Tetramethylrhodamine Isothiocyanate Research Articles

Intermolecular FRET Pairs as An Approach to Visualize Specific Enzyme Activity in Model Biomembranes and Living Cells

Herein, we propose an analytical approach based on intermolecular fluorescent resonant energy transfer (FRET) pairs for the visualization of specific enzyme activity in model biomembranes and in living cells. Cell visualizations with fluorescent confocal laser microscopy usually rely on fluorescent probes, such as Fluorescein isothiocyanate (FITC), Alexa488, Tetramethylrhodamine isothiocyanate (TRITC) and many others. However, for more specific tasks, such as the detection of certain enzymatic activity inside the living cell, the toolbox is quite limited. In the case of enzyme-hydrolases for example, the choice is limited to organic molecules comprising a fluorescent dye (typically, 4-methylumbelliferone (MUmb) or 7-amino-4-methylcoumarin (AMC) derivatives) and a fluorescence quencher, bound via an enzyme-sensitive linker—so that when the linker is degraded, the fluorescent signal increases. Unfortunately, both MUmb and AMC are quenched and have a relatively low quantum yield in cells, and their excitation and emission ranges overlap with that of intracellular fluorophores, often producing a strong background noise. R6G, on the other hand, has excellent quantum yield apart from intracellular fluorophores, but there are no efficient quenchers that could be chemically linked to R6G. Herein, we show that R6G is able to form intermolecular FRET pairs with MUmb or AMC, with the latter serving as fluorescence donors. This yields a combination of R6G’s excellent fluorescence properties with a possibility to use an enzyme-sensitive linker in MUTMAC or AMC derivatives. This phenomenon was initially discovered in a model system, reversed micelles, where the donor, the acceptor, and the enzyme are forced to be in close proximity to each other, so that proximity could serve as an explanation for the intermolecular FRET effect. Surprisingly enough, the phenomenon has been reproduced in living cells. Moreover, we were able to create working intermolecular donor–acceptor FRET pairs for several different enzymes, including chymotrypsin, phosphatase, and asparaginase. This appears counterintuitive, as besides the overlap of the emission spectra of the donor and the absorption spectra of the acceptor, there are other criteria for the FRET effect, including the convergence of two fluorophores at a distance of about 1–10 nm, and the orientation of their dipoles at a certain angle, which is difficult to imagine in a bulk system like a living cell. We hypothesize that FRET-enabling donor–acceptor interaction may be taking place at the inner surface of the lipid bilayer, to which both donor and acceptor molecules would likely have an affinity. This hypothesis would require a more detailed investigation. Therefore, we have shown that the method suggested has good potential in the visualization of enzyme functioning inside living cells, which is often a challenging task. Shifting of the fluorescence signal to the long-wavelength region would increase the signal selectivity, making it easily distinguishable from autofluorescence.

A comparative analysis of gastrointestinal tract barrier function and immune markers in gilt vs. sow progeny at birth and weaning.

Progeny born to primiparous sows (gilt progeny; GP) have lower birth, weaning and slaughter weights than sow progeny (SP). GP also have reduced gastrointestinal tract (GIT) development, as evidenced by lower organ weights. Therefore, the aim of this experiment was to quantify changes in GIT barrier function that occur in birth and weaning, representing two major challenges to the young piglet. The effects of parity (GP vs. SP) in GIT barrier integrity function were quantified at four timepoints: birth (~0h), 24h after birth (24h), 1-d preweaning (PrW), and 1-d postweaning (PoW) in commercially reared piglets. Due to inherent differences between newborn and weanling pigs, the results were analyzed in two cohorts, birth (0 vs. 24h, n = 31) and weaning (PrW vs. PoW, n = 40). Samples of the stomach, jejunum, ileum, and colon were excised after euthanasia and barrier integrity was quantified by measuring transepithelial resistance (TER), macromolecular permeability, the abundance of inflammatory proteins (IL-8, IL-1β, and TNF-α) and tight junction proteins (claudin-2 and -3). Papp was characterized using a dual tracer approach comprising 4 KDa fluorescein isothiocyanate (FD4) and 150kDa tetramethyl rhodamine isothiocyanate (T150)-labeled dextrans. Characteristic effects of the initiation of feeding and weaning were observed on the GIT with the initiation of feeding, such as increasing TER and reducing Papp at 24h, consistent with mucosal growth (P = 0.058) This was accompanied by increased cytokine abundance as evidenced by elevations in TNF-α and IL-1β. However, GP had increased IL-8 abundance (P = 0.011 and 0.063 for jejunum and ileum respectively) at birth than 24h overall. In the weaning cohort, jejunal and ileal permeability to FD4 was higher in GP (P = 0.05 and 0.022, respectively) while only higher ileal T150 was observed in GP (P = 0.032). Ileal claudin-2 abundance tended to be higher in SP overall (P = 0.063), but GP ileal claudin-2 expression was upregulated weaning while no change was observed in SP (P = 0.043). Finally, other than a higher jejunal TNF-α abundance observed in SP (P = 0.016), no other effect of parity was observed on inflammatory markers in the weaning cohort. The results from this study indicate that the GIT of GP have poorer adaptation to early life events, with the response to weaning, being more challenging which is likely to contribute to poorer postweaning growth.

Low sanitary housing conditions increase protein fermentation in piglets but do not aggravate the effects of protein fermentation on intestinal health

Protein fermentation has been identified as a risk factor for the occurrence of post-weaning diarrhea. Associations between low protein digestion – provoking protein fermentation - and low sanitary housing conditions suggest that protein fermentation, low sanitary conditions, and post-weaning diarrhea are interlinked.To this end, an experiment (2×2 factorial treatment arrangement) was performed with 160 weaned female piglets (TN70 х Tempo) divided over 5 batches (4 piglets per pen, 2 pens/treatment/batch). The piglets were kept under high (HSC) or low (LSC) sanitary conditions and were fed a diet low (LiP) or high (HiP) in indigestible protein for 14 days. Fecal consistency was scored daily per piglet and after one week, digesta samples were collected to analyze protein digestibility, colonic flow of protein-derived metabolites, and digesta mean retention time in 2 piglets per pen. Furthermore, we measured intestinal permeability for Fluorescein isothiocyanate-dextran (FITC, 4 kDa) and Tetramethylrhodamine isothiocyanate–dextran (TRITC, 40 kDa), and jejunal absorption capacity for lysine, methionine, and glucose, using the everted gut sac technique.Piglets kept under LSC had 9% units lower apparent total tract protein digestibility than piglets kept under HSC (P < 0.001), mainly explained by 7% units lower apparent ileal protein digestibility in LSC piglets (P < 0.001). Since no relevant correlations were observed between digesta mean retention time and ileal N digestibility and jejunal absorption capacity did not differ between treatment groups, these factors were excluded as underlying causes. The colonic flow of protein-derived metabolites was higher for LSC piglets than for HSC piglets (NH3: +31%, BCFA: +40%, P < 0.001), and higher for HiP piglets than for LiP piglets (NH3: +76%, BCFA: +42% for LSC, +25% for HSC, P < 0.001, SC х Diet P < 0.05). Colonic N disappearance was 5.5% units higher for HiP piglets than for LiP piglets (P < 0.01), but was not affected by SC. Fecal consistency scores were reduced for both HiP (−1.5, P < 0.001) and LSC (−0.5, P < 0.001) piglets compared with LiP and HSC, but no SC х diet interaction was observed. Jejunal permeability was slightly increased for HiP piglets (P < 0.05). No negative effects of feeding HiP on colonic permeability were observed.Low sanitary conditions decrease both ileal and fecal protein digestion to a similar extent and stimulate colonic protein fermentation. It is unlikely that digesta transport or jejunal absorption rates are causal to this effect. There are no indications that housing under low sanitary conditions aggravates the effects of protein fermentation on intestinal health.

Enhanced Uptake and Phototoxicity of C60@albumin Hybrids by Folate Bioconjugation.

Fullerenes are considered excellent photosensitizers, being highly suitable for photodynamic therapy (PDT). A lack of water solubility and low biocompatibility are, in many instances, still hampering the full exploitation of their potential in nanomedicine. Here, we used human serum albumin (HSA) to disperse fullerenes by binding up to five fullerene cages inside the hydrophobic cavities. Albumin was bioconjugated with folic acid to specifically address the folate receptors that are usually overexpressed in several solid tumors. Concurrently, tetramethylrhodamine isothiocyanate, TRITC, a tag for imaging, was conjugated to C60@HSA in order to build an effective phototheranostic platform. The in vitro experiments demonstrated that: (i) HSA disperses C60 molecules in a physiological environment, (ii) HSA, upon C60 binding, maintains its biological identity and biocompatibility, (iii) the C60@HSA complex shows a significant visible-light-induced production of reactive oxygen species, and (iv) folate bioconjugation improves both the internalization and the PDT-induced phototoxicity of the C60@HSA complex in HeLa cells.

LncRNA SNHG15 as a ceRNA modulates Osteoclast Differentiation, Proliferation, and Metastasis by Sponging miR-381-3p/NEK2 Axis.

Background A growing number of studies have shown that long noncoding RNAs play an important role in osteoclast differentiation. However, there are few studies on the roles of lncRNA small nucleolar RNA host gene 15 (SNHG15) in osteoclast differentiation. Methods The expressions of SNHG15, miR-381-3p, and never in mitosis-related kinase 2 (NEK2) mRNA were detected by real-time quantitative polymerase chain reaction (RT-qPCR); Western blot detected NEK2 and osteoclast markers (Cathepsin K, CTSK), matrix metalloproteinase 9 (MMP9), nuclear factor of activated T cell 2 (NFAT2), and tartrate-resistant acid phosphatase (TRAP) protein levels; cell proliferation was detected by Cell Counting Kit-8 (CCK-8), and the formation of osteoclasts was observed by TRAP staining; the F-actin skeleton was stained with tetramethylrhodamine isothiocyanate (TRITC) phalloidin; cell migration rate was detected by Transwell; dual-luciferase reporter gene assay and RNA-binding protein immunoprecipitation (RIP) assay verified the targeting relationship between miR-381-3p, SNHG15, and NEK2. Results The expression of SNHG15 was increased in THP-1 cells stimulated by macrophage colony-stimulating factor (M-CSF)/receptor activator of nuclear factor-kappa B ligand (RANKL). Overexpression of SNHG15 significantly promoted the proliferation, migration, osteoclast differentiation, and expression of osteoclast markers CTSK, MMP9, NFAT2, and TRAP of THP-1 cells induced by M-CSF/RANKL. Knockdown of SNHG15 reversed this effect. Overexpression of SNHG15 downregulated the inhibitory effect of overexpression of miR-381-3p on the proliferation, migration, and differentiation of THP-1 cells induced by M-CSF/RANKL. Knockdown of miR-381-3p reversed the inhibitory effect of knockdown of NEK2 on the proliferation, migration, and differentiation of THP-1 cells induced by M-CSF/RANKL. Conclusion SNHG15 acted as a ceRNA promoted the proliferation, migration, and differentiation of THP-1 cells induced by M-CSF/RANKL through sponging miR-381-3p to promote the expression of NEK2.

Haptenation of Macrophage Migration Inhibitory Factor: A Potential Biomarker for Contact Hypersensitivity.

The immunological response in contact hypersensitivity is incited by small electrophilic compounds, known as haptens, that react with endogenous proteins after skin absorption. However, the identity of hapten-modified proteins seen as immunogenic remains as yet largely unknown. In a recent study, we have for the first time identified a hapten-modified protein in the local lymph nodes of mice treated topically with the model hapten tetramethylrhodamine isothiocyanate (TRITC). The TRITC modification was located on the N-terminal proline of the protein macrophage migration inhibitory factor (MIF). The focus of the current study was to investigate the presence of the same hapten-protein conjugate in blood samples from mice treated topically with TRITC. Furthermore, TRITC modifications of the two major blood proteins, namely hemoglobin (Hb) and albumin (Alb), as well as TRITC modifications of MIF other than the N-terminal proline, were examined. Following incubation with different molar ratios of TRITC, a proteomic approach was applied to characterize conjugate formation of the three aforementioned proteins, using high resolution mass spectrometry (HRMS). The targeted screening of the TRITC-treated mice blood and lymph node samples for these sites led to the identification of only the same TRITC-MIF conjugate previously detected in the lymph nodes. No Hb and Alb conjugates were detected. Quantification of both the TRITC-modified and unmodified N-terminal peptide of MIF in blood and lymph node samples gave interesting insights of MIF’s role in murine contact hypersensitivity. Incubation of MIF with four different haptens encompassing different reactivity mechanisms and potencies, showed adduct formation at different amino acid residues, suggesting that MIF can be the preferred target for a wide variety of haptens. The present study provides essential progress toward understanding of hapten-protein conjugate formation in contact hypersensitivity and identifies hapten-modified MIF as a potential biomarker for this condition. Further investigation of MIF as a target protein can be a next step to determine if MIF is a biomarker that can be used to develop better diagnostic tools and targeted therapeutics for individuals with allergic contact dermatitis.

HopE and HopD Porin-Mediated Drug Influx Contributes to Intrinsic Antimicrobial Susceptibility and Inhibits Streptomycin Resistance Acquisition by Natural Transformation in Helicobacter pylori.

ABSTRACTHelicobacter pylori is a human pathogen competent for natural transformation. Intrinsic and acquired antibiotic resistance contribute to the survival and multiplication of H. pylori under antibiotics. While drug-resistance dissemination by natural transformation (NT)-mediated horizontal gene transfer remains poorly understood in H. pylori. The purpose of the study was to investigate the role of H. pylori porins (HopA, HopB, HopC, HopD, and HopE) in the intrinsic antibiotic resistance and to preliminarily reveal the potential effect of HopE and HopD porins in streptomycin resistance acquisition after NT in the presence of antibiotics. Using traditional antibiotic susceptibility tests and growth curve analysis, we found the MIC values of metronidazole, clarithromycin, levofloxacin, tetracycline, rifampin, and streptomycin in mutants lacking HopE and/or HopD were significantly elevated compare to those in wild-type strain. The quantitative analysis of the tetramethyl rhodamine isothiocyanate (TRITC)-labeled streptomycin accumulation at the single-cell level showed reduced streptomycin intracellular fluorescence in ΔhopE and ΔhopD mutant cells. Furthermore, in the presence of translation-inhibiting antibiotic streptomycin, the resistance acquisition frequency was decreased in the wild-type strain, which could be reversed by mutants lacking HopE and HopD that restored relatively high resistance acquisition frequencies. By transforming a pUC19-rpsLmut-sfgfp linear plasmid carrying a streptomycin conferring mutation, we observed that the impaired ability of rpsLmut synthesis in the wild-type strain was restored in the ΔhopE and ΔhopD mutant transformants. Our study revealed that in the presence of streptomycin, resistance acquisition at least partially relied on the deletion of the hopE and hopD genes, because their loss reduced streptomycin concentration in the cell and thus restored the expression of the resistance-conferring gene, which was inhibited by streptomycin in wild-type strain. The loss of HopE and HopD influx activity may also preserve resistance acquisition by transformation in the presence of antibiotics with other modes of action.IMPORTANCE Helicobacter pylori is constitutively competent for natural transformation (NT) and possesses an efficient system for homologous recombination, which could be utilized to study the NT-mediated horizontal gene transfer induced antibiotic resistance acquisition. Bacterial porins have drawn renewed attention because of their crucial role in antibiotic susceptibility. From the perspective of porin-mediated influx in H. pylori, our study preliminarily revealed the important role of HopE and HopD porins not only in preserving the intrinsic susceptibility to specific antibiotic but also in evading acquired antibiotic resistance by NT in the presence of translation-inhibiting antimicrobial. Therefore, the loss of HopE or HopD porin in H. pylori genomes, combined with the large number of secreted or cell-free genetic elements carrying mutations conferring antibiotic resistance, may raise the possibility that this mechanism plays a potential role in the propagation of antibiotic resistance within H. pylori communities.

Nanoparticle immuno-fluorescent probes as a method for detection of viable E. coli O157:H7

Development of revolutionary sensitive biosensors for detecting the presence of harmful biological species in the environment is a necessity for countering disease outbreaks. This work examined the interaction of fluorescence-labeled antibody on amine functionalized gold nanoparticles (GNP) as a model system. The synthesized tetramethylrhodamine isothiocyanate (TRITC) labeled antibody-amine functionalized GNP interaction was characterized using UV–Vis spectroscopy and Fluorescent Microscopy imaging. Transmission Electron Microscopy (TEM) was also used to observe the morphology of the GNP. In contrast to TEM, the fluorescence microscopy imaging revealed the coating of the TRITC labeled antibody on the surface of the GNP. The signals were measured using a Photon Technology Inc. fluorometer at excitation of 541 nm and emission at 555 nm to 650 nm. Tests were conducted at near real-time with results obtained using the biosensor assay within 5 min. Results indicated that there was a shift of the wavelength from lower to higher wavelength (blue to red shift) when conjugated GNP (anti-E. coliO157:H7; IgY-TRITC-GNP) are compared to free GNP, a difference of about 28 nm. The GNP demonstrated a quenching capability when compared to the TRITC labeled antibody (degree of labeling of 15.41 mol dye per mole of IgY) using fluorometer. The lower and upper detection range of this method was found to be 103–105 CFU/mL with observed fluorescence of about 42,000 counts per seconds as against 24,000 counts per seconds that was observed when the specificity of the sensor was tested using Salmonella enterica.

Organelle targeting using a fluorescent probe that selectively penetrates the zona pellucida

The characteristics of oocytes, which are female germ cells, have not been studied using optical materials. The structural layers (zona pellucida, ZP) around oocytes make it difficult to deliver drugs aimed at treating infertility. Here, we investigated whether the fluorescent probes sulforhodamine, fluorescein 5(6)-isothiocyanate, tetramethylrhodamine isothiocyanate, cyanine 3 carboxylic acid, and cyanine 5 carboxylic acid penetrate oocytes. By targeting the ZP layer of the oocyte, the characteristics of the model drug, a fluorescent probe, were analyzed, and the position of the probe in the oocyte was confirmed for differences in the characteristics.Penetration of the ZP and delivery into the cytoplasm differed between the fluorescent probes. This was due to their different physiochemical properties, including hydrophobicity (contact angle and surface tension), surfactant activity, and electrical charge. Among the fluorescent probes delivered to cytoplasm, unlike TRITC, Cy3 and Cy5 perturbed oocyte development. These results suggest that in oocytes with high physical barriers (cell membrane, zona pellucida), the delivery efficiency can be estimated by considering the properties (molecular weight and structure, solubility and functional structure, etc.) of the drug. In addition, it suggests that an encapsulated or bound carrier of a drug with properties similar to that of a fluorescent probe can be efficiently delivered into oocytes.

Influence of Lysine and TRITC Conjugation on the Size and Structure of Dextran Nanoconjugates with Potential for Biomolecule Delivery to Neurons.

As a potent nonviral system for biomolecular delivery to neurons via their axons, we have studied molecular characteristics of lysinated fluorescent dextran nanoconjugates with degrees of conjugation of 0.54-15.2 mol lysine and 0.25-7.27 mol tetramethyl rhodamine isothiocyanate (TRITC) per mol dextran. We studied the influence of conjugation with lysine and TRITC on the size and structure of different molecular weight dextrans and their mobility within axons. Dynamic light scattering (DLS) and small-angle neutron scattering (SANS) experiments revealed significant differences in the size and structure of unmodified and modified dextrans. Unexpectedly, lower-molecular-weight conjugated dextrans exhibited higher molecular volumes, which we propose is due to fewer intramolecular interactions than in higher-molecular-weight conjugated dextrans. Assessment of retrograde and anterograde movement of lysine- and TRITC-conjugated dextrans in axons in the lumbar spinal cord of chicken embryos showed that lower-molecular-weight dextrans translocate more efficiently than higher-molecular-weight dextrans, despite having larger molecular volumes. This comparative characterization of different molecular weight dextrans will help define optimal features for intracellular delivery.

MUSCULATURE AND NEUROTRANSMITTERS IN THE DIGESTIVE SYSTEM OF TREMATODES

In this paper we analyzed the results of our own and published data concerning the presence of muscle elements in various parts of the digestive system in adult and larval forms of trematodes. The data on the localization of the circular and longitudinal muscle fibers in the pharynx, esophagus, and intestine of various representatives of trematodes are presented. The results of immunocytochemical studies indicate the presence the serotonergic and peptidergic (FMRFamidergic) nerve elements in the parts of the digestive system of trematodes. The available literature date is supplemented by the studies conducted on Prodistomum alaskense, a representative of the family Lepocreadiidae, an intestinal parasite of deep-sea fish (Zaprora silenus and Aptocyclus ventricosus). The localization of the serotoninergic and FMRFamidergic nervous structures was identified using immunocytochemical methods and the confocal scanning laser microscopy. For musculature staining the TRITC (tetramethylrhodamine isothiocyanate) – conjugated phalloidin was used. The preparations were examined using a fluorescence microscope and a confocal scanning laser microscope. The analysis of the data obtained and the information available in the literature suggests that the muscular system of the digestive tract is well developed in trematodes of various taxonomic groups. The musculature of the digestive system of trematodes is innervated by serotonergic and peptidergic (FMRFamidergic) nerve elements, which are involved in the regulation of the contractile activity of various parts of the digestive system of trematodes.

Loss of the disease-associated glycosyltransferase Galnt3 alters Muc10 glycosylation and the composition of the oral microbiome

Loss of the disease-associated glycosyltransferase Galnt3 alters Muc10 glycosylation and the composition of the oral microbiome

Shengui Sansheng Pulvis maintains blood-brain barrier integrity by vasoactive intestinal peptide after ischemic stroke

Background Shengui Sansheng Pulvis (SSP) has about 300 years history used for stroke treatment, and evidences suggest it has beneficial effects on neuro-angiogenesis and cerebral energy metabolic amelioration post-stroke. However, its protective action and mechanisms on blood-brain barrier (BBB) is still unknown.Purpose Based on multiple neuroprotective properties of vasoactive intestinal peptide (VIP) in neurological disorders, we investigate if SSP maintaining BBB integrity is associated with VIP pathway in rat permanent middle cerebral artery occlusion (MCAo) model.Methods Three doses of SSP extraction were administered orally. Evaluations of motor and balance abilities and detection of brain edema were performed, and BBB permeability were assessed by Evans blue (EB) staining. Primary brain microvascular endothelial cells (BMECs) were subjected to oxygen-glucose deprivation, and incubated with high dose SSP drug-containing serum and VIP-antagonist respectively. Transendothelial electrical resistance (TEER) assay and Tetramethylrhodamine isothiocyanate (TRITC)-dextran (4.4 kDa) and fluorescein isothiocyanate (FITC)-dextran (70 kDa) were used to evaluate the features of paracellular junction. Western blot detected the expressions of Claudin-5, ZO-1, Occludin and VE-cadherin, matrix metalloproteinase (MMP) 2/9 and VIP receptors 1/2, and immunofluorescence staining tested VIP and Claudin-5 expressions.Results Our results show that SSP significantly reduces EB infiltration in dose-dependent manner in vivo and attenuates TRITC- dextran and FITC-dextran diffusion in vitro, and strengthens endothelial junctional complexes as represented by decreasing Claudin-5, ZO-1, Occludin and VE-cadherin degradations and MMP 2/9 expression, as well as promoting TEER in BMECs after ischemia. Moreover, it suggests that SSP notably enhances VIP and its receptors 1/2 expressions. VIP-antagonist exacerbates paracellular barrier of BMECs, while the result is reversed after incubation with high dose SSP drug-containing serum. Additionally, SSP also improve brain edema and motor and balance abilities after ischemic stroke.Conclusions we firstly demonstrate that the ameliorated efficacy of SSP on BBB permeability is related to the enhancements of VIP and its receptors, suggesting SSP might be an effective therapeutic agent on maintaining BBB integrity post-stroke.

In-situ disintegration of egg white gels by pepsin and kinetics of nutrient release followed by time-lapse confocal microscopy

Digestion behaviour of food protein-based hydrogels is greatly influenced by the gel characteristics and in particular the microstructure. Using egg white gels (EWGs) as a model food, this study aimed to explore the real time disintegration by pepsin of different microstructures and subsequent nutrient release kinetics. Using thermal treatment at 80 °C, EWGs with two different microstructures but similar protein concentration (10%) were produced by varying the pH conditions (pH 5 and pH 9). The in situ spatiotemporal disintegration of the microstructure during static in vitro gastric digestion was followed using a high resolution confocal microscopic technique. Tetramethylrhodamine isothiocyanate (TRITC)-dextran (4400 Da) was incorporated into the gels as a model fluorescent molecule of peptide-like size, to trace its release due to the pepsin action. The looser microstructure of pH5-EWG caused the gel to disintegrate more quickly and to a greater extent, leading to a higher rate of TRITC-dextran release. In contrast, the compact-dense microstructure of the pH9-EWG showed slower kinetics of disintegration and (TRITC)-dextran release, likely due to a reduced accessibility of pepsin to its substrates. Pepsin activity being highly pH-dependent, high local pH and high buffering capacity of pH9-EWG may also play a pivotal role in the slower disintegration observed for this gel. In both EWGs, spatial degradation was mainly observed at the gel surface while the interior area fractions remained unchanged. Thus, surface erosion is possibly the underlying mechanism of EWG disintegration by pepsin in these experimental conditions.

Probing Deviation of Adhered Membrane Dynamics between Reconstituted Liposome and Cellular System.

The dynamics of cell-cell adhesion are complicated due to complexities in cellular interactions and intra-membrane interactions. In the present work, we have reconstituted a liposome-based model system to mimic the cell-cell adhesion process. Our model liposome system consists of one fluorescein-tagged and one TRITC (tetramethyl-rhodamine isothiocyanate)-tagged liposome, adhered through biotin-neutravidin interaction. We monitored the adhesion process in liposomes using Förster Resonance Energy Transfer (FRET) between fluorescein (donor) and TRITC (acceptor). Occurrence of FRET is confirmed by the decrease in donor lifetime as well as distinct rise time of the acceptor fluorescence. Interestingly, the acceptor's emission exhibits fluctuations in the range of ≈3±1 s. This may be attributed to structural oscillations associated in two adhered liposomes arising from the flexible nature of biotin-neutravidin interaction. We have compared the dynamics in a cell-mimicking liposome system with that in an in vitro live cell system. In the adhered live cell system, we used CPM (7-diethylamino-3-(4-maleimido-phenyl)-4-methylcoumarin, donor) and nile red (acceptor), which are known to stain the membrane of CHO (Chinese Hamster Ovary) cells. The dynamics of the adhered membranes of two live CHO cells were observed through FRET between CPM and nile red. The acceptor fluorescence intensity exhibits an oscillation in the time-scale of ≈1±0.75 s, which is faster compared to the reconstituted liposome system, indicating the contributions and involvement of multiple dynamic protein complexes around the cell membrane. This study offers simple reconstituted model systems to understand the complex membrane dynamics using a FRET-based physical chemistry approach.

N-acetylcysteine Amide Ameliorates Blast-Induced Changes in Blood-Brain Barrier Integrity in Rats.

Traumatic brain injury resulting from exposure to blast overpressure (BOP) is associated with neuropathology including impairment of the blood-brain barrier (BBB). This study examined the effects of repeated exposure to primary BOP and post-blast treatment with an antioxidant, N-acetylcysteine amide (NACA) on the integrity of BBB. Anesthetized rats were exposed to three 110 kPa BOPs separated by 0.5 h. BBB integrity was examined in vivo via a cranial window allowing imaging of pial microcirculation by intravital microscopy. Tetramethylrhodamine isothiocyanate Dextran (TRITC-Dextran, mw = 40 kDa or 150 kDa) was injected intravenously 2.5 h after the first BOP exposure and the leakage of TRITC-Dextran from pial microvessels into the brain parenchyma was assessed. The animals were randomized into 6 groups (n = 5/group): four groups received 40 kDa TRITC-Dextran (BOP-40, sham-40, BOP-40 NACA, and sham-40 NACA), and two groups received 150 kDa TRITC-Dextran (BOP-150 and sham-150). NACA treated groups were administered NACA 2 h after the first BOP exposure. The rate of TRITC-Dextran leakage was significantly higher in BOP-40 than in sham-40 group. NACA treatment significantly reduced TRITC-Dextran leakage in BOP-40 NACA group and sham-40 NACA group presented the least amount of leakage. The rate of leakage in BOP-150 and sham-150 groups was comparable to sham-40 NACA and thus these groups were not assessed for the effects of NACA. Collectively, these data suggest that BBB integrity is compromised following BOP exposure and that NACA treatment at a single dose may significantly protect against blast-induced BBB breakdown.

Gamma secretase orthologs are required for lysosomal activity and autophagic degradation in Dictyostelium discoideum, independent of PSEN (presenilin) proteolytic function

ABSTRACT Mutations in the γ-secretase complex are strongly associated with familial Alzheimer disease. Both proteolytic and non-proteolytic functions for the γ-secretase complex have been previously described in mammalian model organisms, but their relative contributions to disease pathology remain unclear. Here, we dissect the roles of orthologs of the γ-secretase components in the model system Dictyostelium, focusing on endocytosis, lysosomal activity and autophagy. In this model, we show that the orthologs of PSEN (psenA and psenB), Ncstn (nicastrin) and Aph-1 (gamma-secretase subunit Aph-1), are necessary for optimal fluid-phase uptake by macropinocytosis and in multicellular development under basic pH conditions. Disruption of either psenA/B or Aph-1 proteins also leads to disrupted phagosomal proteolysis as well as decreased autophagosomal acidification and autophagic flux. This indicates a general defect in lysosomal trafficking and degradation, which we show leads to the accumulation of ubiquitinated protein aggregates in cells lacking psenA/B and Aph-1 proteins. Importantly, we find that all the endocytic defects observed in Dictyostelium PSEN ortholog mutants can be fully rescued by proteolytically inactive Dictyostelium psenB and human PSEN1 proteins. Our data therefore demonstrates an evolutionarily conserved non-proteolytic role for presenilin, and γ-secretase component orthologs, in maintaining Dictyostelium lysosomal trafficking and autophagy. Abbreviations Atg8: autophagy protein 8a; Aph-1: gamma-secretase subunit Aph-1; crtA: calreticulin; ER: endoplasmic reticulum; GFP: green fluorescent protein; GSK3B: glycogen synthase kinase 3 beta; Ncstn: nicastrin; PSEN1: presenilin 1; psenA and psenB: Dictyostelium presenilin A and B; TRITC; tetramethylrhodamine isothiocyanate.

Dye Aggregate-Mediated Self-Assembly of Bacteriophage Bioconjugates.

One of the central themes of biomolecular engineering is the challenge of exploiting the properties of biological materials. Part of this challenge has been uncovering and harnessing properties of biological components that only emerge following their ordered self-assembly. One biomolecular building block that has received significant interest in the past decade is the M13 bacteriophage. There have been a number of recent attempts to trigger the ordered assembly of M13 bacteriophage into multivirion structures, relying on the innate tendency of M13 to form liquid crystals at high concentrations. These, in general, yield planar two-dimensional materials. Presented here is the production of multivirion assemblies of M13 bacteriophage via the chemical modification of its surface by the covalent attachment of the xanthene-based dye tetramethylrhodamine (TMR) isothiocyanate (TRITC). We show that TMR induces the formation of three-dimensional aster-like assemblies of M13 by providing "adhesive" action between bacteriophage particles through the formation of H-aggregates (face-to-face stacking of dye molecules). We also show that the H-aggregation of TMR is greatly enhanced by covalent attachment to M13 and is enhanced further still upon the ordered self-assembly of M13, leading to the suggestion that M13 could be used to promote the self-assembly of dyes that form J-aggregates, a desirable arrangement of fluorescent dye, which has interesting optical properties and potential applications in the fields of medicine and light harvesting technology.

A new approach to assess the effect of photodamage on corneocyte envelope maturity using combined hydrophobicity and mechanical fragility assays.

The maturity of the corneocyte envelope (CE) provides information about the barrier functionality of the stratum corneum (SC). Corneocytes are enclosed by the CE, a protein-lipid matrix, contributing to mechanical resistance and hydrophobicity of the SC. The aim of the work was to develop a novel and robust approach to characterize CE maturity based on rigidity, hydrophobicity and surface area. This offers an alternative approach to the Nile red staining and antigenicity of involucrin to characterize the CE. The photoexposed (PE) cheek and photoprotected (PP) post-auricular sites were selected for investigation. Nine tape strips were obtained from the cheek and post-auricular sites of healthy Caucasians. CEs on the first and last tape strip were subjected to sonication to assess rigidity, and Nile red staining to determine hydrophobicity per unit surface area. In addition, the presence of involucrin and lipids was assessed to determine CE maturity by examination of the red/green pixel ratio, percentage of involucrin expressing CEs and alternatively the ratio of fluorescence density. The CE rigidity was lower in the deeper SC layers of the cheek, whereas post-auricular CEs were mechanically more resistant. Post-auricular CEs from the superficial SC had a larger surface area with a stronger fluorescence signal than those from the cheek. Interestingly, those CEs from the deeper SC layers had similar surface areas in both anatomical sites but were significantly different in hydrophobicity. These three parameters can be summarized as a relative CE maturity index that expresses CE maturity more precisely with a higher sensitivity than the conventional involucrin and Nile red staining approach. CEs of the cheek surface are more mature than CEs in the deeper SC layer, whereas CEs obtained from the post-auricular surface are more mature than those from the cheek surface. The combined method developed allows characterization of CE maturity based on hydrophobicity per unit surface area and rigidity rather than a simple ratio of lipid to involucrin. A more robust and sensitive measurement has therefore been developed addressing the limitations of earlier protocols.

Elastin Shapes Small Molecule Distribution in Lymph Node Conduits.

The spatial and temporal Ag distribution determines the subsequent T cell and B cell activation at the distinct anatomical locations in the lymph node (LN). It is well known that LN conduits facilitate small Ag distribution in the LN, but the mechanism of how Ags travel along LN conduits remains poorly understood. In C57BL/6J mice, using FITC as a fluorescent tracer to study lymph distribution in the LN, we found that FITC preferentially colocalized with LN capsule-associated (LNC) conduits. Images generated using a transmission electron microscope showed that LNC conduits are composed of solid collagen fibers and are wrapped with fibroblastic cells. Superresolution images revealed that high-intensity FITC is typically colocalized with elastin fibers inside the LNC conduits. Whereas tetramethylrhodamine isothiocyanate appears to enter LNC conduits as effectively as FITC, fluorescently-labeled Alexa-555-conjugated OVA labels significantly fewer LNC conduits. Importantly, injection of Alexa-555-conjugated OVA with LPS substantially increases OVA distribution along elastin fibers in LNC conduits, indicating immune stimulation is required for effective OVA traveling along elastin in LN conduits. Finally, elastin fibers preferentially surround lymphatic vessels in the skin and likely guide fluid flow to the lymphatic vessels. Our studies demonstrate that fluid or small molecules are preferentially colocalized with elastin fibers. Although the exact mechanism of how elastin fibers regulate Ag trafficking remains to be explored, our results suggest that elastin can be a potentially new target to direct Ag distribution in the LN during vaccine design.