Imaging of Oil/Monoglyceride Networks by Polarizing Near-Field Scanning Optical Microscopy

Polarizing near-field scanning optical microscopy (NSOM) was applied for visualization of lipid coagel structures. The technique ensures obtaining polarization contrast images at micro- and nanoscale resolution. Comparison to the polarizing light microscopy images revealed that the same fractal structural organization persists also at submicron scale, at the level of primary ordered structures creation. Many long birefringent needle-shaped primary crystallites were imaged in the corn oil:monoglyceride samples, and lower amount of smaller oval-shaped primary crystallites—in the olive oil:monoglyceride samples. Unlike atomic force microscopy, polarizing NSOM brought direct evidence on the physical state of specific features. Compared to the polarizing light microscopy, polarizing NSOM provided additional information on the structural organization of oil–monoglyceride coagels at the micro- and submicron scale.     

Osmotically Induced Reversible Transitions in Lipid-DNA Mesophases

We follow the effect of osmotic pressure on isoelectric complexes that self-assemble from mixtures of DNA and mixed neutral and cationic lipids. Using small angle x-ray diffraction and freeze-fracture cryo-electron microscopy, we find that lamellar complexes known to form in aqueous solutions can reversibly transition to hexagonal mesophases under high enough osmotic stress exerted by adding a neutral polymer. Using molecular spacings derived from x-ray diffraction, we estimate the reversible osmotic pressure-volume (Π-V) work needed to induce this transition. We find that the transition free energy is comparable to the work required to elastically bend lipid layers around DNA. Consistent with this, the required work is significantly lowered by an addition of hexanol, which is known to soften lipid bilayers. Our findings not only help to resolve the free-energy contributions associated with lipid-DNA complex formation, but they also demonstrate the importance that osmotic stress can have to the macromolecular phase geometry in realistic biological environments.     

EGFR and HER2 exert distinct roles on colon cancer cell functional properties and expression of matrix macromolecules

Background

ErbB receptors, EGFR and HER2, have been implicated in the development and progression of colon cancer. Several intracellular pathways are mediated upon activation of EGFR and/or HER2 by EGF. However, there are limited data regarding the EGF-mediated signaling affecting functional cell properties and the expression of extracellular matrix macromolecules implicated in cancer progression.

Methods

Functional assays, such as cell proliferation, transwell invasion assay and migration were performed to evaluate the impact of EGFR/HER2 in constitutive and EGF-treated Caco-2 cells. Signaling pathways were evaluated using specific intracellular inhibitors. Western blot was also utilized to examine the phosphorylation levels of ERK1/2. Real time PCR was performed to evaluate gene expression of matrix macromolecules.

Results

EGF increases cell proliferation, invasion and migration and importantly, EGF mediates overexpression of EGFR and downregulation of HER2. The EGF–EGFR axis is the main pathway affecting colon cancer's invasive potential, proliferative and migratory ability. Intracellular pathways (PI3K-Akt, MEK1/2-Erk and JAK-STAT) are all implicated in the migratory profile. Notably, MT1- and MT2-MMP as well as TIMP-2 are downregulated, whereas uPA is upregulated via an EGF–EGFR network. The EGF–EGFR axis is also implicated in the expression of syndecan-4 and TIMP-1. However, glypican-1 upregulation by EGF is mainly mediated via HER2.

Conclusions and general significance

The obtained data highlight the crucial importance of EGF on the expression of both receptors and on the EGF–EGFR/HER2 signaling network, reveal the distinct roles of EGFR and HER2 on expression of matrix macromolecules and open a new area in designing novel agents in targeting colon cancer. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

A general platform for antibody purification utilizing engineered-micelles

We introduce a new concept and potentially general platform for antibody (Ab) purification that does not rely on chromatography or specific ligands (e.g., Protein A); rather, it makes use of detergent aggregates capable of efficiently capturing Ab while rejecting hydrophilic impurities. Captured Ab are then extracted from the aggregates in pure form without co-extraction of hydrophobic impurities or aggregate dissolution. The aggregates studied consist of conjugated “Engineered-micelles” built from the nonionic detergent, Tween-20; bathophenanthroline, a hydrophobic metal chelator, and Fe²⁺ions. When tested in serum-free media with or without bovine serum albumin as additive, human or mouse IgGs were recovered with good overall yields (70–80%, by densitometry). Extraction of IgGs with 7 different buffers at pH 3.8 sheds light on possible interactions between captured Ab and their surrounding detergent matrix that lead to purity very similar to that obtained via Protein A or Protein G resins. Extracted Ab preserve their secondary structure, specificity and monomeric character as determined by circular dichroism, enzyme-linked immunosorbent assay and dynamic light scattering, respectively.     

Morphological analysis of young and old pellicles of Salmonella Typhimurium

A wide variety of microorganisms are able to form biofilms at the interface between air and liquid (pellicles). In this study changes during the maturation of the pellicle of Salmonella Typhimurium were analysed and the role of cellulose in the pellicle structure and morphology evaluated. The morphology of both sides of the pellicle was characterised using atomic force microscopy and scanning electron microscopy. Overall, there was a marked difference in the morphology of the water-facing (WF) and air-facing (AF) biofilm surfaces. While the AF side appeared to be uniform, and extensively covered with an exocellular coating, cells in the WF side were distributed into clusters and were less covered. However, the similarity in size and shape of single cells from both sides of the pellicle may indicate that the bacterial cells across the pellicle have a similar physiological status. During maturation, porous structures with multiple cracks and channels were created in the pellicle, leading to disintegration. By comparison with the structure of pellicles of a cellulose-deficient mutant, it was demonstrated that the observed disintegration of mature pellicles probably occurred in part by self-hydrolysis of components of the matrix.     

Morphological analysis of young and old pellicles of Salmonella Typhimurium

Abstract

A wide variety of microorganisms are able to form biofilms at the interface between air and liquid (pellicles). In this study changes during the maturation of the pellicle of Salmonella Typhimurium were analysed and the role of cellulose in the pellicle structure and morphology evaluated. The morphology of both sides of the pellicle was characterised using atomic force microscopy and scanning electron microscopy. Overall, there was a marked difference in the morphology of the water-facing (WF) and air-facing (AF) biofilm surfaces. While the AF side appeared to be uniform, and extensively covered with an exocellular coating, cells in the WF side were distributed into clusters and were less covered. However, the similarity in size and shape of single cells from both sides of the pellicle may indicate that the bacterial cells across the pellicle have a similar physiological status. During maturation, porous structures with multiple cracks and channels were created in the pellicle, leading to disintegration. By comparison with the structure of pellicles of a cellulose-deficient mutant, it was demonstrated that the observed disintegration of mature pellicles probably occurred in part by self-hydrolysis of components of the matrix.     

Metastasis to the breast from an adenocarcinoma of the lung with extensive micropapillary component: a case report and review of the literature

Intranodal palisaded myofibroblastoma (IPM) is a benign mesenchymal neoplasm originating from smooth muscle cells and myofibroblasts. It is characterized by spindle cells, amianthoid fibers, and by the proliferation of hemosiderin-containing histiocytes in the lymph node. A nodular lesion was excised from the inguinal region of an 80-year-old male patient. Macroscopic examination of a section of the lesion demonstrated a solid appearance with hemorrhagic areas. Microscopic examination revealed spindle cell proliferation, amianthoid fibers, hemosiderin pigment, and extravasated erythrocytes. Nuclei of the spindle cells displayed a palisaded appearance. Compressed lymphoid tissue was observed around the lesion. With Masson's trichrome, spindle cells stained as smooth muscle, whereas collagen staining was observed in homogeneous eosinophilic accumulations. Neoplastic cells were identified by the presence of vimentin and SMA. The Ki67 index was less than 1%. In light of these results, the case was diagnosed as "intranodal palisaded myofibroblastoma." IPM is an uncommon neoplasm originating from the stromal component of the lymph node. Although IPM is benign, it is frequently confused with metastatic lesions.     

Controlled micelle conjugation via charged peptide amphiphiles

Abstract We report the first demonstration of nonionic detergent micelle conjugation and phase separation using purpose‐synthesized, peptide amphiphiles, C₁₀‐(Asp)₅ and C₁₀‐(Lys)₅. Clustering is achieved in two different ways. Micelles containing the negatively charged peptide amphiphile C₁₀‐(Asp)₅ are conjugated (a) via a water‐soluble, penta‐Lys mediator or (b) to micelles containing the C₁₀‐(Lys)₅ peptide amphiphile. Both routes lead to phase separation in the form of oil‐rich globules visible in the light microscope. The hydrophobic nature of these regions leads to spontaneous partitioning of hydrophobic dyes into globules that were found to be stable for weeks to months. Extension of the conjugation mechanism to micelles containing a recently discovered, light‐driven proton pump King Sejong 1‐2 (KS1‐2) demonstrates that a membrane protein may be concentrated using peptide amphiphiles while preserving its native conformation as determined by characteristic UV absorption. The potential utility of these peptide amphiphiles for biophysical and biomedical applications is discussed.     

Fluorinated amphiphiles control the insertion of α-hemolysin pores into lipid bilayers

The insertion of fully folded and assembled ion channels and pores into planar lipid bilayers for electrical recording has been facilitated by the use of conventional detergents at a final concentration below the critical micelle concentration (CMC). After the desired number of channels or pores (often one) has been incorporated into a bilayer, it is important to prevent further insertion events, which is often done by awkward techniques such as perfusion. Here, we show that the addition of single-chain fluorinated amphiphiles (F-amphiphiles) with zwitterionic, simple neutral, and neutral oligomeric headgroups at a concentration above the CMC prevents the further insertion of staphylococcal α-hemolysin pores, MspA pores, and Kcv potassium channels into lipid bilayers. We found the commercially available F(6)FC (fluorinated fos-choline with a C(6)F(13)C(2)H(4) chain) to be the least perturbing and most effective agent for this purpose. Bilayers are known to be resistant to F-amphiphiles, which in this case we suppose sequester the pores and channels within amphiphile aggregates. We suggest that F-amphiphiles might be useful in the fabrication of bilayer arrays for nanopore sensor devices and the rapid screening of membrane proteins.