin to block fibrin accumulation or a Gi2α gain-of-function mutation to expand the thrombus shell. Collectively, these observations describe a novel approach for studying vessel sealing after injury in real time in vivo and show that (1) the core/shell architecture previously observed in arterioles also occurs in venules, (2) plasma leakage persists well beyond red cell escape and mature thrombus formation, (3) the most critical events for limiting plasma extravasation are the stable accumulation of platelets, ADP-dependent signaling, and the emergence of a densely packed core, not the accumulation of fibrin, and (4) drugs that affect platelet accumulation and packing can delay vessel sealing, permitting protein escape to continue.

Visualizing Trimming Dependence of Biodistribution and Kinetics with Homo- and Heterogeneous N-Glycoclusters on Fluorescent Albumin.

A series of N-glycans, each sequentially trimmed from biantennary sialoglycans, were homo- or heterogeneously clustered efficiently on fluorescent albumin using a method that combined strain-promoted alkyne-azide cyclization and 6π-azaelectrocyclization. Noninvasive in vivo kinetics and dissection analysis revealed, for the first time, a glycan-dependent shift from urinary to gall bladder excretion mediated by sequential trimming of non-reducing end sialic acids. N-glycoalbumins that were trimmed further, in particular, GlcNAc- and hybrid biantennary-terminated congeners, were selectively taken up by sinusoidal endothelial and stellate cells in the liver, which are critical for diagnosis and treatment of liver fibrillation. Our glycocluster strategy can not only reveal the previously unexplored extracellular functions of N-glycan trimming, but will be classified as the newly emerging glycoprobes for diagnostic and therapeutic applications.

Loss of Endothelial Barrier in Marfan Mice (mgR/mgR) Results in Severe Inflammation after Adenoviral Gene Therapy.

ObjectivesMarfan syndrome is an autosomal dominant inherited disorder of connective tissue. The vascular complications of Marfan syndrome have the biggest impact on life expectancy. The aorta of Marfan patients reveals degradation of elastin layers caused by increased proteolytic activity of matrix metalloproteinases (MMPs). In this study we performed adenoviral gene transfer of human tissue inhibitor of matrix metalloproteinases-1 (hTIMP-1) in aortic grafts of fibrillin-1 deficient Marfan mice (mgR/mgR) in order to reduce elastolysis.MethodsWe performed heterotopic infrarenal transplantation of the thoracic aorta in female mice (n = 7 per group). Before implantation, mgR/mgR and wild-type aortas (WT, C57BL/6) were transduced ex vivo with an adenoviral vector coding for human TIMP-1 (Ad.hTIMP-1) or β-galactosidase (Ad.β-Gal). As control mgR/mgR and wild-type aortas received no gene therapy. Thirty days after surgery, overexpression of the transgene was assessed by immunohistochemistry (IHC) and collagen in situ zymography. Histologic staining was performed to investigate inflammation, the neointimal index (NI), and elastin breaks. Endothelial barrier function of native not virus-exposed aortas was evaluated by perfusion of fluorescent albumin and examinations of virus-exposed tissue were performed by transmission electron microscopy (TEM).ResultsIHC and ISZ revealed sufficient expression of the transgene. Severe cellular inflammation and intima hyperplasia were seen only in adenovirus treated mgR/mgR aortas (Ad.β-Gal, Ad.hTIMP-1 NI: 0.23; 0.43), but not in native and Ad.hTIMP-1 treated WT (NI: 0.01; 0.00). Compared to native mgR/mgR and Ad.hTIMP-1 treated WT aorta, the NI is highly significant greater in Ad.hTIMP-1 transduced mgR/mgR aorta (p = 0.001; p = 0.001). As expected, untreated Marfan grafts showed significant more elastolysis compared to WT (p = 0.001). However, elastolysis in Marfan aortas was not reduced by adenoviral overexpression of hTIMP-1 (compared to untreated Marfan aorta: Ad.hTIMP-1 p = 0.902; control Ad.β-Gal. p = 0.165). The virus-untreated and not transplanted mgR/mgR aorta revealed a significant increase of albumin diffusion through the endothelial barrier (p = 0.037). TEM analysis of adenovirus-exposed mgR/mgR aortas displayed disruption of the basement membrane and basolateral space.ConclusionsMurine Marfan aortic grafts developed severe inflammation after adenoviral contact. We demonstrated that fibrillin-1 deficiency is associated with relevant dysfunction of the endothelial barrier that enables adenovirus to induce vessel-harming inflammation. Endothelial dysfunction may play a pivotal role in the development of the vascular phenotype of Marfan syndrome.

Impaired Albumin Uptake and Processing Promote Albuminuria in OVE26 Diabetic Mice.

The importance of proximal tubules dysfunction to diabetic albuminuria is uncertain. OVE26 mice have the most severe albuminuria of all diabetic mouse models but it is not known if impaired tubule uptake and processing are contributing factors. In the current study fluorescent albumin was used to follow the fate of albumin in OVE26 and normal mice. Compared to normal urine, OVE26 urine contained at least 23 times more intact fluorescent albumin but only 3-fold more 70 kD fluorescent dextran. This indicated that a function other than size selective glomerular sieving contributed to OVE26 albuminuria. Imaging of albumin was similar in normal and diabetic tubules for 3 hrs after injection. However 3 days after injection a subset of OVE26 tubules retained strong albumin fluorescence, which was never observed in normal mice. OVE26 tubules with prolonged retention of injected albumin lost the capacity to take up albumin and there was a significant correlation between tubules unable to eliminate fluorescent albumin and total albuminuria. TUNEL staining revealed a 76-fold increase in cell death in OVE26 tubules that retained fluorescent albumin. These results indicate that failure to process and dispose of internalized albumin leads to impaired albumin uptake, increased albuminuria, and tubule cell apoptosis.

New 3-(heteroaryl)-2-iminocoumarin-based borate complexes: synthesis, photophysical properties, and rational functionalization for biosensing/biolabeling applications.

Members of a series of boron difluoride complexes with 3-(heteroaryl)-2-iminocoumarin ligands bearing both a phenolic hydroxyl group (acting as a fluorogenic center) and an N-aryl substituent (acting as a stabilizing moiety) have been synthesized in good yields by applying a straightforward two-step method. These novel fluorogenic dyes belong to the family of "Boricos" (D. Frath et al., Chem. Commun.- 2013, 49, 4908-4910) and are the first examples of phenol-based fluorophores of which the photophysical properties in the green-yellow spectral range are dramatically improved by N,N-chelation of a boron atom. Modulation of their fluorescence properties through reversible chemical modification of their phenol moieties has been demonstrated by the preparation of the corresponding 2,4-dinitrophenyl (DNP) ethers, which led to a dramatic "OFF-ON" fluorescence response upon reaction with thiols. Additionally, to expand the scope of these "7-hydroxy-Borico" derivatives, particularly in biolabeling, amine or carboxylic acid functionalities amenable to (bio)conjugation have been introduced within their scaffold. Their utility has been demonstrated in the preparation of fluorescent bovine serum albumin (BSA) conjugates and "Borico"-DOTA-like scaffolds in an effort to design novel monomolecular multimodal fluorescence- radioisotope imaging agents.

Fluorescence Resonance Energy Transfer Detection of Cobalt Ions by Silver Triangular Nanoplates and Rhodamine 6G

In the present paper, the authors studied fluorescence resonance energy transfer (FRET) phenomenon between silver triangular nanoplates and bovine serum albumin (BSA)/Rhodamine 6G fluorescence complex, and established a fluorescence method for the detection of cobalt ions. We found that when increasing the silver triangular nanoplates added to certain concentrations of fluorescent bovine serum albumin (BSA)/Rhodamine 6G complex, the fluorescence of Rhodamine 6G would be quenched up to 80% due to the FRET between the quencher and donor. However, in the presence of cobalt ions, the disassociation of the fluorescent complex from silver triangular nanoplates occurred and the fluorescence of the Rhodamine 6G recovered. The recovery of fluorescence intensity rate (I/I0) has a good relationship with the cobalt ion concentration (cCO2+) added. Thus, the authors developed a fluorescence method for the detection of cobalt ions based on the FRET of silver triangular nanoplates and Rhodamine 6G.

A Novel Isolated Glomerular Albumin Permeability Assay Based on Quantitative Fluorescence Microscopy

Vascular permeability is disrupted in several pathological conditions. We've taken advantage of the microscope densitometric technique to develop a new method to measure the solute permeability coefficient (Ps) of fluorescently labelled molecules ex vivo in glomeruli. Calibration experiments have been carried out to test the validity of this assay. Glomeruli were pre-perfused with Octadecyl-Rhodamine-B Chloride and Alexa Fluor 488 BSA (AF488-BSA) via whole-organ kidney perfusion in vivo. Following exsanguination and nephrectomy, glomeruli were sieved from the cortex of each kidney. Glomeruli were then secured in an imaging chamber and exposed to an albumin gradient, which drives efflux of fluorescent albumin from glomerular capillaries that can be quantified by time-lapse confocal imaging. Analysis and acquisition parameters were chosen using specific gain values. The fluorescence intensity values were calculated by selecting specific regions of interest inside different loops and in the perfusion bath. Using a similar protocol we also assessed the contribution of the z-stack position, the photobleaching effect, the free AF488 present in our solution, and differences between acellular and intact glomeruli on Ps values. There was no significant difference in photobleaching between AF488-BSA exposed to the laser for 90 min or 0min (n=4, Paired T Test = 0.20). Neither free dye present in our solution (Ps=0 n=4[1]), or z-stack position effected. Ps values were significantly higher in acellular glomeruli compared to non treated glomeruli. This technique is powerful to sensitively measure changes in albumin across a single glomerular capillary wall and has important implications, especially for isolated human glomeruli.

Abstract 16789: Loss of Endothelial Barrier in Fibrillin-1-Deficient Marfan Mice (mgR/mgR) Results in Severe Inflammation After Adenoviral Gene Therapy

Introduction: Nowadays surgery still remains option of choice for therapy of aneurysm in Marfan syndrome. Aortic tissue of Marfan patients shows elastolysis enhanced by proteolytic activity of Matrix Metalloproteinases (MMPs). In this study we performed adenoviral gene transfer of Tissue Inhibitor of MMP-1 (TIMP-1) in aortic grafts of Fibrillin-1-deficient Marfan mice (mgR/mgR) in order to reduce aortic elastolysis. Methods: We performed heterotopic infrarenal transplantation of thoracic aorta in female mice (n=35, n=7/group). MgR/mgR and wild-type (WT) aorta (C57BL/6) was either incubated ex vivo with adenoviral vector coding for human TIMP-1 or ß-Galactosidase (5x10^9pfu/ml) or as control with culture medium. 30 days after surgery overexpression of transgene was assessed by immunohistochemistry (IHC) and collagen in situ zymography (ISZ). Histologic staining was performed to outline elastin breaks, signs of inflammation and Neointimal Index (NI). Endothelial barrier was evaluated with the use of transmission electron microscope (TEM) and perfusion of fluorescent albumin. Results: IHC and ISZ revealed sufficient expression of transgene. Severe intimal hyperplasia and distinctive signs of cellular inflammation were solely observed in mgR/mgR aorta after adenoviral transduction (NI: ß-Gal 0.23; TIMP-1 0.43), but not in WT aorta (NI: Native 0.01; TIMP-1 0.00). Compared to native mgR/mgR and TIMP-1 treated WT aorta, NI in TIMP-1 treated mgR/mgR aorta was highly significantly larger (p=0.001; p=0.001). Native mgR/mgR grafts presented with severe elastolysis compared to WT (p=0.001). Regardless of the vector transduced elastolysis in mgR/mgR aorta after adenoviral gene therapy was more pronounced compared to WT aorta (ß-Gal p=0.001; TIMP-1 p=0.001). Albumin diffusion through the endothelial barrier was increased in native mgR/mgR aorta compared to native WT aorta (p=0.037). TEM analysis of mgR/mgR aorta displayed reconstruction of the basement membrane and basolateral space. Conclusions: Murine Marfan aortic grafts developed severe inflammation after adenoviral exposure. Fibrillin-1-deficiency is associated with dysfunction of endothelial barrier which could be a target for alternative therapies in Marfan syndrome.

Microscopic validation of macroscopic in vivo images enabled by same-slide optical and nuclear fusion.

It is currently difficult to determine the molecular and cellular basis for radioscintigraphic signals obtained during macroscopic in vivo imaging. The field is in need of technology that helps bridge the macroscopic and microscopic regimes. To solve this problem, we developed a fiducial marker (FM) simultaneously compatible with 2-color near-infrared (NIR) fluorescence (700 and 800 nm), autoradiography, and conventional hematoxylin-eosin (HE) histology. The FM was constructed from an optimized concentration of commercially available human serum albumin, 700- and 800-nm NIR fluorophores, (99m)Tc-pertechnetate, dimethyl sulfoxide, and glutaraldehyde. Lymphangioleiomyomatosis cells coexpressing the sodium iodide symporter and green fluorescent protein were labeled with 700-nm fluorophore and (99m)Tc-pertechnatate and then administered intratracheally into CD-1 mice. After in vivo SPECT imaging and ex vivo SPECT and NIR fluorescence imaging of the lungs, 30-μm frozen sections were prepared and processed for 800-nm NIR fluorophore costaining, autoradiography, and HE staining on the same slide using the FMs to coregister all datasets. Optimized FMs, composed of 100 μM unlabeled human serum albumin, 1 μM NIR fluorescent human serum albumin, 15% dimethyl sulfoxide, and 3% glutaraldehyde in phosphate-buffered saline (pH 7.4), were prepared within 15 min, displayed homogeneity and stability, and were visible by all imaging modalities, including HE staining. Using these FMs, tissue displaying high signal by SPECT could be dissected and analyzed on the same slide and at the microscopic level for 700-nm NIR fluorescence, 800-nm NIR fluorescence, autoradiography, and HE histopathologic staining. When multimodal FMs are combined with a new technique for simultaneous same-slide NIR fluorescence imaging, autoradiography, and HE staining, macroscopic in vivo images can now be studied unambiguously at the microscopic level.

PH-sensitive tubular polymersomes: formation and applications in cellular delivery.

Optimizing the shape of a nanovector influences its interaction with a cell and determines the internalization kinetics. Block copolymer amphiphiles self-assemble into monodisperse structures in aqueous solutions and have been explored extensively as drug delivery vectors. However, the structure of self-assembled block copolymers has mainly been limited to spherical vesicles or spherical and worm-like micelles. Here we show the controlled formation and purification of tubular polymersomes, long cylindrical vesicles. Tubular polymersomes are purified from other structures, and their formation is manipulated by incorporating the biocompatible membrane components cholesterol and phospholipids. Finally we show that these tubular polymersomes have different cellular internalization kinetics compared with spherical polymersomes and can successfully encapsulate and deliver fluorescent bovine serum albumin protein intracellularly.

T-PA, but not desmoteplase, induces plasmin-dependent opening of a blood-brain barrier model under normoxic and ischaemic conditions

Tissue-type plasminogen activator (t-PA) is the only thrombolytic treatment available for patients with acute ischaemic stroke. However, t-PA can increase permeability of the blood-brain barrier (BBB). Desmoteplase is a plasminogen activator derived from the common vampire bat, currently under clinical development for ischaemic stroke. We compared how t-PA and desmoteplase influenced BBB permeability using a human in vitro model where primary brain endothelial cells (BEC) and astrocytes are co-cultured on the opposite sides of a porous membrane. Permeability changes were evaluated 6 or 24h post-stimulation by passage of fluorescent albumin across the membrane. Under normoxic conditions, t-PA, but not desmoteplase, increased BBB permeability. Surprisingly, the ability of t-PA to affect the barrier was lost under conditions of oxygen-glucose deprivation (OGD). Addition of plasminogen re-sensitised the BBB to the action of t-PA under both normoxia and OGD, but did not affect the inert behaviour of desmoteplase, even when digested fibrinogen was added to ensure optimal plasmin generation. These observations coincided with plasmin-dependent changes in astrocyte and BEC morphology and disruption of tight junction proteins in BECs, specifically initiated by t-PA but not by desmoteplase. Finally, inhibition of plasmin post-stimulation with t-PA and plasminogen, especially within 2h, protected the BBB against t-PA-mediated barrier opening. Hence t-PA, but not desmoteplase, increases BBB permeability under both normoxic and OGD conditions in a reversible, plasmin-dependent process. The inability of desmoteplase to increase permeability despite its capacity to generate plasmin provides further support for its use as thrombolytic in patients with ischaemic stroke.

Photophysics, RET Probes and Imaging Applications for Fluorescent BSA Au Nanoclusters

Protein protected fluorescent metal nanoclusters are attracting great deal of attention from fluorescence community. This work explores photo-physics, fluorescence polarization properties; resonance energy transfer (RET) probes, and cellular imaging applications of novel fluorescent probe bovine serum albumin protected gold (BSA Au) nanoclusters. Important findings were long fluorescence lifetime (> 1µs) and polarized fluorescence emission studied in solvents of different viscosity. The BSA Au cluster, due to long lifetime and high polarization, can potentially be used in developing fluorescence polarization immunoassays. Moreover, we describe an approach by developing RET of BSA Au clusters with the near infra-red (NIR) dye Dylight 750 (Dy750), where BSA Au cluster act as a donor to Dy750, showing 46% energy transfer efficiency to the NIR dye Dy750 with long lifetime component in acceptor decay through RET. Such RET probe with narrow emission spectrum and long lifetime component can be explored in microscopy imaging applications. Furthermore, we describe the use of these long lived BSA Au clusters in cellular and tissue imaging applications. We have shown using multiple pulse excitation (MPE) scheme that one can increase the intensity of long lived BSA Au cluster in cells without increasing the short lived auto-fluorescence background thereby increasing signal to background ratio at least by 15 times. Moreover, by applying time gated detection strategy to MPE imaging experiment we increased the signal to noise ratio to 30, a dramatic improvement in contrast for low fluorescence quantum yield dye. In summary, BSA Au clusters can be used as a fluorescent cellular stain advancing the bioimaging technology via their long fluorescence lifetime.

A novel core-satellite CdTe/Silica/Au NCs hybrid sphere as dual-emission ratiometric fluorescent probe for Cu2+

Herein, we synthesized a novel core-satellite CdTe/Silica/Au NCs hybrid sphere by covalently linking the separately synthesized highly fluorescent bovine serum albumin (BSA) stabilized gold nanoclusters (Au@BSA NCs) to the surface of the amino functionalized CdTe@SiO2 spheres by using the EDC chemistry. Numerous “satellites” of Au NCs were linked on the surface of the CdTe@SiO2 by the way of amide bonding. The synthesized dual-emission hybrid spheres were further characterized by the transmission electron microscopy (TEM), energy dispersive X-ray spectroscopy (EDS), UV–vis absorption spectroscopy, Fourier transform infrared (FTIR) spectroscopy, photoluminescence (PL), etc. Finally, the CdTe/Silica/Au NCs hybrid spheres were developed as ratiometric fluorescence probe for the determination of Cu2+ with high sensitivity and selectivity. The fluorescence intensity ratio (F545nm/F655nm) of the probe against the concentration of Cu2+ showed a good linear relationship from 6.0×10−7molL−1 to 100.0×10−7molL−1. It showed an excellent reproducibility (0.67% relative standard deviation for 10 replicate measurements of Cu2+ at 40.0×10−7molL−1) and low detection limit (4.1×10−7molL−1). Furthermore, the ratiometric fluorescent probe was successfully applied in the determination of Cu2+ in vegetable samples with satisfactory results.

Circulating Mononuclear Progenitor Cells: Differential Roles for Subpopulations in Repair of Retinal Vascular Injury

We examined effect on retinal vascular homing of exogenous CD34(+) and CD14(+) progenitor cells using mouse models of chronic (streptozotocin [STZ]-induced diabetes) and acute (ischemia-reperfusion [I/R]) ocular vascular injury. STZ-treated mice of short or long duration (≤4, ≥11 months) diabetes, along with age- and sex-matched controls, were given intravitreous injections of human CD34(+) and CD14(+) cells isolated from healthy or diabetic donors alone or in combination. I/R injured mice were given diabetic or nondiabetic CD34(+) cells with mesenchymal stem cells (MSCs) or diabetic CD34(+) cells manipulated by ex vivo fucosylation with ASC-101. Injected cells were localized by fluorescent immunocytochemistry, and the degree of retinal vascular colocalization quantified morphometrically. Permeability was assessed by fluorescent albumin leakage. Diabetic CD14(+) cells associated with vessels to a greater degree than diabetic CD34(+) cells. Vascular permeability was reduced only by nondiabetic cells and only at the highest number of cells tested. Diabetic CD34(+) cells consistently demonstrated reduced migration. There was a 2-fold or 4-fold increase over control in the specific localization of diabetic CD34(+) cells within the vasculature when these cells were co-administered with MSCs or ex vivo fucosylated prior to injection, respectively. Diabetic CD14(+) cells, unlike diabetic CD34(+) cells, retain robust homing characteristics. CD34(+) or CD14(+) subsets rather than whole bone marrow or peripheral blood cells may prove more beneficial in autologous cell therapy for diabetics. Co-administration with MSCs or ex vivo fucosylation may enhance utility of CD34(+) cells in cell therapy for diabetic ocular conditions like macular ischemia and retinal nonperfusion.

Quantifying Glomerular Permeability of Fluorescent Macromolecules Using 2-Photon Microscopy in Munich Wistar Rats

Kidney diseases involving urinary loss of large essential macromolecules, such as serum albumin, have long been thought to be caused by alterations in the permeability barrier comprised of podocytes, vascular endothelial cells, and a basement membrane working in unison. Data from our laboratory using intravital 2-photon microscopy revealed a more permeable glomerular filtration barrier (GFB) than previously thought under physiologic conditions, with retrieval of filtered albumin occurring in an early subset of cells called proximal tubule cells (PTC)(1,2,3). Previous techniques used to study renal filtration and establishing the characteristic of the filtration barrier involved micropuncture of the lumen of these early tubular segments with sampling of the fluid content and analysis(4). These studies determined albumin concentration in the luminal fluid to be virtually non-existent; corresponding closely to what is normally detected in the urine. However, characterization of dextran polymers with defined sizes by this technique revealed those of a size similar to serum albumin had higher levels in the tubular lumen and urine; suggesting increased permeability(5). Herein is a detailed outline of the technique used to directly visualize and quantify glomerular fluorescent albumin permeability in vivo. This method allows for detection of filtered albumin across the filtration barrier into Bowman's space (the initial chamber of urinary filtration); and also allows quantification of albumin reabsorption by proximal tubules and visualization of subsequent albumin transcytosis(6). The absence of fluorescent albumin along later tubular segments en route to the bladder highlights the efficiency of the retrieval pathway in the earlier proximal tubule segments. Moreover, when this technique was applied to determine permeability of dextrans having a similar size to albumin virtually identical permeability values were reported(2). These observations directly support the need to expand the focus of many proteinuric renal diseases to included alterations in proximal tubule cell reclamation.

Receptor-mediated endocytosis and endosomal acidification is impaired in proximal tubule epithelial cells of Dent disease patients

Receptor-mediated endocytosis, involving megalin and cubilin, mediates renal proximal-tubular reabsorption and is decreased in Dent disease because of mutations of the chloride/proton antiporter, chloride channel-5 (CLC-5), resulting in low-molecular-weight proteinuria, hypercalciuria, nephrolithiasis, and renal failure. To facilitate studies of receptor-mediated endocytosis and the role of CLC-5, we established conditionally immortalized proximal-tubular epithelial cell lines (ciPTECs) from three patients with CLC-5 mutations (30:insH, R637X, and del132-241) and a normal male. Confocal microscopy using the tight junction marker zona occludens-1 (ZO-1) and end-binding protein-1 (EB-1), which is specific for the plus end of microtubules demonstrated that the ciPTECs polarized. Receptor-mediated endocytic uptake of fluorescent albumin and transferrin in 30:insH and R637X ciPTECs was significantly decreased, compared with normal ciPTECs, and could be further reduced by competition with 10-fold excess of unlabeled albumin and transferrin, whereas in the del132-241 ciPTEC, receptor-mediated endocytic uptake was abolished. Investigation of endosomal acidification by live-cell imaging of pHluorin-VAMP2 (vesicle-associated membrane protein-2), a pH-sensitive-GFP construct, revealed that the endosomal pH in normal and 30:insH ciPTECs was similar, whereas in del132-241 and R637X ciPTECs, it was significantly more alkaline, indicating defective acidification in these ciPTECs. The addition of bafilomycin-A1, a V-ATPase inhibitor, raised the pH significantly in all ciPTECs, demonstrating that the differences in acidification were not due to alterations in the V-ATPase, but instead to abnormalities of CLC-5. Thus, our studies, which have established human Dent disease ciPTECs that will facilitate studies of mechanisms in renal reabsorption, demonstrate that Dent disease-causing CLC-5 mutations have differing effects on endosomal acidification and receptor-mediated endocytosis that may not be coupled.