Subsequent Fluorescence Microscopy Research Articles

Differences in Biofilm Formation of Listeria monocytogenes and Their Effects on Virulence and Drug Resistance of Different Strains.

Listeria monocytogenes is recognized as one of the primary pathogens responsible for foodborne illnesses. The ability of L. monocytogenes to form biofilms notably increases its resistance to antibiotics such as ampicillin and tetracycline, making it exceedingly difficult to eradicate. Residual bacteria within the processing environment can contaminate food products, thereby posing a significant risk to public health. In this study, we used crystal violet staining to assess the biofilm-forming capacity of seven L. monocytogenes strains and identified ATCC 19112 as the strain with the most potent biofilm-forming. Subsequent fluorescence microscopy observations revealed that the biofilm-forming capacity was markedly enhanced after two days of culture. Then, we investigated into the factors contributing to biofilm formation and demonstrated that strains with more robust extracellular polymer secretion and self-agglutination capabilities exhibited a more pronounced ability to form biofilms. No significant correlation was found between surface hydrophobicity and biofilm formation capability. In addition, we found that after biofilm formation, the adhesion and invasion of cells were enhanced and drug resistance increased. Therefore, we hypothesized that the formation of biofilm makes L. monocytogenes more virulent and more difficult to remove by antibiotics. Lastly, utilizing RT-PCR, we detected the expression levels of genes associated with biofilm formation, including those involved in quorum sensing (QS), flagellar synthesis, and extracellular polymer production. These genes were significantly upregulated after biofilm formation. These findings underscore the critical relationship between extracellular polymers, self-agglutination abilities, and biofilm formation. In conclusion, the establishment of biofilms not only enhances L. monocytogenes' capacity for cell invasion and adhesion but also significantly increases its resistance to drugs, presenting a substantial threat to food safety.

Mechanical and biocompatible properties of the poly(lactide-co-glycolide) matrices produced by antisolvent 3D printing

Three-dimensional scaffolds were made from a solution of poly(lactide-co-glycolide) mixed with tetraglycol using antisolvent 3D printing. The elastic properties and the structure of the obtained matrices were studied. MTT-test and staining with PKH-26, Calcein-AM, DAPI with subsequent fluorescence microscopy were used to study biological properties. The three-dimensional scaffolds had good mechanical properties. Young’s modulus value was 18±2 MPa, tensile strength was 0.43±0.05 MPa. The relative survival rate of cells after the first day was 99.58±2.28%, on the 14th day – 98.14±2.22%. The structure of the scaffold promoted cell adhesion and spreading on its surface. The poly(lactide-co-glycolide) matrices produced by antisolvent printing have high porosity, biocompatibility and good mechanical properties. It is allowed to use them in the future as a basis for personalized constructions for the replacement of extensive bone defects.

Gut-microbiota-derived phenylacetylglutamine induces oxidative stress in endothelial cells via a NOX2-dependent pathway

Abstract Introduction The gut microbiota-derived metabolite phenylacetylglutamine (PAG) is clinically and mechanistically linked to cardiovascular disease (CVD)(1). Here, we aim to identify the effect of PAG on endothelial cells and elucidate the underlying molecular pathways. Methods We treated human aortic endothelial cells (HAEC) at a dose of 100 µM PAG (a concentration which can be observed in vivo) for 72h. As a positive control, cells were treated with H2O2 for 1h. For the determination of reactive oxygen species (ROS) formation, we used the CellROX Green assay with subsequent fluorescence microscopy. For signaling studies, protein expression levels of the NADPH oxidases NOX2 and NOX4 as well as the transcription factor NF-kB were determined by western blot analyses. Results Our studies showed that PAG treatment for 72h significantly increased the fluorescence intensity after CellROX Green staining (p<0.0001). Concomitantly, our immunoblotting results revealed that NOX2 (p<0.01) and NF-kB (p<0.001) protein levels were upregulated, whereas NOX4 remained unchanged in response to PAG. Conclusion We conclude that PAG-induced oxidative stress in endothelial cells is mediated by a NOX2 and NF-kB but not by a NOX4 dependent pathway. This study is a first step towards better understanding the molecular mechanisms of cellular responses to the gut microbiota-derived metabolite PAG.

Influence of Mesenchymal Stem Cells Seeding on Morphology of Tissue-Engineered Vascular Graft Based on Poly (L-lactide) Scaffold

Introduction - The problem of small-diameter vascular grafts still exists in modern vascular surgery. Autologous or synthetic vascular grafts are used routinely for arterial bypass in patients with cardiovascular diseases. However, some patients either lack suitable autologous tissue or cannot receive synthetic grafts. Such patients could benefit from a vascular graft produced by tissue engineering. The aim of the study is to evaluate the effects of adipose-derived mesenchymal stem cells (AD-MSC) on the formation of tissue-engineered vascular graft (TEVG). Methods - Tubular scaffolds 1.1 mm inner diameter were obtained by the electrospinning method on the basis of nano- and microfibers of poly(L-lactide) (PLLA). AD-MSC were seeded on the scaffolds by the developed filtration method. The subsequent culturing for 14 days was carried out in the constructed flow bioreactor. Cell distribution in the scaffold wall was assessed by the fluorescence microscopy. Obtained grafts were implanted into rat aorta: group 1 without AD-MSC (n=36) with follow-up till 16 months, group 2 with seeded AD-MSC (n=36) with follow-up till 16 months. The material was subjected to histological examination, electron microscopy, immunohistochemistry CD 31+, aSMA and morphometric analysis (cell counting, neointima and neoadventia thickness). To track AD-MSC in vivo PKH-26 labeling and subsequent fluorescence microscopy were performed in 2, 7 and 14 days. Results - Filtration seeding and subsequent cultivation in the flow bioreactor led to a uniform distribution of AD-MSC in the scaffold wall. Graft patency in the first group was 86%, in the second 96%. All grafts formed neointima, with no signs of hyperplasia in the area of anastomoses. The total resorption of PLLA fibers in group 1 was observed by histological examination, new vascular wall consisted of endothelial lining and connective tissue without smooth muscle cells (SMC); in all cases aneurysm formation was detected. Total cells number of implant wall was more in group 2, including SMC that formed neomedia. Also the outer connective tissue layer was much thicker. Histology identified the uniform formation of new tissues in group 2 with no signs of aneurysm. Conclusion - AD-MSC seeding leads to the formation of TEVG, morphologically similar to the natural structure of the vessels. The study was supported by Russian Science Foundation (project 14-33-00003)

Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells.

The ability to study the intracellular localization of proteins is essential for the understanding of many cellular processes. In turn, this requires the ability to obtain single cells for fluorescence microscopy, which can be particularly challenging when imaging cells that exist within bacterial communities. For example, the human pathogen Vibrio parahaemolyticus exists as short rod-shaped swimmer cells in liquid conditions that upon surface contact differentiate into a subpopulation of highly elongated swarmer cells specialized for growth on solid surfaces. This paper presents a method to perform single cell fluorescence microscopy analysis of V. parahaemolyticus in its two differential states. This protocol very reproducibly induces differentiation of V. parahaemolyticus into a swarmer cell life-cycle and facilitates their proliferation over solid surfaces. The method produces flares of differentiated swarmer cells extending from the edge of the swarm-colony. Notably, at the very tip of the swarm-flares, swarmer cells exist in a single layer of cells, which allows for their easy transfer to a microscope slide and subsequent fluorescence microscopy imaging of single cells. Additionally, the workflow of image analysis for demographic representation of bacterial societies is presented. As a proof of principle, the analysis of the intracellular localization of chemotaxis signaling arrays in swimmer and swarmer cells of V. parahaemolyticus is described.

Hydrophilic trans-Cyclooctenylated Noncanonical Amino Acids for Fast Intracellular Protein Labeling.

Introduction of bioorthogonal functionalities (e.g., trans-cyclooctene-TCO) into a protein of interest by site-specific genetic encoding of non-canonical amino acids (ncAAs) creates uniquely targetable platforms for fluorescent labeling schemes in combination with tetrazine-functionalized dyes. However, fluorescent labeling of an intracellular protein is usually compromised by high background, arising from the hydrophobicity of ncAAs; this is typically compensated for by hours-long washout to remove excess ncAAs from the cellular interior. To overcome these problems, we designed, synthesized, and tested new, hydrophilic TCO-ncAAs. One derivative, DOTCO-lysine was genetically incorporated into proteins with good yield. The increased hydrophilicity shortened the excess ncAA washout time from hours to minutes, thus permitting rapid labeling and subsequent fluorescence microscopy.

An Optogenetic Test for the Functional Specificity of Bulbospinal Sympathoexcitatory Neurons

Sympathetic preganglionic neurons are functionally specialised, each belonging to one of a limited number of functional classes (e.g. muscle vasoconstrictor, cutaneous vasoconstrictor, sudomotor). How far this functional specificity applies to the premotor neurons that drive them is uncertain. We devised an optogenetic test for the existence of premotor neurons that are collateralised to drive more than one functional class of sympathetic neuron.Bulbospinal neurons were transduced by a retrogradely transported viral vector (canine adenovirus) encoding Cre recombinase (CAV‐CMV‐Cre; provided by Eric Kremer, Montpellier). This was injected around sympathetic preganglionic neurons at the T12‐L1 spinal levels. At the same time, a Cre‐activated adeno‐associated virus (AAV) encoding Channelrhodopsin‐2 (ChR2) and the fluorescent label mCherry (AAV1.EF1.dflox.hChR2(H134R)‐mCherry.WPRE.hGH; provided by Deisseroth, Stanford via UPenn) was injected into the left RVLM. Only neurons transduced by both viral vectors should then express the ChR2 and mCherry. Spinal injections were made at T12‐L1 because they were considered unlikely to overlap with the preganglionic supply to the heart. The bulbospinal presynaptic terminals infected with CAV‐Cre were thus presumably on preganglionic neurons that supplied end organs such as hindlimb muscle, but not the heart.Terminal functional experiments under urethane anaesthesia were performed 2–8 weeks after the viral injections. Activity was simultaneously recorded from the left cardiac and lumbar sympathetic nerves, whilst pulses of blue laser light (473 nm, 10–12 mW, 20 ms) were flashed onto the left RVLM through a 100 μm diameter fibre‐optic probe.In about one‐third of experiments, we were able to show functional activation of sympathetic nerves that was time‐locked to the flashed laser light. In all successful experiments, we found cardiac sympathetic activation as well as lumbar sympathetic activation (peak response latencies 135–150 ms and 215–230 ms, respectively). Subsequent fluorescence microscopy on brainstem sections confirmed the presence of small numbers of mCherry expressing bulbospinal neurons in those rats.This study demonstrates that at least some bulbospinal neurons send excitatory synaptic inputs to preganglionic neurons of more than one functional type. It also demonstrates the feasibility of using anatomically‐based optogenetic transduction as a test for functional collateralisation in central pathways.Support or Funding InformationAustralian Research Council DP13014661

PET imaging of tumor associated macrophages using mannose coated 64Cu liposomes

Macrophages within the tumor microenvironment (TAMs) have been shown to play a major role in the growth and spread of many types of cancer. Cancer cells produce cytokines that cause macrophages to express scavenger receptors (e.g. the mannose receptor) and factors that facilitate tissue and blood vessel growth, suppress T cell mediated anti-tumor activity, and express enzymes that can break down the extracellular matrix, thereby promoting metastasis. We have designed a mannosylated liposome (MAN-LIPs) and show that it accumulates in TAMs in a mouse model of pulmonary adenocarcinoma. These liposomes are loaded with 64Cu to allow tracking by PET imaging, and contain a fluorescent dye in the lipid bilayer permitting subsequent fluorescence microscopy. We injected these liposomes into a mouse model of lung cancer. In vivo PET images were acquired 6 h after injection followed by the imaging of select excised organs. MAN-LIPs accumulated in TAMs and exhibited little accumulation in remote lung areas. MAN-LIPs are a promising new vehicle for the delivery of imaging agents to lung TAMs. In addition to imaging, MAN-LIPs hold the potential for delivery of therapeutic agents to the tumor microenvironment.

Effective Collection of Airborne Micro-Organisms by Direct Impaction on Silicone Sealants—Comparison of Different Adherent Surfaces

For effective sampling of airborne micro-organisms different techniques such as impingement, filtration and impaction are commonly used. Impaction is the method which allows best for direct collection on adhesive surfaces for immediate microscopic analysis. The efficiency of collection largely depends on the quality of the sampling surface to retain particles. This article compares the suitability of some traditional and new adhesive coatings to collect and retain airborne micro-organisms efficiently for subsequent fluorescence microscopy. Sacharomyces cerevisiae yeast cells were aerosolized in a test tubing system in the laboratory. Samples were collected by a round jet impactor in a defined round deposition area on glass slides coated with different adhesive materials. Particle distribution in the deposition area and collection efficiency as well as particle losses were estimated. Fluorescent staining with Mycoval and DAPI was used to quantify the amount of collected cells and cell losses due to the sta...

Abstract 5521: IL13 linked lipsomal doxorubicin for malignant peripheral nerve sheath tumors

Abstract We have previously demonstrated the therapeutic effect of interleukin-13 (IL13) linked liposomal doxorubicin (IL13LDXR) on glioma tumors. These tumors selectively overexpress IL13 receptor α2, a receptor for IL13. In our subsequent study, we found that malignant peripheral nerve sheath tumors (MPNST) also express the receptor for IL13. This observation prompted us to investigate the therapeutic value of IL13LDXR in a MPNST model. IL13LDXR was formulated and characterized in our laboratory using our previously established method. In vitro binding and internalization studies and subsequent fluorescence microscopy demonstrated the effective nuclear delivery of the doxorubicin in the MPNST cells expressing IL13Rα2 receptor. The cytotoxic potential of IL13LDXR was demonstrated in MPNST cell lines sNF96.2, ST88-14 and #215. Immunohistochemistry was performed on malignant and benign peripheral nerve sheath tumor (PNST) sections ex vivo to determine the expression of IL13Rα2 receptor. In addition we also demonstrated the expression of P-glycoprotein, a multidrug resistant protein in MPNST cell lines by immunocytochemistry. We also studied the effect of IL-13 protein in altering the expression of Pgp expression in established MPNST cell lines. We confirmed by an immunoblot that IL13 protein at 5µM concentration was able to decrease the Pgp expression in ST88-14 and sNF96.2 cells. This may explain the mechanism by which the IL-13 conjugated liposomes can deliver the encapsulated doxorubicin in a much more effective manner to drug resistant MPNST cell lines. We investigated the correlation of IL13Rα2 receptor expression with P-glycoprotein a drug resistance associated protein by immunocytochemistry. We confirmed the expression of IL13Rα2 in PNST tumors and established MPNST cell lines. We attempted to develop a MPNST tumor model in athymic nude mice by matrigel implantation method. We administered a single injection of 15 mg/kg of IL13 linked liposomal doxorubicin in a mouse tumor model by intraperitoneal route and observed the selective accumulation of doxorubicin in the tumor by endogenous fluorescence of doxorubicin, whereas no fluorescence was observed in the normal skin region as observed under fluorescence microscope. In conclusion consistent expression of IL13Ra2 receptors in PNST and MPNST cells and tissues indicate a viable approach to use this receptor as a target for MPNST therapy. The doxorubicin accumulation property and its cytotoxic potential on MPNST cells when delivered though the targeted liposomes envisage IL13LDXR as a potential drug for MPNST. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5521.

Iron oxide crystal formation on a substrate modified with the Mms6 protein from magnetotactic bacteria

AbstractMms6 is a small acidic protein which is tightly bound to magnetite in the bacterium Magnetospirillum magneticum AMB-1. Mms6 has been previously shown to promote iron-binding capacity as well as modulate the size and morphology of magnetic iron oxide crystals in vitro. In this study, we synthesized iron oxide crystals by using a monolayer-modified substrate. A self-assembled monolayer of octadecyltrimethoxysilane was modified on a silicon substrate. Recombinant Mms6 protein was attached to the substrate through the hydrophobic interactions between the protein molecules and the monolayer. The immobilization of protein molecules on the substrate surface was confirmed by fluorescent labeling of these molecules and subsequent fluorescence microscopy. This protein-modified substrate was then used as a template for iron oxide crystal formation in a ferrous solution. Scanning electron microscopy revealed site-specific formation of iron oxide crystals in substrate regions with immobilized proteins. This use of proteins might provide an alternative method for the bottom-up fabrication of nano-sized magnetic particles.

The novel protein PTPIP51 is expressed in human keratinocyte carcinomas and their surrounding stroma

Background: The novel protein PTPIP51 (SwissProt accession code Q96SD6) is known to interact with two non-transmembrane protein-tyrosine phosphatases, PTP1B and TCPTP in vitro. Overexpression of the full-length protein induces apoptosis in HeLa and HEK293T cells (Lv et al. 2006). PTPIP51 shows a tissue-specific expression pattern and is associated with cellular differentiation and apoptosis in some mammalian tissues, especially in human follicular and interfollicular epidermis. PTPIP51 protein is expressed in all suprabasal layers of normal epidermis, whereas the basal layer contains PTPIP51 mRNA only but lacks the protein. Objectives: The expression of PTPIP51 was investigated in keratinocyte carcinomas, that is human basal cell carcinomas (BCCs) and squamous cell carcinomas (SCCs) as well as Bowen's disease (BD) and keratoacanthomas (KAs) on a transcriptional (mRNA) and translational (immunohistochemical) level. Methods: Formalin-fixed, paraffin-embedded sections of BCCs, SCCs, KAs and BD, respectively, were analysed by RT-PCR, as well as immunohistochemistry and subsequent fluorescence microscopy. PTPIP51-positive cells of the tumour and the surrounding stroma were identified on the basis of specific morphological features by means of H & E staining. To obtain further information about a putative function of PTPIP51, a possible association of PTPIP51 with apoptotic cells, as well as an assumed negative correlation with proliferating cells was investigated by means of an in-situ TUNEL assay and Ki67/MIB-1 antigen staining, respectively. Co-immunostainings with PTPIP51 were performed for the following antigens: TCPTP, PTP1B and β-catenin. Results: PTPIP51-expression was detected in BCCs and SCCs of the skin, as well as in KAs and BD. Both types of keratinocyte carcinoma revealed a specific localization pattern of PTPIP51 in malignant keratinocytes. Whereas PTPIP51 -positive cells of BCC were found to form two cluster types with a different subcellular localization of the protein, i.e. cytoplasmic and nuclear or predominantly membranous, investigation of SCC revealed a meshwork-like appearance of PTPIP51-positive malignant keratinocytes, created by a mainly membranous localization. BD and KA resembled the findings of PTPIP51-expression in SCC. Furthermore, we observed a partial co-localization of PTP1B and PTPIP51 in BCC. SCC and BCC showed a co-expression and partial co-localization of PTPIP51 with β-catenin. Some PTPIP51-positive cells were found to undergo apoptosis. PTPIP51 was also expressed in cells comprising the surrounding stromal microenvironment. This was particularly noticed for endothelial cells lining peritumoural vessels as well as for infiltrating cells of both, the innate and the adaptive immune system. Conclusions: The results showed a distinct mainly membranous expression pattern of PTPIP51 in BCCs and SCCs. Since PTPIP51 was also detected in the peritumoural tissue, the protein may play a crucial role in keratinocyte tumour development.

Synthesis and characterization of oxazinone and oxazoline substituted pyrroles: Towards electrically conducting bi-functional copolymers

The synthesis of N-(3-aminopropyl)pyrrole and N-(2-carboxyethyl)pyrrole was reviewed and repeated. Both compounds were utilized as pre-cursors for the synthesis of oxazinone and oxazoline modified pyrroles by different synthetic pathways. The N-substituted pyrroles were characterized by 1H NMR, ATR-FTIR, and MALDI-TOF MS. The chemical oxidative copolymerization of pyrrole and its derivatives was carried out in various solvent systems using FeCl 3 and (NH 4) 2S 2O 8 as oxidants. Furthermore, sodium poly(styrene sulfonate) was used as dopant leading to core-shell-like structures, and, after the copolymerization of pyrrole with oxazinone or oxazoline functionalized monomers, to exceptionally increased conductivities. The produced (co-)polymers were characterized in terms of their chemical composition, morphology, and electrical conductivity. Oxazinone and oxazoline moieties in the copolymers were confirmed by ATR-FTIR. Furthermore, the retained reactivity of oxazinone and oxazoline functions after chemical oxidative copolymerization was evidenced by model reactions with 1-pyrenemethylamine and 1-pyreneacetic acid, respectively, and subsequent fluorescence microscopy and spectroscopy.

A New Type of Peroxisomal Acyl-Coenzyme A Synthetase from Arabidopsis thaliana Has the Catalytic Capacity to Activate Biosynthetic Precursors of Jasmonic Acid

Arabidopsis thaliana contains a large number of genes that encode carboxylic acid-activating enzymes, including nine long-chain fatty acyl-CoA synthetases, four 4-coumarate:CoA ligases (4CL), and 25 4CL-like proteins of unknown biochemical function. Because of their high structural and sequence similarity with bona fide 4CLs and their highly hydrophobic putative substrate-binding pockets, the 4CL-like proteins At4g05160 and At5g63380 were selected for detailed analysis. Following heterologous expression, the purified proteins were subjected to a large scale screen to identify their preferred in vitro substrates. This study uncovered a significant activity of At4g05160 with medium-chain fatty acids, medium-chain fatty acids carrying a phenyl substitution, long-chain fatty acids, as well as the jasmonic acid precursors 12-oxo-phytodienoic acid and 3-oxo-2-(2'-pentenyl)-cyclopentane-1-hexanoic acid. The closest homolog of At4g05160, namely At5g63380, showed high activity with long-chain fatty acids and 12-oxo-phytodienoic acid, the latter representing the most efficiently converted substrate. By using fluorescent-tagged variants, we demonstrated that both 4CL-like proteins are targeted to leaf peroxisomes. Collectively, these data demonstrate that At4g05160 and At5g63380 have the capacity to contribute to jasmonic acid biosynthesis by initiating the beta-oxidative chain shortening of its precursors.

Local circuit interactions between oriens/alveus interneurons and CA1 pyramidal cells in hippocampal slices: electrophysiology and morphology

Electrophysiological and anatomical techniques were used to determine the role, in the hippocampal circuitry, of local circuit neurons located at the oriens/alveus border (O/A interneurons). Intracellular recording from these cells showed that their response characteristics were clearly nonpyramidal: high input resistance, short membrane time constant, short-duration action potential, pronounced, brief afterhyperpolarizations (AHP), and nondecremental firing during intrasomatic depolarizing current pulses. Intracellular Lucifer yellow (LY) injection and subsequent fluorescence microscopy confirmed their nonpyramidal nature. O/A interneuron somata were bipolar or multipolar; their dendrites projected mostly parallel to the alveus, except for 1 or 2 processes that turned perpendicularly, and ascended through stratum oriens and pyramidale and into radiatum. Their axons were seen to branch profusely in stratum oriens and pyramidale. Simultaneous intracellular recordings from O/A interneurons and CA 1 pyramidal cells showed that pyramidal cells directly excite these interneurons. Major hippocampal afferents also directly excited the O/A interneurons. In a small number of interneuron-pyramidal pairs, stimulation of the O/A interneuron directly inhibited pyramidal cells. In one case, reciprocal connections were observed: The pyramidal cell excited the interneuron, and the interneuron inhibited the pyramidal cell. In 1 interneuron-to-interneuron pair, an inhibitory connection from O/A interneuron to stratum pyramidale interneuron was also observed. With intracellular HRP injections into O/A interneurons and subsequent electron microscopy, we observed that O/A interneuron axons made contacts with pyramidal and nonpyramidal cells. HRP-filled symmetric synaptic contacts were found on pyramidal cell dendrites and somata. HRP-filled axons also made contacts with pyramidal cell initial segments. HRP-filled O/A interneuron axon contacts were also found on nonpyramidal cell dendrites in stratum oriens. These electrophysiological and anatomical results suggest that O/A interneurons make synaptic contact with pyramidal cells and may mediate feedforward and feedback inhibition onto CA 1 pyramidal cells.

Location of histological structure of the brain for subsequent monoamine fluorescence with the help of illumination by phase contrast condenser.

Various histological structures from rat brain prepared by the formaldehyde induced fluorescence method can be visualized with the help of illumination in tungsten light by using phase contrast condenser (without phase contrast objective). The procedure was found useful for the location of areas to be studied by subsequent fluorescence microscopy in order to relate the monoamine fluorescence to the histological structure.