Immunocytochemical localization of tubulin with colloidal gold in cilia of rabbit tracheal epithelial cultures

Ciliated tracheal epithelia cell cultures were investigated immunocytochemically with anti-tubulin and colloidal gold. When rabbit tracheal cultures were fixed in paraformaldehyde, treated with acetone, anti-tubulin and a second antibody coupled to FITC, fluorescence was associated with cytoskeletal and axonemal microtubules. Cilia covering the apical surface of the ciliated tracheal cells fluoresced very brightly thus facilitating identification of this cell type. Electron microscopy of tracheal cultures fixed as above, treated with Triton-X 100 and incubated in anti-tubulin and protein A coupled to colloidal gold resulted in the highly specific localization of tubulin in ciliary axonemes and basal bodies. Omission of primary or secondary antibody resulted in extremely low levels of fluorescence while no colloidal gold particles could be detected in cultures at the electron microscopy level when rabbit anti-tubulin was omitted.     

A comparative Proteomics Analysis Identified Differentially Expressed Proteins in Pancreatic Cancer–Associated Stellate Cell Small Extracellular Vesicles

Human pancreatic stellate cells (HPSCs) are an essential stromal component and mediators of pancreatic ductal adenocarcinoma (PDAC) progression. Small extracellular vesicles (sEVs) are membrane-enclosed nanoparticles involved in cell-to-cell communications and are released from stromal cells within PDAC. A detailed comparison of sEVs from normal pancreatic stellate cells (HPaStec) and from PDAC-associated stellate cells (HPSCs) remains a gap in our current knowledge regarding stellate cells and PDAC. We hypothesized there would be differences in sEVs secretion and protein expression that might contribute to PDAC biology. To test this hypothesis, we isolated sEVs using ultracentrifugation followed by characterization by electron microscopy and Nanoparticle Tracking Analysis. We report here our initial observations. First, HPSC cells derived from PDAC tumors secrete a higher volume of sEVs when compared to normal pancreatic stellate cells (HPaStec). Although our data revealed that both normal and tumor-derived sEVs demonstrated no significant biological effect on cancer cells, we observed efficient uptake of sEVs by both normal and cancer epithelial cells. Additionally, intact membrane-associated proteins on sEVs were essential for efficient uptake. We then compared sEV proteins isolated from HPSCs and HPaStecs cells using liquid chromatography–tandem mass spectrometry. Most of the 1481 protein groups identified were shared with the exosome database, ExoCarta. Eighty-seven protein groups were differentially expressed (selected by 2-fold difference and adjusted p value ≤0.05) between HPSC and HPaStec sEVs. Of note, HPSC sEVs contained dramatically more CSE1L (chromosome segregation 1–like protein), a described marker of poor prognosis in patients with pancreatic cancer. Based on our results, we have demonstrated unique populations of sEVs originating from stromal cells with PDAC and suggest that these are significant to cancer biology. Further studies should be undertaken to gain a deeper understanding that could drive novel therapy.     

Development and evaluation of different strategies for the clean synthesis of silver nanoparticles using Yarrowia lipolytica and their antibacterial activity

An environment-friendly, cheap method, biogenic synthesis of silver nanoparticles (AgNPs) is interesting as compared to physical and chemical synthesis methods. The aim of the present study was to utilize the inherent capability of Yarrowia lipolytica as a novel biocatalyst for green production of AgNPs using different strategies, including growing cells, resting cells, and cell-free extracts (CFE) under optimized reaction conditions. The produced AgNPs were evaluated with UV–vis spectroscopy, field emission scanning electron microscopy, energy dispersive X-ray analysis, X-ray diffraction, and Fourier transform infrared spectrometry. In the growing cells strategy, Y. lipolytica produced spherical AgNPs under the optimized conditions, 2.5 mM of silver ions, 7.5 g/l of yeast biomass, a temperature of 30 °C, a pH of 6, and a shaking rate of 50 rpm after 48 h. The sizes and monodispersity of the AgNPs in the resting cells strategy were better than those in the other two. However, the AgNPs were produced faster in the CFE strategy. The antibacterial activity and minimal inhibitory concentration of the AgNPs against certain Gram-positive and Gram-negative bacteria were determined by the agar well diffusion and broth microdilution methods. The AgNPs had a considerable antibacterial effect compared to chloramphenicol as a broad-spectrum antibiotic.     

Nanovaccine: A novel approach in immunization

Despite great advances in the field of vaccination, there are still needs for novel and effective vaccines because still no effective vaccines have been produced for some diseases such as malaria, acquired immune deficiency syndrome (AIDS), and tuberculosis. Furthermore, many of the existing vaccines have disadvantages such as failure to stimulate completely the immune system, in vivo instability, high toxicity, the need for cold chain, and multiple administrations. Nanotechnology has been raised as a powerful tool for solving these problems in this regard. Generally, nanovaccines are a new generation of vaccines using nanoparticles (NPs) as carriers and/or adjuvants. Due to the similar scale (size) between the NPs and pathogens, the immune system can be stimulated well, resulting in triggered cellular and humoral immunity responses. Other benefits of the nanovaccines include their better stability in blood flow to increase the shelf life in blood, enhanced immune system stimulation, no need for booster doses, no need to maintain the cold chain, and ability to create active targeting. In addition, nanovaccines have raised the hope to treat diseases such as rheumatoid arthritis, AIDS, malaria, and chronic autoimmune, and so forth.     

Synthesis of isochroman-4-ones and 2H-pyran-3(6H)-ones by gold-catalyzed oxidative cycloalkoxylation of alkynes

Gold catalysis is a convenient tool to oxidatively functionalize alkyne into a range of valuable compounds. In this article, we report a new access to isochroman-4-one and 2H-pyran-3(6H)-one derivatives that involves a gold-catalyzed oxidative cycloalkoxylation of an alkyne in the presence of a pyridine N-oxide. The reaction proceeds under mild conditions, is relatively efficient and exhibits a high functional group compatibility.     

Gold Toning Improves the Visualization of Nucleolar Organizer Regions in Paraffin Embedded Tissues

A modification of the silver colloid technique for staining nucleolar organizer regions in paraffin embedded tissues is described. This modification involves the application of a gold toning step with subsequent gold reduction, if necessary, following incubation of sections in the standard silver colloid solution. Silver stained nucleolar organizer regions (AgNORs) in toned sections are more sharply delineated when compared to untoned controls. in high grade tumors the addition of the toning step results in significantly higher AgNOR counts due to the ability to discriminate more easily individual AgNORs in argyrophilic aggregates within the nucleus. It is recommended, because of enhanced visualization, that this modification of the silver colloid technique be used in studies involving quantification of AgNORs in tissue sections.     

Effect of redox-responsive DTSSP crosslinking on poly(l-lysine)-grafted-poly(ethylene glycol) nanoparticles for delivery of proteins

Therapeutic proteins are utilized in a variety of clinical applications, but side effects and rapid in vivo clearance still present hurdles. An approach that addresses both drawbacks is protein encapsulation within in a polymeric nanoparticle, which is effective but introduces the additional challenge of destabilizing the nanoparticle shell in clinically relevant locations. This study examined the effects of crosslinking self-assembled poly(l -lysine)-grafted-poly(ethylene glycol) nanoparticles with redox-responsive 3,3′-dithiobis(sulfosuccinimidyl propionate) (DTSSP) to achieve nanoparticle destabilization in a reductive environment. The polymer-protein nanoparticles (DTSSP NPs) were formed through electrostatic self-assembly and crosslinked with DTSSP, which contains a glutathione-reducible disulfide. As glutathione is upregulated in various cancers, DTSSP NPs could display destabilization within cancer cells. A library of DTSSP NPs was formed with varying copolymer to protein (C:P) and crosslinker to protein (X:P) mass ratios and characterized by size and encapsulation efficiency. DTSSP NPs with a 7:1 C:P ratio and 2:1 X:P ratio were further characterized by stability in the presence proteases and reducing agents. DTSSP NPs fully encapsulated the model protein and displayed 81% protein release when incubated with 5 mM dithiothreitol for 12 hr. This study contributes to understanding stimulus-responsive crosslinking of polymeric nanoparticles and could be foundational to clinical administration of therapeutic proteins.     

A biomolecular recognition approach for the functionalization of cellulose with gold nanoparticles

Materials with new and improved functionalities can be obtained by modifying cellulose with gold nanoparticles (AuNPs) via the in situ reduction of a gold precursor or the deposition or covalent immobilization of pre‐synthesized AuNPs. Here, we present an alternative biomolecular recognition approach to functionalize cellulose with biotin‐AuNPs that relies on a complex of 2 recognition elements: a ZZ‐CBM3 fusion that combines a carbohydrate‐binding module (CBM) with the ZZ fragment of the staphylococcal protein A and an anti‐biotin antibody. Paper and cellulose microparticles with AuNPs immobilized via the ZZ‐CBM3:anti‐biotin IgG supramolecular complex displayed an intense red color, whereas essentially no color was detected when AuNPs were deposited over the unmodified materials. Scanning electron microscopy analysis revealed a homogeneous distribution of AuNPs when immobilized via ZZ‐CBM3:anti‐biotin IgG complexes and aggregation of AuNPs when deposited over paper, suggesting that color differences are due to interparticle plasmon coupling effects. The approach could be used to functionalize paper substrates and cellulose nanocrystals with AuNPs. More important, however, is the fact that the occurrence of a biomolecular recognition event between the CBM‐immobilized antibody and its specific, AuNP‐conjugated antigen is signaled by red color. This opens up the way for the development of simple and straightforward paper/cellulose‐based tests where detection of a target analyte can be made by direct use of color signaling.     

Gold Toning Improves the Visualization of Nucleolar Organizer Regions in Paraffin Embedded Tissues

A modification of the silver colloid technique for staining nucleolar organizer regions in paraffin embedded tissues is described. This modification involves the application of a gold toning step with subsequent gold reduction, if necessary, following incubation of sections in the standard silver colloid solution. Silver stained nucleolar organizer regions (AgNORs) in toned sections are more sharply delineated when compared to untoned controls. in high grade tumors the addition of the toning step results in significantly higher AgNOR counts due to the ability to discriminate more easily individual AgNORs in argyrophilic aggregates within the nucleus. It is recommended, because of enhanced visualization, that this modification of the silver colloid technique be used in studies involving quantification of AgNORs in tissue sections.