三维电子显微学自动化数据采集的进展

生物三维电子显微学在过去几年取得了巨大的突破,一些具有高对称性的病毒颗粒获得了准原子分辨率的结构,非对称性的生物大分子及其复合体的结构分辨率也有快速的提高。而要获得高分辨率的结构,获取足够多的高质量电子显微照片是其中的一个关键因素。近年来,自动化数据采集技术在电子断层成像术和单颗粒方法中都取得了很大的进展。其广泛应用将使结构测定更加快速并使结构分辨率提高到更高的层次。     

Quantifying the Mechanical Properties of the Endothelial Glycocalyx with Atomic Force Microscopy

Our understanding of the interaction of leukocytes and the vessel wall during leukocyte capture is limited by an incomplete understanding of the mechanical properties of the endothelial surface layer. It is known that adhesion molecules on leukocytes are distributed non-uniformly relative to surface topography ³, that topography limits adhesive bond formation with other surfaces ⁹, and that physiological contact forces (≈ 5.0 − 10.0 pN per microvillus) can compress the microvilli to as little as a third of their resting length, increasing the accessibility of molecules to the opposing surface ^{3, 7}. We consider the endothelium as a two-layered structure, the relatively rigid cell body, plus the glycocalyx, a soft protective sugar coating on the luminal surface ⁶. It has been shown that the glycocalyx can act as a barrier to reduce adhesion of leukocytes to the endothelial surface ⁴. In this report we begin to address the deformability of endothelial surfaces to understand how the endothelial mechanical stiffness might affect bond formation. Endothelial cells grown in static culture do not express a robust glycocalyx, but cells grown under physiological flow conditions begin to approximate the glycocalyx observed in vivo ². The modulus of the endothelial cell body has been measured using atomic force microscopy (AFM) to be approximately 5 to 20 kPa ⁵. The thickness and structure of the glycocalyx have been studied using electron microscopy ⁸, and the modulus of the glycocalyx has been approximated using indirect methods, but to our knowledge, there have been no published reports of a direct measurement of the glycocalyx modulus in living cells. In this study, we present indentation experiments made with a novel AFM probe on cells that have been cultured in conditions to maximize their glycocalyx expression to make direct measurements of the modulus and thickness of the endothelial glycocalyx.     

Three-Dimensional Immunoelectron Microscopy of Scorpion Hemocyanin Labeled with a Monoclonal Fab Fragment

An immunocomplex of the 4 × 6-meric hemocyanin of the scorpion Androctonus australis with the monoclonal Fab fragment L104 was reconstructed from electron micrographs of a negatively stained specimen, using the double-carbon-layer technique. The resulting structure enables a clear visualization of the Fab fragments bound to the four copies of the Aa6 subunit and directly confirms a previous localization of the L104 epitope deduced from two-dimensional image processing. Despite a strong flattening effect produced by the negative-staining technique the orientations of the Fab fragments are well characterized. Moreover, the observation of a central hole within the elbow bends of the Fab fragments provides information about the disposition of the Fabs around their main axis.     

Scanning Electron Microscopy of Cell-Surface Changes in Methylnitrosurea (MNU)-Treated Rat Bladders in vivo and in vitro

Scanning electron microscopy has been used (1) to characterize epithelial cells of bladders from normal rats and from rats treated with a single initiating but non-carcinogenic dose of 2 mg methylnitrosurea (MNU), 24 h and 6 weeks after treatment; and (2) to compare morphological aspects of epithelial differentiation in organ culture of bladder explants taken from untreated and MNU-treated rats at these time intervals.
There are marked differences in vivo between the surface organization of normal urothelium and urothelium undergoing reversible hyperplasia following MNU treatment. Maturation of the normal rat bladder epithelium in vivo is shown to be related to a series of well-defined cell-surface changes readily identified by SEM. By contrast the maturation response is perturbed in the hyperplastic epithelium; the cells lose their ability to differentiate normally and form instead an excess of stubby globular microvilli which project from the cell surface.
In organ culture, maturation of normal bladder epithelium (both in re-epithelialized areas of the explant and in areas of epithelial outgrowth over cellulose acetate substrates) can be also related to a series of cell surface changes showing close similarities to those in vivo. However, epithelial maturation remains defective in organ cultures of bladders from MNU-treated animals. The closely parallel behaviour of the bladder epithelium in vivo and in vitro in both normal and treated tissues underlines the potential value of the bladder organ culture system for studying the comparative biology of hyperplastic development produced by a single initiating dose of MNU and suggests it will be useful with which to study carcinogenesis following multiple doses of MNU.     

A New Histochemical Double-Stain Method Using Three-Dimensional Analysis with Confocal laser Scanning Microscopy

We describe a new technique for immunohistochemical and enzyme-histochemi-cal double staining using confocal laser scanning microscopy in the reflection mode. As an example, we investigated the immunoreactivity for Spot 35-calbindin-D_28K, a vitamin D-dependent calcium binding protein, and the enzyme activity for ma⁺-ATPase in the rat kidney. The lead precipitation method for Ca²⁺-ATPase was initially used to process kidney slices. Each specimen was then dehydrated and embedded in a water soluble resin. Thin sections were cut from the resin block, and an indirect immunocolloidal gold method with silver enhancement for Spot 35-calbindin-D_28K antigen was carried out on the glass slides. Results were then observed by confocal laser scanning microscopy in the reflection mode. The three-dimensional distribution of the reaction products was detected by serial optic slice images. Lead phosphate particles, which represented the location of Ca²⁺-ATPase, were distributed deep in the section. The most intense signals from the silver partkles were detected from the surface slice of the section. A stereoscopic image generated from the serial optic slices clearly showed the differences in their distribution.     

Spexin Expression in Normal Rat Tissues

Spexin is a highly conserved peptide which was recently identified through the bioinformatics approach. Immunohistochemical analysis of its expression has not yet been performed. Thus, in this study, we examined spexin location in a wide range of rat organs by both RT-PCR and IHC. RT-PCR identified spexin mRNA in all tissues examined. Spexin immunoreaction was mainly cytoplasmic. Spexin was immunohistochemically detected, although with different staining intensities, in epithelia and glands of skin and respiratory, digestive, urinary, and reproductive systems. Smooth muscle cells showed weak immunostaining, and connective tissue was negative. In the central nervous system, neuronal groups showed different intensities for reaction product. Immunoreaction was also found in ganglionic cells of both trigeminal and superior cervical ganglia and in photoreceptor, inner nuclear, and ganglionic layers of the retina. In the endocrine system, spexin immunoreaction was detected in the hypothalamic paraventricular and supraoptic nuclei; adenohypophysis, thyroid, and parathyroid glands; adrenal cortex and medulla (mainly ganglionic cells); Leydig cells; and thecal, luteal, and interstitial cells of the ovary. Because of its widespread expression, spexin is probably involved in many different physiological functions; in particular, location of spexin in neurons and endocrine cells suggests its roles as neurotransmitter/neuromodulator and endocrine factor. (J Histochem Cytochem 58:825–837, 2010)     

Staining of Different Tissues in Thick Epoxy Resin-Impregnated Sections of Human Fetuses

Sections of undecalcified human fetuses, fixed in formaldehyde, embedded in the epoxy resin Biodur E 12 and cut on a diamond-wire saw were stained according to a slight modification of the method described by Laczko and Levai. The sections were immersed in a methylene blue/azure II solution at 90 C for at least 3 min and counterstained with a basic fuchsin solution at the same temperature. Differential staining was as follows: bone stained pinkish; cartilage, violet; collagen fibers, blue-violet; elastic fibers, red and muscle fibers, green-blue. Most other tissues were stained blue-violet against the transparent background of the embedding epoxy resin. Thanks to the distinct and differential staining of each tissue, contrast is sufficient for black and white as well as for color photography.     

Quantifying fibronectin adhesion with nanoscale spatial resolution on glycosaminoglycan doped polypyrrole using Atomic Force Microscopy

Background

The interaction of ECM proteins is critical in determining the performance of materials used in biomedical applications such as tissue regeneration, implantable bionics and biosensing.

Methods

To improve our understanding of ECM protein–conducting polymer interactions, we have used Atomic Force Microscopy (AFM) to elucidate the interactions of fibronectin (FN) on polypyrrole (PPy) doped with different glycosaminoglycans.

Results

We were able to classify four main types of FN interactions, including those related to 1) non-specific adhesion, 2) protein unfolding and subsequent unbinding from the surface, 3) desorption and 4) interactions with no adhesion. FN adhesion on PPy/hyaluronic acid showed a significantly lower density of surface adhesion with the adhesion restricted to nodule structures, as opposed to their peripheries, of the polymer morphology. In contrast, PPy/chondroitin sulfate showed a significantly higher density of surface adhesion to the point where the distribution of adhesion effectively masked the topography. Through conductive AFM imaging, we found that the conductive regions correlated with regions of FN adhesion.

Conclusions

Given that the conductivity requires doping of the polymer, these findings suggest that FN adhesion is mediated by interactions with chondroitin sulfate and hyaluronic acid at the polymer surface and may be indicative of specific interactions due to contributions from electrostatic attraction between the FN and sulfate/anionic groups of the dopants.

General significance

This study demonstrates the ability of AFM to resolve the protein–conducting polymer interactions at the molecular and nanoscale level, which will be important for interfacing these polymer materials with biological systems. This article is part of a Special Issue entitled Organic Bioelectronics — Novel Applications in Biomedicine.     

Three-Dimensional High-Resolution Second-Harmonic Generation Imaging of Endogenous Structural Proteins in Biological Tissues

We find that several key endogenous protein structures give rise to intense second-harmonic generation (SHG)—nonabsorptive frequency doubling of an excitation laser line. Second-harmonic imaging microscopy (SHIM) on a laser-scanning system proves, therefore, to be a powerful and unique tool for high-resolution, high-contrast, three-dimensional studies of live cell and tissue architecture. Unlike fluorescence, SHG suffers no inherent photobleaching or toxicity and does not require exogenous labels. Unlike polarization microscopy, SHIM provides intrinsic confocality and deep sectioning in complex tissues. In this study, we demonstrate the clarity of SHIM optical sectioning within unfixed, unstained thick specimens. SHIM and two-photon excited fluorescence (TPEF) were combined in a dual-mode nonlinear microscopy to elucidate the molecular sources of SHG in live cells and tissues. SHG arose not only from coiled-coil complexes within connective tissues and muscle thick filaments, but also from microtubule arrays within interphase and mitotic cells. Both polarization dependence and a local symmetry cancellation effect of SHG allowed the signal from species generating the second harmonic to be decoded, by ratiometric correlation with TPEF, to yield information on local structure below optical resolution. The physical origin of SHG within these tissues is addressed and is attributed to the laser interaction with dipolar protein structures that is enhanced by the intrinsic chirality of the protein helices.     

Claudins蛋白在胃癌组织中的表达

紧密连接（tightjunctions,TJs）对于维持上皮细胞或内皮细胞的正常结构和发挥生理功能起着重要作用。Claudins（CLDNs）蛋白是构成细胞膜紧密连接的主要成分之一,其异常表达导致上皮细胞或内皮细胞结构破坏、功能受损,与多种疾病的发生、发展关系密切。近年来,越来越多的文献报道了claudins蛋白在胃癌组织中的异常表达。不同claudin蛋白亚型在胃癌组织中的表达降低或增高所起作用亦不相同。Claudins蛋白与肿瘤形成、癌细胞的增殖、转移、以及预后关系密切。因此,深入研究claudins蛋白与胃癌发生、发展的关系具有重要意义。     

Changes in Biomarker Expression in the Development of Prostatic Adenocarcinoma

In spite of the prevalence of prostatic adenocarcinoma, the development and natural history of this malignancy is poorly understood. This paper reviews the current knowledge of biomarker expression during the development and progression of prostatic adenocarcinoma. Emphasis is placed on the comparison of biomarker expression in benign prostatic epithelium, intraepithelial neoplasia (PIN), a putative preinvasive lesion, and prostatic adenocarcinoma. Within the benign epithelium, the proliferative potential is restricted to the basal cells as demonstrated by the expression of proliferating cellular nuclear antigen (PCNA). The strong expression of the bcl-2 protein, an inhibitor of apoptosis, supports the concept that the basal cells or a subpopulation of the basal cells represent the stem cell of the epithelium. In addition, the strong expression of growth factor receptors such as the epidermal growth factor receptor (EGFr), p185^erbB-2, p180^erbB-3, and c-met suggests that the growth of the basal cells is regulated by autocrine or paracrine factors. The luminal cells express secretory products such as prostate specific antigen and prostatic acid phosphatase, but demonstrate little expression of PCNA as well as growth factor receptors and proto-oncogene products. These observations are consistent with the theory that the luminal cell population is derived from the differentiation of the basal cells. In contrast to the normal epithelium, PCNA expression is frequently detected in the dysplastic luminal cells of the PIN lesion. Likewise, strong expression of p185^erbB-2, p180^erbB-3 and the c-met proto-oncogene product is also detected in the luminal cells of PIN lesions. Other factors which are strongly expressed by the dysplastic luminal cells include the nm23-Hl gene product, tumor associated glycoprotein-72 (TAG-72), fatty acid synthetase (FASE) and proteolytic enzymes. These findings suggest that PIN lesions are derived from an impairment of the differentiation of basal cells. The majority of biomarkers such as PCNA, p185^erbB-2, p180^erbB-3, TAG-72, nm23-Hl and FASE which are strongly expressed in PIN lesions are also expressed in prostatic adenocarcinoma supporting the concept that PIN is a preinvasive lesion. Mutations of the p53 tumor suppressor gene, as well as strong expression of transforming growth factor a and bcl-2 typically occur in advanced stage prostatic adenocarcinomas and therefore likely represent late events in the development of prostatic adenocarcinoma.     

Prediction of Biomarker Expression on Primary Pancreatic Ductal Adenocarcinoma Tissues Using Fine-Needle Biopsies: Paving the Way for a Patient-Tailored Molecular Imaging Approach

Molecular Diagnosis & Therapy - Targeted molecular imaging may improve tumor cell identification during diagnosis and resection of pancreatic ductal adenocarcinoma (PDAC). Although many...     

miRNA Expression Analyses in Prostate Cancer Clinical Tissues

A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, diagnosis, prognosis and treatment. In recent years, microRNAs (miRNAs) have emerged as promising alternate biomarkers for prostate cancer diagnosis and prognosis. However, the development of miRNAs as effective biomarkers for prostate cancer heavily relies on their accurate detection in clinical tissues. miRNA analyses in prostate cancer clinical specimens is often challenging owing to tumor heterogeneity, sampling errors, stromal contamination etc. The goal of this article is to describe a simplified workflow for miRNA analyses in archived FFPE or fresh frozen prostate cancer clinical specimens using a combination of quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH). Within this workflow, we optimize the existing methodologies for miRNA extraction from FFPE and frozen prostate tissues and expression analyses by Taqman-probe based miRNA RT-PCR. In addition, we describe an optimized method for ISH analyses formiRNA detection in prostate tissues using locked nucleic acid (LNA)- based probes. Our optimized miRNA ISH protocol can be applied to prostate cancer tissue slides or prostate cancer tissue microarrays (TMA).