Epifluorescence Microscopy Imaging Research Articles

Primary cortical cell tri-culture to study effects of amyloid-β on microglia function and neuroinflammatory response.

Microglia play a critical role in neurodegenerative disorders, such as Alzheimer's disease, where alterations in microglial function may result in pathogenic amyloid-β (Aβ) accumulation, chronic neuroinflammation, and deleterious effects on neuronal function. However, studying these complex factors in vivo, where numerous confounding processes exist, is challenging, and until recently, in vitro models have not allowed sustained culture of critical cell types in the same culture. We employed a rat primary tri-culture (neurons, astrocytes, and microglia) model and compared it to co-culture (neurons and astrocytes) and mono-culture (microglia) to study microglial function (i.e., motility and Aβ clearance) and proteomic response to exogenous Aβ. The cultures were exposed to fluorescently-labeled Aβ (FITC-Aβ) particles for varying durations. Epifluorescence microscopy images were analyzed to quantify the number of FITC-Aβ particles and assess cytomorphological features. Cytokine profiles from conditioned media were obtained. Live-cell imaging was employed to extract microglia motility parameters. FITC-Aβ particles were more effectively cleared in the tri-culture compared to the co-culture. This was attributed to microglia engulfing FITC-Aβ particles, as confirmed via epifluorescence and confocal microscopy. FITC-Aβ treatment significantly increased microglia size, but had no significant effect on neuronal surface coverage or astrocyte size. Upon FITC-Aβ treatment, there was a significant increase in proinflammatory cytokines in tri-culture, but not in co-culture. Aβ treatment altered microglia motility evident as a swarming-like motion. The results suggest that neuron-astrocyte-microglia interactions influence microglia function and highlight the utility of the tri-culture model for studies of neuroinflammation, neurodegeneration, and cell-cell communication.

Epifluorescence microscopy study of a quadruple node of triple junctions of grain boundaries in a Eu2+-decorated highly textured composite of (Cl, Br)(K, Rb) and I(K, Rb) solid solutions.

The structural nature and geometry, as well as the lattice-relative orientation, of an arrangement of crystal defects in a highly textured Eu2+-doped composite of two alkali-halide solid solutions was studied by epifluorescence microscopy (EFM) using the doping ion as a fluorochrome. A three-dimensional reconstruction and a skeleton type model, as built from a sequence of EFM images of different optical cross-sections of this arrangement, are presented. Structurally, this arrangement is a quadruple node (QN) of triple junctions of grain boundaries. The QN core geometry is that of a tetragonal tristetrahedron (TTTH), centred at the QN site, whose tetrahedron vertices and edges are on the QN triple junctions and grain boundaries, respectively, whereas the tristetrahedron tetragonal axis is nearly parallel to the lattice [001]-axis. The measured values of the angles between triple junctions and between the grain boundaries forming them are reported. The distinct chemical compositions of the composite solid solutions are discussed to be responsible, in last instance, for the tristetrahedron departure from a cubic configuration. Collaterally, certain families of translationally periodic almost-parallel (TPAP)-wall-like regions which consist of TPAP-columns of TPAP-spindle-like singularities, as well as certain zigzag arrays of columns of this like, existing into the QN grains, are reported to be observed. Three-dimensional reconstructions of typical individuals of these families and arrays as well as of their constituent parts are presented and geometrically analysed. These families and arrays are discussed to be families of tilt subboundaries, whose constituent dislocations are decorated by cylindrical second-phase europium di-halide precipitates, and regularly faceted tilt subboundaries, respectively. Crystal growing and sample preparation, composite structural characterisation by powder and single-slab X-ray diffraction (PXRD and SSXRD, respectively), microscopy and fluorescence-cube unit optics, image processing, electronic three-dimensional reconstruction and measuring methodologies, are all described in detail.

Fluorescence based HTS-compatible ligand binding assays for dopamine D3 receptors in baculovirus preparations and live cells.

Dopamine receptors are G-protein-coupled receptors that are connected to severe neurological disorders. The development of new ligands targeting these receptors enables gaining a deeper insight into the receptor functioning, including binding mechanisms, kinetics and oligomerization. Novel fluorescent probes allow the development of more efficient, cheaper, reliable and scalable high-throughput screening systems, which speeds up the drug development process. In this study, we used a novel Cy3B labelled commercially available fluorescent ligand CELT-419 for developing dopamine D3 receptor-ligand binding assays with fluorescence polarization and quantitative live cell epifluorescence microscopy. The fluorescence anisotropy assay using 384-well plates achieved Z' value of 0.71, which is suitable for high-throughput screening of ligand binding. The assay can also be used to determine the kinetics of both the fluorescent ligand as well as some reference unlabeled ligands. Furthermore, CELT-419 was also used with live HEK293-D3Rcells in epifluorescence microscopy imaging for deep-learning-based ligand binding quantification. This makes CELT-419 quite a universal fluorescence probe which has the potential to be also used in more advanced microscopy techniques resulting in more comparable studies.

Lamina-specific immunohistochemical signatures in the olfactory bulb of healthy, Alzheimer\u2019s and Parkinson\u2019s disease patients

Traditional neuroanatomy immunohistology studies involve low-content analyses of a few antibodies of interest, typically applied and compared across sequential tissue sections. The efficiency, consistency, and ultimate insights of these studies can be substantially improved using high-plex immunofluorescence labelling on a single tissue section to allow direct comparison of many markers. Here we present an expanded and efficient multiplexed fluorescence-based immunohistochemistry (MP-IHC) approach that improves throughput with sequential labelling of up to 10 antibodies per cycle, with no limitation on the number of cycles, and maintains versatility and accessibility by using readily available commercial reagents and standard epifluorescence microscopy imaging. We demonstrate this approach by cumulatively screening up to 100 markers on formalin-fixed paraffin-embedded sections of human olfactory bulb sourced from neurologically normal (no significant pathology), Alzheimer’s (AD), and Parkinson’s disease (PD) patients. This brain region is involved early in the symptomology and pathophysiology of AD and PD. We also developed a spatial pixel bin analysis approach for unsupervised analysis of the high-content anatomical information from large tissue sections. Here, we present a comprehensive immunohistological characterisation of human olfactory bulb anatomy and a summary of differentially expressed biomarkers in AD and PD using the MP-IHC labelling and spatial protein analysis pipeline.

NMDA receptor expression during cell transformation process at early stages of liver cancer in rodent models.

Hepatocellular carcinoma (HCC) is the most common primary liver cancer, which is not sensitive to radiotherapy and chemotherapy and very often experiences postoperative relapse. In this regard, effective screening of liver cancer is considered as the most important and urgent task. The aim of our study was to determine whether N-methyl-D-aspartate receptor (NMDAR) and, in particular, its subunits, can serve as biomarkers to distinguish the precancerous liver at early stages of liver fibrosis. We assessed the development of HCC after 10, 15, and 22 wk using a HCC rat model. The expression of NMDAR subunits was monitored at different stages of HCC by means of immunohistochemistry combined with epifluorescence microscopy imaging, Western blotting, and direct bisulfite sequencing. NMDAR subunits were not found in healthy liver tissues. In contrast, NMDAR subunits, in particular NR1 and NR2B, appeared at the stage of severe liver fibrosis (precancerous liver disease) in rats and were expressed during the development of HCC in rats and mice. Using the direct bisulfite sequencing, we detected that increased expression of NMDAR directly correlated with the demethylation of CpG islands in the promoter region of genes encoding receptor subunits. The obtained results confirmed that NMDAR subunits can serve as new biomarkers of precancerous liver disease, severe fibrosis, and its progression towards HCC.NEW & NOTEWORTHY We have shown NMDAR expression in cell transformation process at early stages of cancer, specifically HCC. The aim of our study was to define the disease stages from precancerous liver disease towards liver cancer progression when NMDAR subunits were expressed/detected. A fibrosis/HCC rat model, immunohistochemistry combined with epifluorescence microscopy imaging, Western blotting was used. The dynamics of appearance of NMDAR subunits, their expression and methylation status during the development of HCC were shown and discussed.

Dermal Penetration Analysis of Curcumin in an ex vivo Porcine Ear Model Using Epifluorescence Microscopy and Digital Image Processing

Introduction: Curcumin is a promising drug candidate, but its use for dermal application is limited due to its poor aqueous solubility. Thus, formulations that increase the solubility of curcumin are needed to fully exploit the therapeutic potential of curcumin. Various previous studies address this issue, but a comparison of the efficacy between these formulations remains difficult. The reason for this is a missing standard formulation as benchmark control and an easy-to-use skin penetration model that allows for a fast discrimination between different formulations. Objective: Thus, the aims of this study were the development of a curcumin standard formulation and a screening tool that allows for a fast discrimination between the dermal penetration efficacies of curcumin from different formulations. Methods: Ethanolic curcumin solutions were selected as simple and easy to produce standard formulations, and the ex vivo porcine ear model, coupled with epifluorescence microscopy and subsequent digital image analysis, was utilized to determine the dermal penetration efficacy of curcumin from the different formulations. Results: Results show that the utilized skin penetration model is a suitable and versatile tool that enables not only a fast determination of the dermal penetration efficacy of curcumin from different formulations but also a detailed and mechanistic information on the fate of chemical compounds after dermal penetration. Ethanolic solutions containing 0.25% curcumin were found to be the most suitable standard formulation. Conclusions: Results of the study provide a new, effective screening tool for the development of dermal formulations for improved dermal delivery of curcumin.

Phenylalanine intercalation parameters for liquid-disordered phase domains \u2013 a membrane model study

BackgroundPropensity of phenylalanine (Phe) for nonpolar environments drives its intercalation into phospholipid membranes, which has been implicated in metabolic and neurological disorders. The knowledge of Phe intercalation parameters can be instrumental in understanding various membrane processes triggered by interactions with Phe, in particular the early events leading to the formation of nucleation/docking sites for the self-assembly of Phe amyloid fibrils at the membrane surface.ResultsIn this study, we used monolayers of phosphatidylethanolamine (DPPE) and phosphatidylcholine (DPPC) to mimic the membrane outer leaflet. Its initial interaction with Phe was modeled by injecting Phe into the aqueous phase underneath the monolayer. Constant pressure insertion assays augmented with epifluorescence microscopy imaging were used to monitor Phe intercalation. Our primary goal was to determine the Phe intercalation area, APhe. Two values were obtained for APhe, 33 ± 2 and 48 ± 3 Å2.ConclusionsPhe appeared to discriminate between DPPE and DPPC packing, and use two modes of intercalation. The area of APhe 33 ± 2 Å2 is consistent with a Phe monomer intercalating into membrane by inserting the phenyl ring nearly normal to the membrane surface. This mode has been found to operate in DPPE membranes. For DPPC membranes however, the value of APhe = 48 ± 3 Å2 suggests that, from saline, Phe tends to intercalate as a larger species plausibly dragging along a counterion, Na+, in a Na+(Phe) complex.

Patch-Based Semantic Segmentation for Detecting Arterioles and Venules in Epifluorescence Imagery.

Segmentation and quantification of microvasculature structures are the main steps toward studying microvasculature remodeling. The proposed patch based semantic architecture enables accurate segmentation for the challenging epifluorescence microscopy images. Our pixel-based fast semantic network trained on random patches from different epifluorescence images to learn how to discriminate between vessels versus nonvessels pixels. The proposed semantic vessel network (SVNet) relies on understanding the morphological structure of the thin vessels in the patches rather than considering the whole image as input to speed up the training process and to maintain the clarity of thin structures. Experimental results on our ovariectomized - ovary removed (OVX) - mice dura mater epifluorescence microscopy images shows promising results in both arteriole and venule part. We compared our results with different segmentation methods such as local, global thresholding, matched based filter approaches and related state of the art deep learning networks. Our overall accuracy (> 98%) outperforms all the methods including our previous work (VNet). [1].

Automated Microscopic Analysis of Metal Sulfide Colonization by Acidophilic Microorganisms.

Industrial biomining processes are currently focused on metal sulfides and their dissolution, which is catalyzed by acidophilic iron(II)- and/or sulfur-oxidizing microorganisms. Cell attachment on metal sulfides is important for this process. Biofilm formation is necessary for seeding and persistence of the active microbial community in industrial biomining heaps and tank reactors, and it enhances metal release. In this study, we used a method for direct quantification of the mineral-attached cell population on pyrite or chalcopyrite particles in bioleaching experiments by coupling high-throughput, automated epifluorescence microscopy imaging of mineral particles with algorithms for image analysis and cell quantification, thus avoiding human bias in cell counting. The method was validated by quantifying cell attachment on pyrite and chalcopyrite surfaces with axenic cultures of Acidithiobacillus caldus, Leptospirillum ferriphilum, and Sulfobacillus thermosulfidooxidans. The method confirmed the high affinity of L. ferriphilum cells to colonize pyrite and chalcopyrite surfaces and indicated that biofilm dispersal occurs in mature pyrite batch cultures of this species. Deep neural networks were also applied to analyze biofilms of different microbial consortia. Recent analysis of the L. ferriphilum genome revealed the presence of a diffusible soluble factor (DSF) family quorum sensing system. The respective signal compounds are known as biofilm dispersal agents. Biofilm dispersal was confirmed to occur in batch cultures of L. ferriphilum and S. thermosulfidooxidans upon the addition of DSF family signal compounds.IMPORTANCE The presented method for the assessment of mineral colonization allows accurate relative comparisons of the microbial colonization of metal sulfide concentrate particles in a time-resolved manner. Quantitative assessment of the mineral colonization development is important for the compilation of improved mathematical models for metal sulfide dissolution. In addition, deep-learning algorithms proved that axenic or mixed cultures of the three species exhibited characteristic biofilm patterns and predicted the biofilm species composition. The method may be extended to the assessment of microbial colonization on other solid particles and may serve in the optimization of bioleaching processes in laboratory scale experiments with industrially relevant metal sulfide concentrates. Furthermore, the method was used to demonstrate that DSF quorum sensing signals directly influence colonization and dissolution of metal sulfides by mineral-oxidizing bacteria, such as L. ferriphilum and S. thermosulfidooxidans.

Endocytic recycling via the TGN underlies the polarized hyphal mode of life.

Intracellular traffic in Aspergillus nidulans hyphae must cope with the challenges that the high rates of apical extension (1μm/min) and the long intracellular distances (>100 μm) impose. Understanding the ways in which the hyphal tip cell coordinates traffic to meet these challenges is of basic importance, but is also of considerable applied interest, as fungal invasiveness of animals and plants depends critically upon maintaining these high rates of growth. Rapid apical extension requires localization of cell-wall-modifying enzymes to hyphal tips. By combining genetic blocks in different trafficking steps with multidimensional epifluorescence microscopy and quantitative image analyses we demonstrate that polarization of the essential chitin-synthase ChsB occurs by indirect endocytic recycling, involving delivery/exocytosis to apices followed by internalization by the sub-apical endocytic collar of actin patches and subsequent trafficking to TGN cisternae, where it accumulates for ~1 min before being re-delivered to the apex by a RAB11/TRAPPII-dependent pathway. Accordingly, ChsB is stranded at the TGN by Sec7 inactivation but re-polarizes to the apical dome if the block is bypassed by a mutation in geaAgea1 that restores growth in the absence of Sec7. That polarization is independent of RAB5, that ChsB predominates at apex-proximal cisternae, and that upon dynein impairment ChsB is stalled at the tips in an aggregated endosome indicate that endocytosed ChsB traffics to the TGN via sorting endosomes functionally located upstream of the RAB5 domain and that this step requires dynein-mediated basipetal transport. It also requires RAB6 and its effector GARP (Vps51/Vps52/Vps53/Vps54), whose composition we determined by MS/MS following affinity chromatography purification. Ablation of any GARP component diverts ChsB to vacuoles and impairs growth and morphology markedly, emphasizing the important physiological role played by this pathway that, we propose, is central to the hyphal mode of growth.

An automated epifluorescence microscopy imaging assay for the identification of phospho-AKT level modulators in breast cancer cells

AKT is an enzyme of the PI3K/pAKT pathway, regulating proliferation and cell survival. High basal levels of active, phosphorylated AKT (pAKT) are associated with tumor progression and therapeutic resistance in some breast cancer subtypes, including HER2 positive breast cancers. Various stimuli can increase pAKT levels and elevated basal pAKT levels are a feature of PTEN-deficient breast cancer cell lines. The aim of this study was to develop an assay able to identify modulators of pAKT levels using an automated epifluorescence microscope and high content analysis. To develop this assay, we used HCC-1569, a PTEN-deficient, HER2-overexpressing breast cancer cell line with elevated basal pAKT levels. HCC-1569 cells were treated with a selective pharmacological inhibitor of AKT (MK-2206) to reduce basal pAKT levels or EGF to increase pAKT levels. Immunofluorescence images were acquired using an automated epifluorescence microscope and integrated intensity of cytoplasmic pAKT staining was calculated using high content analysis software. Mean and median integrated cytoplasmic intensity were normalized using fold change and standard score to assess assay quality and to identify most robust data analysis. The highest z′ factor was achieved for median data normalization using the standard score method (z′ = 0.45). Using our developed assay we identified the calcium homeostasis regulating proteins TPRV6, STIM1 and TRPC1 as modulators of pAKT levels in HCC-1569 cells. Calcium signaling controls a diverse array of cellular processes and some calcium homeostasis regulating proteins are involved in modulating pAKT levels in cancer cells. Thus, these identified hits present promising targets for further assessment.

Cytochrome-c-assisted escape of cardiolipin from a model mitochondrial membrane

Binding of cytochrome c (Cytc) to cardiolipin (CL) in the inner mitochondrial membrane is involved with the onset of apoptosis. In this study, we used CL-containing phospholipid monolayers to mimic the inner mitochondrial membrane. Constant pressure insertion assay was employed to monitor the Cytc-induced expansion of membrane area. Simultaneous epifluorescence microscopy imaging afforded the in-situ visualization of phospholipid demixing and sorting in the membrane. The formation of a CL-rich Ld phase has been observed to prelude the insertion of Cytc. Based on the relative expansion of membrane area, a cluster of a few amino acid residues of Cytc with an area of 117±7Å2 has been found to insert into the membrane. The insertion of Cytc disrupted the membrane in a way facilitating the escape of CL. When the exclusion of Cytc was induced by compression, CL molecules appeared to escape the membrane together with the protein, which resulted in a loss of more than a half of CL content from the membrane. These findings may aid in understanding the early events leading to the remodeling of inner mitochondrial membrane and loss of its function during apoptosis.

Cospreading of Anionic Phospholipids with Peptides of the Structure (KX)4K at the Air-Water Interface: Influence of Lipid Headgroup Structure and Hydrophobicity of the Peptide on Monolayer Behavior.

Mixtures of anionic phospholipids (PG, PA, PS, and CL) with cationic peptides were cospread from a common organic solvent at the air-water interface. The compression of the mixed film was combined with epifluorescence microscopy or infrared reflection adsorption spectroscopy (IRRAS) to gain information on the interactions of the peptide with the different lipids. To evaluate the influence of the amino acid X of peptides with the sequence (KX)4K on the binding, 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) was mixed with different peptides with increasing hydrophobicity of the uncharged amino acid X. The monolayer isotherms of DPPG/(KX)4K mixtures show an increased area for the lift-off due to incorporation of the peptide into the liquid-expanded (LE) state of the lipid. The surface pressure for the transition from LE to the liquid-condensed (LC) state is slightly increased for peptides with amino acids X with moderate hydrophobicity. For the most hydrophobic peptide (KL)4K two plateaus are seen at a charge ratio PG to K of 5:1, and a strongly increased transition pressure is observed for a charge ratio of 1:1. Epifluorescence microscopy images and infrared spectroscopy show that the lower plateau corresponds to the LE-LC phase transition of the lipid. The upper plateau is connected with a squeeze-out of the peptide into the subphase. To test the influence of the lipid headgroup structure on peptide binding (KL)4K was cospread with different anionic phospholipids. The shift of the isotherm to larger areas for lift-off and to higher surface pressure for the LE-LC phase transition was observed for all tested anionic lipids. Epifluorescence microscopy reveals the formation of LC domains with extended filaments indicating a decrease in line tension due to accumulation of the peptides at the LC-domain boundaries. This effect depends on the size of the headgroup of the anionic phospholipid.

HOS cell adhesion on TiO2 nanotubes texturized by laser engraving

Due to its outstanding properties, the titanium and its alloys have been widely used in the dental and orthopaedic fields as biomaterials. The TiO2 nanotubes surface and the texturized process by laser engraving enables significantly accelerated osteoblast adhesion on the biomaterial. For this reason in this paper, the HOS cell responses on TiO2 nanotubes fabricated on Ti6Al4V alloy and texturized by laser engraving were evaluated. The test surfaces were carried out on smooth Ti6Al4V as control, TiO2 nanotubes (NT) and surfaces with micropoints obtained by laser engraving, with 1mm spacing (NTP1) and 0.5mm (NTP2). The results show that the texturized process enables decreases the contact angle thus improving wettability of the TiO2 nanotubes surface. The NTP1 and NTP2 surfaces show excellent cell adhesion and spreading on the surface, which is evident in epifluorescence microscopy images. Furthermore, the NTP1 and NTP2 surfaces improved the cell proliferation at 18% and 16% respectively in relation with NT surface, showing that the laser texturing improves cell response of TiO2 nanotubes.

Photosynthetic activity detected in the seed epidermis of Thalassia testudinum

Seagrasses are marine angiosperms that have evolved from terrestrial ancestors. Photosynthetic activity on seed has been studied before in Posidonia genus. T. testudinum and P. oceanica share some evolutionary aspects as they live in more stable environments. As in P. oceanica, T. testudinim seed is also green-coloured suggesting the presence of photosynthetic pigments. Here, the photosynthetic activity in the T. testudinum seed was examined. Measurements of photosynthetic production, pigment content and maximum PSII photochemical efficiency (Fv: Fm) were determined. Epifluorescence microscopy analysis was used to obtain evidence of photosynthetic structures within the seed. This study revealed photosynthetic activity in seed epidermis which increased linearly with increasing irradiance. T. testudinum seed displayed a higher Photosynthetic efficiency (α), and lower light saturated photosynthesis (P. max), saturation irradiance (Ek), and compensation irradiance (Ec) compared to adult leaves. Epifluorescence microscopy images of the seeds showed an epidermis composed of relatively small, regular cells, tending towards a rectangular shape, containing abundant chloroplasts. The photobiological behaviour results in the T. testudinum seed being strongly adapted to colonize environments with reduced light availability. Photosynthetic activity in T. testudinum seed shed some light on seagrass dispersal and expansion mechanisms.

Charge-Mediated Localization of Conjugated Polythiophenes in Zwitterionic Model Cell Membranes.

The selective engineering of conjugated polyelectrolyte (CPE)-phospholipid interfaces is poised to play a key role in the design of advanced biomedical and biotechnological devices. Herein, we report a strategic study to investigate the relationship between the charge of the CPE side group and their association with zwitterionic phospholipid bilayers. The interaction of dipalmitoylphosphatidylcholine (DPPC) phospholipid vesicles with a series of poly(thiophene)s bearing zwitterionic, cationic, or anionic terminal groups (P3Zwit, P3TMAHT and P3Anionic, respectively) has been probed. Although all CPEs showed an affinity for the zwitterionic vesicles, the calculated partition coefficients determined using photoluminescence spectroscopy suggested preferential incorporation within the lipid bilayer in the order P3Zwit > P3Anionic ≫ P3TMAHT. The polarity probe Prodan was used to further qualify the position of the CPE inside the vesicle bilayers via Förster resonance energy transfer (FRET) studies. The varying proximity of the CPEs to Prodan was reflected in the Stern-Volmer quenching constants and decreased in the order P3Anionic > P3TMAHT ≫ P3Zwit. Dynamic light scattering measurements showed an increase in the hydrodynamic diameter of the DPPC vesicles upon addition of each poly(thiophene), but to the greatest extent for P3Anionic. Small-angle neutron scattering studies also revealed that P3Anionic specifically increased the thickness of the headgroup region of the phospholipid bilayer. Epifluorescence and atomic force microscopy imaging showed that P3TMAHT formed amorphous agglomerates on the vesicle surface, P3Zwit was buried throughout the bilayer, and P3Anionic formed a shell of protruding chains around the surface, which promoted vesicle fusion. The global data indicate three distinctive modes of interaction for the poly(thiophene)s within DPPC vesicles, whereby the nature of the association is ultimately controlled by the pendant charge group on each CPE chain. Our results suggest that charge-mediated self-assembly may provide a simple and effective route to design luminescent CPE probes capable of specific localization within phospholipid membranes.

PEGylation of novel hydroxyapatite/PEG/Ag nanocomposite particles to improve its antibacterial efficacy.

Hydroxyapatite (HAp) nanocomposite particles were prepared simply in the presence of polyethylene glycol (PEG) and fabricated with silver via a sol-gel route and the physico-chemical and biological properties of these materials were investigated. The objective of this study is to inspect the crystallinity and antibacterial activity of these composite materials. PEG has been used to greatly promote biocompatibility and biodegradability of HAp. Silver nanoparticles were used for improving its bactericidal efficacy while applying composites. Nano-sized HAp composite particles with PEG and nano-silver was incorporated to increase the crystalline nature of the nanocomposite. The structure of nanocomposite particles was studied by XRD, FTIR, HR-SEM, EDS and TEM analyses. Silver nanoparticles loaded on the synthesized HAp-PEG showed a synergistic antibacterial effect against Gram-negative bacterium Escherichia coli (E. coli). The controlled release of Ag(+) ion from HAp-PEG-Ag nanocomposite has given good antibacterial efficacy evidenced by epi-fluorescence microscopy images during different hours.

Structure and anti-inflammatory capacity of peptidoglycan from Lactobacillus acidophilus in RAW-264.7 cells

Probiotic bacteria, especially the Lactobacillus species, can potentially play a significant role in the antiviral and antimicrobial activity of a host's immune system. Lactobacillus acidophilus, an intestinal bacterium, is involved in the intestinal epithelial cell response when a pathogen adheres to a cell. In this study, the structure of peptidoglycan (PGN) isolated from L. acidophilus was determined with the help of HPLC, NMR, FT-IR and MALDI-TOF/TOF MS. The molecular mass of PGN is 875.260Da. The anti-inflammatory capacity of PGN was evaluated in model RAW 264.7 cells. Epifluorescence microscopy images and western blot analysis provided compelling evidence of PGN's anti-inflammatory capacity on LPS-induced macrophages. A significant decrease in inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) levels when PGN was added up to 200μg/ml. These data provide new insight into the cellular and molecular mechanisms by which probiotic bacteria can contribute to maintaining good health.

Silver colloidal nanoparticles: effect on matrix composition and structure ofCandida albicansandCandida glabratabiofilms

The aim of this study was to assess the effect of different silver nanoparticles (SN) concentrations on the matrix composition and structure of Candida albicans and Candida glabrata biofilms. Candida biofilms were developed in 6-well microtiter plates during 48 h. After, these biofilms were exposed to 13.5 or 54 μg SN ml(-1) for 24 h. Then, extracellular matrices were extracted from biofilms and analysed chemically in terms of proteins, carbohydrates and DNA. To investigate the biofilm structure, scanning electron microscopy (SEM) and epifluorescence microscopy were used. SN interfered with the matrix composition of Candida biofilms tested in terms of protein, carbohydrate and DNA, except for the protein content of C. albicans biofilm. By SEM, Candida biofilms treated with SN revealed structural differences, when compared with the control groups. Further, SN showed a trend of agglomeration within the biofilms. Epifluorescence microscopy images suggest that SN induced damage on cell walls of the Candida isolates tested. In general, irrespective of concentration, SN affected the matrix composition and structure of Candida biofilms and these findings may be related to the mechanisms of biocide action of SN. This study reveals new insights about the behaviour of SN when in contact with Candida biofilms. SN may contribute to the development of therapies to prevent or control Candida infections.

Image Analysis of Phase Separated Langmuir Monolayers Containing Polyunsaturated Fatty Acids

Results from epifluorescence microscopy studies and image analysis of phase separated Langmuir monolayers of ternary mixtures containing polyunsaturated fatty acids (PUFAs), sphingomyelin, and cholesterol will be presented. Experiments were done using a Langmuir trough and inverted microscope under a sealed chamber providing an inert atmosphere. We will focus on the results and implications from measurements of domain size distribution and area fraction for four different mixed acyl phospholipid species with varying degrees of unsaturation in the acyl chain (1, 2, 4, 6). We have applied a recently developed technique to measure the line tension of these systems using the size distribution [1]. This experimental approach allows us to investigate the relationship between the miscibility phase transition, line tension, and degree of unsaturation even for systems with small domains not otherwise amenable to line tension studies. Experiments described above were combined with more traditional Langmuir film-balance techniques including pressure-area isotherms.[1] Lee et at., Relating Domain Size Distribution to Line Tension and Molecular Dipole Density in Model Cytoplasmic Myelin Lipid Monolayers. PNAS 108, 9425-9430.