Leakage Of Fluorescent Dye Research Articles

Effect of diabetes on microvascular morphology and permeability of rat skeletal muscle: in vivo imaging using two-photon laser scanning microscopy.

This investigation evaluated the microvascular permeability and ultrastructure of skeletal muscle capillaries in the skeletal muscle of diabetic (DIA) rats using two-photon laser scanning microscopy (TPLSM) and transmission electron microscopy (TEM). Microvascular permeability was assessed in the tibialis anterior muscle of control (CON) and DIA (streptozocin) male Wistar rats (n = 20, 10-14 wk) by in vivo imaging using TPLSM after fluorescent dye intravenous infusion. Fluorescent dye leakage was quantified to determine microvascular permeability. The ultrastructure was imaged by TEM ex vivo to calculate the size and number of intercellular clefts between capillary endothelial cells and also intracellular vesicles. Compared with control, the volumetrically determined interstitial fluorescent dye leakage, the endothelial cell thickness, and the number of intercellular clefts per capillary perimeter were significantly higher, and the cleft width was significantly narrower in tibialis anterior (TA) of DIA (interstitial fluorescent dye leakage, 2.88 ± 1.40 vs. 10.95 ± 1.41 µm3 × min × 106; endothelial thickness, 0.28 ± 0.02 vs. 0.45 ± 0.03 µm; number of intercellular clefts per capillary perimeter, 6.3 ± 0.80 vs. 13.6 ± 1.7/100 µm; cleft width, 11.92 ± 0.95 vs. 8.40 ± 1.03 nm, CON vs. DIA, respectively, all P < 0.05). The size of intracellular vesicles in the vascular endothelium showed an increased proportion of large vesicles in the DIA group compared with the CON group (P < 0.05). Diabetes mellitus enhances the microvascular permeability of skeletal muscle microvessels due, in part, to a higher density and narrowing of the endothelial intercellular clefts, and larger intracellular vesicles.NEW & NOTEWORTHY Microvascular permeability in diabetic muscle was investigated using our original two-photon scanning laser microscopy method. Compared with controls, the leakage volume was increased in diabetic muscle, which was atrophic with smaller capillary diameter, endothelial cell thickening, and the appearance of more endothelial intercellular gaps or clefts, and large vesicles. Hyperpermeability was closely related to ultrafine structural changes of the capillary endothelial cell junctions.

EY@UiO-67-CDC-(CH3)2 as an ultra-sensitive ratiometric fluorescence sensor for visible and selective detecting of nitrite

Nitrite is widely used in production and daily life, and detecting this substance has always been crucial for human health or environment monitoring. Here, a dual-emitting dye@MOF composite, EY@UiO-67-CDC-(CH3)2, has been constructed and demonstrated that this material could detect nitrite ions with highly selective and anti-interference properties. By incorporating an organic anionic dye (Eosin Y) within a cationized EY@UiO-67-CDC-(CH3)2 framework through the post-synthetic method, the composite presents an effective combination due to electrostatic attraction. It features a dual emission effect, which belongs to the blue emission of MOF at 428 nm and yellow emission of EY at 559 nm, respectively. After adding NO2−, the fluorescence emission peaks of EY and MOF showed opposite changes due to the inner filter effect of NO2− and MOF, as well as effective excitation of the dye's fluorescence and slight dye leakage. Therefore, Idye/IMOF is used as the detection signal value instead of absolute fluorescence intensity, effectively reducing the detection limit to 0.224 μM and improving its detection accuracy. In addition, during the detection process, the fluorescence sensor showed noticeable color changes, so a smartphone-based analytical device without additional instruments was developed for the quantitative, visual analysis of nitrite by identifying the RGB values of the fluorescence sample solution, with a detection limit of 6.74 μM. The application of the sensor visualizes nitrite detection and reduces the cost of detection, providing a feasible method for on-site detection of nitrite in water samples.

Ocular features associated with mutations in ATOH7 gene overlap those with familial exudative vitreoretinopathy.

To report the ocular findings in 3 patients with a mutation in the ATOH7 gene. The clinical findings were collected from the medical records including those for magnetic resonance imaging (MRI). Genetic testing of the ATOH7 gene was performed. Three patients of two families who had poor vision since infancy were studied. The 3 patients had varying degrees of intraocular vascular proliferation associated with advanced retinal detachments as falciform retinal folds or total retinal detachments. This state is referred to as congenital retinal nonattachment. One eye of a sibling had fluorescein angiographic findings of excessive branching of the retinal vessels and fluorescent dye leakage that were consistent with those of familial exudative vitreoretinopathy (FEVR). Bilateral hypoplasia of the optic nerve was found in all three patients by MRI. Genetic analysis showed a known in-frame deletion of the ATOH7 gene in all three patients. This is the first report of a patient with a mutation in the ATOH7 gene that had typical vascular patterns of FEVR in the peripheral retina. The ocular features associated with mutations in the ATOH7 gene overlap those with FEVR at both the early and advanced stages.

Thromboxane prostanoid signaling in macrophages attenuates lymphedema and facilitates lymphangiogenesis in mice : TP signaling and lymphangiogenesis.

Accumulating evidence suggests that prostaglandin E2, an arachidonic acid (AA) metabolite, enhances lymphangiogenesis in response to inflammation. However, thromboxane A2 (TXA2), another AA metabolite, is not well known. Thus, this study aimed to determine the role of thromboxane prostanoid (TP) signaling in lymphangiogenesis in secondary lymphedema. Lymphedema was induced by the ablation of lymphatic vessels in mouse tails. Compared with wild-type mice, tail lymphedema in Tp-deficient mice was enhanced, which was associated with suppressed lymphangiogenesis as indicated by decreased lymphatic vessel area and pro-lymphangiogenesis-stimulating factors. Numerous macrophages were found in the tail tissues of Tp-deficient mice. Furthermore, the deletion of TP in macrophages increased tail edema and decreased lymphangiogenesis and pro-lymphangiogenic cytokines, which was accompanied by increased numbers of macrophages and gene expression related to a pro-inflammatory macrophage phenotype in tail tissues. In vivo microscopic studies revealed fluorescent dye leakage in the lymphatic vessels in the wounded tissues. The results suggest that TP signaling in macrophages promotes lymphangiogenesis and prevents tail lymphedema. TP signaling may be a therapeutic target for improving lymphedema-related symptoms by enhancing lymphangiogenesis.

Interfacial effect on the ability of peptide-modified gold nanoclusters to inhibit hIAPP fibrillation and cytotoxicity

Deposit of amyloid peptides in the cells is related to various amyloidosis diseases. A variety of nanomaterials have been developed to resist amyloid deposit. Most of the research on the inhibition of nanomaterials against amyloid aggregation are undertaken in solution, while the membranes that may mediate fibrillar aggregation and affect interaction of inhibitors with amyloid peptides in biotic environment are little taken into account. In this study, we synthesized three kinds of gold nanoclusters modified with cysteine (C@AuNCs), glutathione (GSH@AuNCs) and a peptide derived from the core region of hIAPP fibrillation (C-HL-8P@AuNCs), and investigated their inhibitory activities against hIAPP fibrillation in the absence and presence of lipid vesicles (POPC/POPG 4:1 LUVs) by the experiments of ThT fluorescence kinetics, AFM and CD. We also explored the inhibitions of hIAPP-induced membrane damage and cytotoxicity by peptide@AuNCs using fluorescent dye leakage and cell viability assays. Our study revealed that the inhibitory efficiency of these peptide@AuNCs against hIAPP fibrillation follows C-HL-8P@AuNCs≅GSH@AuNCs>C@AuNCs in lipid-free solution and C-HL-8P@AuNCs≫GSH@AuNCs>C@AuNCs in lipid membrane environment. Compared with the results obtained in lipid-free solution, the inhibitions of hIAPP fibrillation observed in lipid membrane environment were more associated with the inhibitions of hIAPP-induced damages of lipid vesicles and INS-1 cells (C-HL-8P@AuNCs≫GSH@AuNCs>C@AuNCs). An additional hydrophobic interaction with the homologous core region of hIAPP, which is only provided by C-HL-8P@AuNCs and largely suppressed in lipid-free solution, enhanced in the membrane environment and therefore made C-HL-8P@AuNCs much more powerful than GSH@AuNCs and C@AuNCs in the inhibitions of hIAPP fibrillation and cytotoxicity.

APP/PS1 Gene-Environmental Cadmium Interaction Aggravates the Progression of Alzheimer's Disease in Mice via the Blood-Brain Barrier, Amyloid-β, and Inflammation.

There is limited information about gene-environment interaction on the occurrence and the progression of Alzheimer's disease. To explore the effect of environmental low-dose cadmium (Cd) exposure on the progress of Alzheimer's disease and the underlining mechanism. We administered 1 mg/L, 10 mg/L cadmium chloride (treated groups), and water (control group) to C57BL/6J and APP/PS1 mice through drinking water, from one week before mating, until the offspring were sacrificed at 6 months of age. The behaviors, Cd level, blood-brain barrier (BBB) leakage, Aβ1-42 deposition, and inflammation expression were evaluated in these mice. Mice of both genotypes had similar blood Cd levels after exposure to the same dose of Cd. The toxic effects of Cd on the two genotypes differed little in terms of neuronal histomorphology and BBB permeability. Cd caused a series of pathological morphological changes in the mouse brains and more fluorescent dye leakage at higher doses. Furthermore, the APP/PS1 mice had more severe damage than the C57BL/6J mice, based on the following five criteria. They were increasing anxiety-like behavior and chaos movement, spatial reference memory damage, Aβ plaque deposition in mouse brains, increasing microglia expression in the brain, and IL-6 higher expression in the cortex and in the serum. Low-dose Cd exposure for 6 months increases Aβ plaque deposition and BBB permeability, exacerbates inflammatory responses, and activates microglia, in APP/PS1 mice. APP/PS1 gene-environmental Cd interaction aggravates the progression of Alzheimer's disease in mice.

De novo design of peptides that form transmembrane barrel pores killing antibiotic resistant bacteria.

De novo design of peptides that self-assemble into transmembrane barrel-like nanopores is challenging due to the complexity of several competing interactions involving peptides, lipids, water, and ions. We have develop a computational approach for the de novo design of α-helical peptides that self-assemble into stable transmembrane barrel pores with a central nano-sized functional channel. We formulate the previously missing design guidelines and report 52 sequence patterns that can be tuned for specific applications using the identified role of each residue. Atomic force microscopy, fluorescent dye leakage, and cryo-EM experiments confirm that the designed peptides form leaky membrane nanopores in vitro. Customized designed peptides act as antimicrobial agents able to kill even antibiotic-resistant ESKAPE bacteria at micromolar concentrations, while exhibiting low toxicity to human cells. The peptides and their assembled nanopore structures can be similarly fine-tuned for other medical and biotechnological applications.

New insights on the mechanism of polyethylenimine transfection and their implications on gene therapy and DNA vaccines

Polyethylenimine (PEI) has been demonstrated as an efficient DNA delivery vehicle both in vitro and in vivo. There is a consensus that PEI-DNA complexes enter the cells by endocytosis and escape from endosomes by the so-called “proton sponge” effect. However, little is known on how and where the polyplexes are de-complexed for DNA transcription and replication to occur inside the cell nucleus. To better understand this issue, we (i) tracked the cell internalization of PEI upon transfection to human epithelial cells and (ii) studied the interaction of PEI with phospholipidic layers mimicking nuclear membranes. Both the biological and physicochemical experiments provided evidence of a strong binding affinity between PEI and the lipidic bilayer. Firstly, confocal microscopy revealed that PEI alone could not penetrate the cell nucleus; instead, it arranged throughout the cytoplasm and formed a sort of aureole surrounding the nuclei periphery. Secondly, surface tension measurements, fluorescence dye leakage assays, and differential scanning calorimetry demonstrated that a combination of hydrophobic and electrostatic interactions between PEI and the phospholipidic monolayers/bilayers led to the formation of stable defects along the model membranes, allowing the intercalation of PEI through the monolayer/bilayer structure. Results are also supported by molecular dynamics simulation of the pore formation in PEI-lipidic bilayers. As discussed throughout the text, these results might shed light on a the mechanism in which the interaction between PEI and the nucleus membrane might play an active role on the DNA release: on the one hand, the PEI-membrane interaction is anticipated to facilitate the DNA disassembly from the polyplex by establishing a competition with DNA for the PEI binding and on the other hand, the forming defects are expected to serve as channels for the entrance of de-complexed DNA into the cell nucleus. A better understanding of the mechanism of transfection of cationic polymers opens paths to development of more efficiency vectors to improve gene therapy treatment and the new generation of DNA vaccines.

An Aggregation-Induced Emission Optical Highlighter for the Studies of Endoplasmic Reticulum-Lipid Droplet Content Dynamics

An Aggregation-Induced Emission Optical Highlighter for the Studies of Endoplasmic Reticulum-Lipid Droplet Content Dynamics

Vascular permeability of skeletal muscle microvessels in rat arterial ligation model: in vivo analysis using two-photon laser scanning microscopy.

Peripheral artery disease (PAD) in the lower limb compromises oxygen supply due to arterial occlusion. Ischemic skeletal muscle is accompanied by capillary structural deformation. Therefore, using novel microscopy techniques, we tested the hypothesis that endothelial cell swelling temporally and quantitatively corresponds to enhanced microvascular permeability. Hindlimb ischemia was created in male Wistar rat's by iliac artery ligation (AL). The tibialis anterior (TA) muscle microcirculation was imaged using intravenously infused rhodamine B isothiocyanate dextran fluorescent dye via two-photon laser scanning microscopy (TPLSM) and dye extravasation at 3 and 7 days post-AL quantified to assess microvascular permeability. The TA microvascular endothelial ultrastructure was analyzed by transmission electron microscopy (TEM). Compared with control (0.40 ± 0.15 μm3 × 106), using TPLSM, the volumetrically determined interstitial leakage of fluorescent dye measured at 3 (3.0 ± 0.40 μm3 × 106) and 7 (2.5 ± 0.8 μm3 × 106) days was increased (both P < 0.05). Capillary wall thickness was also elevated at 3 (0.21 ± 0.06 μm) and 7 (0.21 ± 0.08 μm) days versus control (0.11 ± 0.03 μm, both P < 0.05). Capillary endothelial cell swelling was temporally and quantitatively associated with elevated vascular permeability in the AL model of PAD but these changes occurred in the absence of elevations in protein levels of vascular endothelial growth factor (VEGF) its receptor (VEGFR2 which decreased by AL-7 day) or matrix metalloproteinase. The temporal coherence of endothelial cell swelling and increased vascular permeability supports a common upstream mediator. TPLSM, in combination with TEM, provides a sensitive and spatially discrete technique to assess the mechanistic bases for, and efficacy of, therapeutic countermeasures to the pernicious sequelae of compromised peripheral arterial function.

New Insights on the Mechanism of Polyethylenimine Transfection and Their Implications on Gene Therapy and DNA Vaccines

Polyethylenimine (PEI) has been demonstrated as an efficient DNA delivery vehicle both in vitro and in vivo. There is a consensus that PEI-DNA complexes enter the cells by endocytosis and escape from endosomes by the so-called “proton sponge” effect. However, little is known on how and where the polyplexes are de-complexed for DNA transcription and replication to occur inside the cell nucleus. To better understand this issue, we (i) tracked the cell internalization of PEI upon transfection to human epithelial cells and (ii) studied the interaction of PEI with phospholipidic layers mimicking nuclear membranes. Both the biological and physicochemical experiments provided evidence of a strong binding affinity between PEI and the lipidic bilayer. Firstly, confocal microscopy revealed that PEI alone could not penetrate the cell nucleus; instead, it arranged throughout the cytoplasm and formed a sort of aureole surrounding the nuclei periphery. Secondly, surface tension measurements, fluorescence dye leakage assays, and differential scanning calorimetry demonstrated that a combination of hydrophobic and electrostatic interactions between PEI and the phospholipidic monolayers/bilayers led to the formation of stable defects along the model membranes, allowing the intercalation of PEI through the monolayer/bilayer structure. Results are also supported by molecular dynamics simulation of the pore formation in PEI-lipidic bilayers. As discussed throughout the text, these results might shed light on a the mechanism in which the interaction between PEI and the nucleus membrane might play an active role on the DNA release: on the one hand, the PEI-membrane interaction is anticipated to facilitate the DNA disassembly from the polyplex by establishing a competition with DNA for the PEI binding and on the other hand, the forming defects are expected to serve as channels for the entrance of de-complexed DNA into the cell nucleus. A better understanding of the mechanism of transfection of cationic polymers opens paths to development of more efficiency vectors to improve gene therapy treatment and the new generation of DNA vaccines that has been used during the SARS-CoV-2 pandemic.

Interaction of graphene oxide with artificial cell membranes: Role of anionic phospholipid and cholesterol in nanoparticle attachment and membrane disruption

A mechanistic understanding of the interaction of graphene oxide (GO) with cell membranes is critical for predicting the biological effects of GO following accidental exposure and biomedical applications. We herein used a quartz crystal microbalance with dissipation monitoring (QCM-D) to probe the interaction of GO with model cell membranes modified with anionic lipids or cholesterol under biologically relevant conditions. The attachment efficiency of GO on supported lipid bilayers (SLBs) decreased with increasing anionic lipid content and was unchanged with varying cholesterol content. In addition, the incorporation of anionic lipids to the SLBs rendered the attachment of GO partially reversible upon a decrease in solution ionic strength. These results demonstrate the critical role of lipid bilayer surface charge in controlling GO attachment and release. We also employed the fluorescent dye leakage technique to quantify the role of anionic lipids and cholesterol in vesicle disruption caused by GO. Notably, we observed a linear correlation between the amount of dye leakage from the vesicles and the attachment efficiencies of GO on the SLBs, confirming that membrane disruption is preceded by GO attachment. This study highlights the non-negligible role of lipid bilayer composition in controlling the physicochemical interactions between cell membranes and GO.

Membrane properties of amacrocyclic tetraether bisphosphatidylcholine lipid: Effect of a single membrane-spanning polymethylene cross-linkage between two head groups of ditetradecylphosphatidylcholine membrane

The plasma membranes of archaea are abundant in macrocyclic tetraether lipids that contain a single or double long transmembrane hydrocarbon chains connecting the two glycerol backbones at both ends. In this study, a novel amacrocyclic bisphosphatidylcholine lipid bearing a single membrane-spanning octacosamethylene chain, 1,1’-O-octacosamethylene-2,2′-di-O-tetradecyl-bis-(sn-glycero)-3,3′-diphosphocholine (AC-(di-O-C14PC)2), was synthesized to elucidate effects of the interlayer cross-linkage on membrane properties based on comparison with its corresponding diether phosphatidylcholine, 1,2-di-O-tetradecyl-sn-glycero-3-phosphocholine (DTPC), that forms bilayer membrane. Several physicochemical techniques demonstrated that while AC-(di-O-C14PC)2 monolayer, which adopts a particularly high-ordered structure in the gel phase, shows remarkably high thermotropic transition temperature compared to DTPC bilayer, the fluidity of both phospholipids above the transition temperature is comparable. Nonetheless, the fluorescent dye leakage from inside the AC-(di-O-C14PC)2 vesicles in the fluid phase is highly suppressed. The origin of the membrane properties characteristic of AC-(di-O-C14PC)2 monolayer is discussed in terms of the single long transmembrane hydrophobic linkage and the diffusional motion of the lipid molecules.

Photolipid Bilayer Permeability is Controlled by Transient Pore Formation.

Controlling the release or uptake of (bio-) molecules and drugs from liposomes is critically important for a range of applications in bioengineering, synthetic biology, and drug delivery. In this paper, we report how the reversible photoswitching of synthetic lipid bilayer membranes made from azobenzene-containing phosphatidylcholine (azo-PC) molecules (photolipids) leads to increased membrane permeability. We show that cell-sized, giant unilamellar vesicles (GUVs) prepared from photolipids display leakage of fluorescent dyes after irradiation with UV-A and visible light. Langmuir-Blodgett and patch-clamp measurements show that the permeability is the result of transient pore formation. By comparing the trans-to-cis and cis-to-trans isomerization process, we find that this pore formation is the result of area fluctuations and a change of the area cross-section between both photolipid isomers.

Penetration of phospholipid membranes by poly-l-lysine depends on cholesterol and phospholipid composition

Clusters of positively-charged basic amino acid residues, particularly lysine, are known to promote the interaction of many peripheral membrane proteins with the cytoplasmic surface of the plasma membrane via electrostatic interactions. In this work, cholesterol's effects on the interaction between lysine residues and membranes have been studied. Using poly-l-lysine (PLL) and vesicles as models to mimic the interaction between lysine-rich protein domains and the plasma membrane, light scattering measurements indicated cholesterol enhanced the electrostatic interaction through indirectly affecting the negatively charged phospholipid dioleoylphosphatidylserine, DOPS. Addition of PLL to lipid vesicles containing DOPS showed an initial increase in static light scattering (SLS), attributed to binding of PLL to the vesicle surface, followed by a slower continuously declining SLS signal, which, from comparison with fluorescent dye leakage studies could be attributed to vesicle lysis. Although electrostatic interactions between PLL and the membrane were not necessary for penetration to occur, cholesterol promoted membrane disruption of negatively charged vesicles, possibly by increasing the electrostatic interactions between PLL and the membrane. In contrast, cholesterol lowered the susceptibility of uncharged vesicles (formed using dioleoylphosphatidylcholine, DOPC) to PLL penetration. This can be explained by the absence of electrostatic interactions and cholesterol's known ability to increase membrane thickness and mechanical strength. Thus, the ability of cationic peptides to penetrate membranes including cholesterol is likely to depend on the membrane's PS:PC ratio.

Nucleation and growth of pores in 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) / cholesterol bilayer by antimicrobial peptides melittin, its mutants and cecropin P1.

Antimicrobial peptides are one of the most promising alternatives to antibiotics for targeting pathogens without developing resistance. In this study, pore formation in 1,2-Dimyristoyl-snglycero-3-phosphocholine (DMPC) / cholesterol liposome induced by native melittin, its two mutant variants (G1I and I17 K), and cecropin P1 was investigated by monitoring the dynamics of fluorescence dye leakage. A critical peptide concentration was required for dye leakage with the rate of leakage being dependent on peptide concentration above a critical value. A lag time was required for dye leakage for low peptide concentrations that are above the critical value, which decreased at higher peptide concentrations eventually approaching zero. Lag time was found to be in the order I17 K mutant with lower hydrophobicity and higher net charge > G1I with higher hydrophobicity > melittin > cecropin P1. Cecropin P1 exhibited the highest rate of dye leakage followed by melittin, G1I, and I17 K. Size distribution and transmission electron microscopy (TEM) of liposomes exposed to peptides of different concentrations indicated pore formation with accompanied stretching of liposomes at low peptide concentrations for both melittin and cecropin P1. At much higher concentrations, however, size distribution indicated three peaks for both peptides. In both cases, TEM images show that the middle and small peaks are shown to be due to stretched liposome and broken stretched liposome respectively. For melittin, the large peak is due to peptide aggregates as well as aggregates of liposome. For cecropin P1, however, the large peak indicates cecropin P1 aggregates with solubilized lipids thus suggesting carpet mechanism.

Understanding the antimicrobial activity of water soluble γ-cyclodextrin/alamethicin complex.

In our previous investigation (Zhang et. al., J. Functional Foods 40 (2018) 700-706), we have proposed a method of complexation of alamethicin (ALM) with γ-cyclodextrin (γ-CD) to increase the solubility and demonstrated an enhancement in its antimicrobial activity against Listeria monocytogenes. In this study, transmission electron microscopy of L. monocytogenes treated with γ-CD/ALM complex indicated cell lysis due to pore formation. This was further corroborated by fluorescent dye leakage from DMPC/cholesterol liposomes when exposed to γ-CD/ALM complex for molar ratios of 1, 5 and 10. The extent of dye leakage increased with ALM-lipid ratio in the range of 0.00015 to 0.16. Dye leakage of γ-CD/ALM complex was found to be the highest for molar ratio of 5, consistent with our earlier results of antimicrobial activity of the complex against L. monocytogenes. All atom molecular dynamics (MD) simulation showed that γ-CD/ALM complex can effectively bind to the 3:1 POPE/POPG bilayer, a mimic of bacterial cell membrane. In addition, circular dichroism spectrum indicated that ALM in the complex has a helical conformation in solution as well as in the presence of liposome. Transmembrane MD simulation of six γ-CD/ALM complex aggregates in α helical conformation showed water channel with barrel stave like pore structure.

Microvascular permeability of skeletal muscle after eccentric contraction-induced muscle injury: in vivo imaging using two-photon laser scanning microscopy.

Via modulation of endothelial integrity and vascular permeability in response to damage, skeletal muscle microvessels play a crucial permissive role in tissue leukocyte invasion. However, direct visual evidence of altered microvascular permeability of skeletal muscle has not been technically feasible, impairing mechanistic understanding of these responses. Two-photon laser scanning microscopy (TPLSM) allows three-dimensional in vivo imaging of skeletal muscle microcirculation. We hypothesized that the regulation of microvascular permeability in vivo is temporally related to acute inflammatory and regenerative processes following muscle injury. To test our hypothesis, tibialis anterior muscles of anesthetized male Wistar rats were subjected to eccentric contractions (ECCs) via electrical stimulation. The skeletal muscle microcirculation was imaged by an intravenously infused fluorescent dye (rhodamine B isothiocyanate-dextran) to assess microvascular permeability via TPLSM 1, 3, and 7 days after ECC. Immunohistochemistry on serial muscle sections was performed to determine the proportion of VEGF-A-positive muscle fibers in the damaged muscle. Compared with control rats, the volumetrically determined interstitial leakage of fluorescent dye (5.1 ± 1.4, 5.3 ± 1.2 vs. 0.51 ± 0.14 μm3 × 106; P < 0.05, days 1 and 3, respectively, vs. control) and percentage of VEGF-A-positive fibers in the damaged muscle (10 ± 0.4%, 22 ± 1.1% vs. 0%; days 1 and 3, respectively, vs. control) were significantly higher on days 1 and 3 after ECC. The interstitial leakage volume returned to control by day 7. These results suggest that microvascular hyperpermeability assessed by in vivo TPLSM imaging is associated with ECC-induced muscle damage and increased VEGF expression. NEW & NOTEWORTHY This investigation employed a novel in vivo imaging technique for skeletal muscle microcirculation using two-photon laser scanning microscopy that enabled microvascular permeability to be assessed by four-dimensional image analysis. By combining in vivo imaging and histological analysis, we found the temporal profile of microvascular hyperpermeability to be related to that of eccentric contraction-induced skeletal muscle injury and pronounced novel myocyte VEGF expression.

Skeletal Muscle Microvascular Permeability After Eccentric Contraction-Induced Muscle Injury

Within injured skeletal muscle the capillary bed plays a crucial role in leukocyte invasion through modulations of the endothelial integrity, associated with increased permeability. However, direct observation of altered microvascular permeability and compromised capillary integrity has not been technically feasible. Two-photon laser scanning microscopy (TPLSM) allows three-dimensional in vivo imaging which, given the depth of penetration and high resolution of TPLSM, will facilitate measurement of microvascular leakage. PURPOSE: We hypothesized that the regulation of capillary permeability in vivo, as assessed by real-time TPLSM, is temporally related to acute inflammatory and regenerative processes following muscle injury. METHODS: Tibialis anterior muscles of anesthetized male Wistar rats (n=57) were subjected to eccentric contractions (ECC) via electrical stimulation. The skeletal muscle microcirculation was imaged by an intravenously infused fluorescent dye (rhodamine b isothiocyanate dextran, molecular weight 70,000 Daltons) to assess microvascular permeability via TPLSM 1, 3 and 7 days after ECC. Immunohistochemistry on serial muscle sections was performed to determine the percentage of VEGF-A positive muscle fibers in the damaged muscle. RESULTS: Compared with non-ECC control, the volumetrically-determined interstitial leakage of fluorescent dye had increased significantly on days 1 and 3 post-ECC (5.1±1.4, 5.3±1.2 vs. 0.51±0.14 μm3x106, P<0.05 respectively days 1 and 3 vs. control). However, by post-ECC day 7 interstitial leakage had returned to control values. Damaged muscle fibers were evident on days 1 and 3 (% damaged muscle fiber: 11.7±4.7, 48.4±12.4% vs. 0% P<0.05 respectively days 1 and 3 vs. control). Percentage of VEGF-A positive muscle fiber in damaged muscle fiber was significantly higher on days 1 and 3 compared to control (24.9±9.8, 39.3±16.7 vs. 0%, P<0.05 respectively days 1 and 3 vs. control). Regenerated skeletal muscle fibers were found only at 7 days post- ECC. CONCLUSION:In vivo TPLSM imaging represents a powerful investigative technique for skeletal muscle microcirculatory research. Microvascular hyperpermeability is associated with ECC-induced muscle damage and increased VEGF expression.

Control of Cell-Selective Activity of Membrane-Active Polyleucine-Based Peptides using Database-Guided High-throughput Screening

We have developed a new 14-residue antimicrobial peptide template sequence containing four residues: L, D, K, and A. This template, called LDKA, which was designed using atomic detail structural information derived from unbiased long-timescale equilibrium molecular dynamics folding, partitioning, and pore assembly simulations, shows low micro-molar antibacterial activity against several gram-positive and gram-negative bacteria. Furthermore, LDKA was shown to form pores in microbial and eukaryotic membrane models using fluorescent dye leakage assays. A library of 2,916 peptides was designed using this template sequence and high-throughput screened using an orthogonal vesicle leakage assay. Dyes of different sizes were embedded into vesicles with varying lipid composition to simultaneously screen for both pore size and affinity to charged and neutral membranes. From this screen, 9 different LDKA variants were selected, sequenced, synthesized, and further characterized biophysically. Remarkably, even though the selected sequences displayed only minor mutational changes, each of these peptides has unique functional properties, forming either small or large pores and being selective for either neutral or anionic charged bilayers. Further screening of these peptides for bacterial and haemolytic activity revealed good correlation between charged membrane model leakage and antibacterial activity, as well as neutral membrane leakage and haemolytic activity. Antimicrobial activity is different for each peptide and depends on the bacterial species. Analysis of the sequences reveals no clear motifs or explanation for these strong preferences, but suggests that hydrophobic moment and sequence arrangement are more important for membrane selectivity than net charge. The results reveal that simple sequence-function relationships for peptides that can form a large repertoire of functional structures may remain elusive and that it may be the peptide sequences propensity to aggregate and assemble in a given environment that holds the key to functional prediction.