Demonstration of pseudorabies virus DNA in the mouse inner ear by an in situ nucleic acid hybridization technique in plastic embedded bony material

This investigation is concerned with the possibility of identifying viral DNA using the in situ DNA hybridization method in methylmethacrylate-embedded material. As an experimental model we chose viral labyrinthitis produced by intranasal infection of the mouse with pseudorabies virus. Fixation and embedding methods specially adapted to this procedure and bony histology preparation technique (specimens by grinding or micromilling) made it possible to identify viral DNA directly morphologically and virologically in the inner ear. Quantitative microphotometric analyses of trans-sagittal sections of the entire skull after in situ DNA hybridization are presented and discussed here as an explicit method of investigating the path of distribution of viral DNA in the brain and the inner ear.     

Chromosome morphology after long-term storage investigated by scanning near-field optical microscopy

Fluorescence in situ hybridization coupled with far‐field fluorescence microscopy is a commonly used technique to visualize chromosomal aberrations in diseased cells. To obtain the best possible results, chromatin integrity must be preserved to ensure optimal hybridization of fluorescence in situ hybridization probes. However, biological samples are known to degrade and storage conditions can be critical. This study concentrates its investigation on chromatin stability as a function of time following fluorescence in situ hybridization type denaturing protocols. This issue is extremely important because chromatin integrity affects the fluorescence response of the chromosome. To investigate this, metaphase chromosome spreads of human lymphocytes were stored at both ?20 and ?80 °C, and were then imaged using scanning near‐field optical microscopy over a nine month period. Using the scanning near‐field optical microscope's topography mode, chromosome morphology was analysed before and after the application of fluorescence in situ hybridization type protocols, and then as a function of storage time. The findings revealed that human chromosome samples can be stored at ?20 °C for short periods of time (～ several weeks), but storage over 3 months compromises chromatin stability. Topography measurements clearly show the collapse of the stored chromatin, with variations as large as 60 nm across a chromosome. However, storage at ?80 °C considerably preserved the integrity with variations in topography significantly reduced. We report studies of the fluorescent response of stored chromosomes using scanning near‐field optical microscopy and their importance for gaining further understanding of chromosomal aberrations.     

Static and dynamic experiments in cryo-electron microscopy: comparative observations using high-vacuum, low-voltage and low-vacuum SEM

We carried out a unique comparative study between three modes of cryo‐scanning electron imaging: high‐vacuum, low‐voltage and low‐vacuum, using ice cream as a model system. Specimens were investigated both with and without a conductive coating (Au/Pd) and at temperatures for which ice either remains fully frozen (< ?110 °C) or undergoes sublimation (?110 to ?90 °C). At high magnification, high‐vacuum imaging of coated specimens gave the best results for ‘static’ specimens (i.e. containing fully frozen ice). Low voltages, such as 1 kV, could be used for imaging uncoated specimens at high vacuum, although slight ‘classical’ charging artefacts remained an issue, and the reduced electron beam penetration tended to decrease the definition between different microstructural features. However, this mode was useful for observing in situ sublimation from uncoated specimens. Low‐vacuum mode, involving small partial pressures of nitrogen gas, was particularly suited to in situ sublimation work: when sublimation was carried out in low vacuum in the absence of an anti‐contaminator plate, sublimation rates were significantly reduced. This is attributed to a small partial pressure of sublimated water vapour remaining near the specimen surface, enhancing thermodynamic stability.     

Influence of calcitonin administration on ultimobranchial and parathyroid glands of pigeon,Columba livia

Pigeons were divided into two numerically equal groups (A and B) containing 30 specimens each. Birds from group A were intraperitoneally injected daily with 0.1 mL/100 g body wt of vehicle (0.1 M acetate buffer, pH 4.3 containing 0.1% gelatin). Specimens from group B were intraperitoneally injected daily with 1 μg/100 g body wt of salmon calcitonin. Six birds were sacrificed from each group 2 h after the last injection on 1st, 3rd, 5th, 10th, and 15th day of the experiment. After collection of blood samples, ultimobranchial and parathyroid glands were fixed for histological studies. In calcitonin-treated C. livia, the plasma calcium levels exhibit a progressive decline from day 1 till day 5. On day 15, the levels become more or less similar to the control value. No change has been noticed on day 1 in the plasma phosphate levels of calcitonin-treated C. livia. The levels decrease progressively from day 3 to day 5; thereafter, it exhibits an elevation so that on day 15, normal plasma phosphate levels is achieved. The ultimobranchial gland of C. livia exhibits no change up to day 5 following calcitonin treatment. The nuclear volume of ultimobranchial cells exhibits a decrease on day 10. This response progresses up to day 15. Few degenerating cells are also discerned following 15 days calcitonin treatment. The parathyroid gland of calcitonin-treated C. livia exhibits no histological alteration up to day 3. The nuclear volume of parathyroidal cells exhibits a progressive increase from day 3 till the close of the experiment (day 15). Moreover, the gland exhibits more compactness on day 10 and day 15. Few degenerating cells are encountered after day 15 following calcitonin treatment. Microsc. Res. Tech. 2009. © 2008 Wiley-Liss, Inc.     

Expression of the male reproduction‐related gene in spermatic ducts of the blue swimming crab,Portunus pelagicus,and transfer of modified protein to the sperm acrosome

Expression of a sex‐specific gene in Macrobrachium rosenbergii (Mr‐Mrr), encoding a male reproduction‐related (Mrr) protein, has been identified in the spermatic ducts (SDs) and postulated to be involved in sperm maturation processes. M. rosenbergii is the only decapod that the expression and fate of the Mrr protein has been studied. To determine that this protein was conserved in decapods, we firstly used cloning techniques to identify the Mrr gene in two crabs, Portunus pelagicus (Pp‐Mrr) and Scylla serrata (Ss‐Mrr). We then investigated expression of Pp‐Mrr by in situ hybridization, and immunolocalization, as well as phosphorylation and glycosylation modifications, and the fate of the protein in the male reproductive tract. Pp‐Mrr was shown to have 632 nucleotides, and a deduced protein of 110 amino acids, with an unmodified molecular weight of 11.79 kDa and a mature protein with molecular weight of 9.16 kDa. In situ hybridization showed that Pp‐Mrr is expressed in the epithelium of the proximal, middle, distal SDs, and ejaculatory ducts. In Western blotting, proteins of 10.9 and 17.2 kDa from SDs were all positive using anti‐Mrr, antiphosphoserine/threonine, and antiphosphotyrosine. PAS staining showed they were also glycosylated. Immunolocalization studies showed Pp‐Mrr in the SD epithelium, lumen, and on the acrosomes of spermatozoa. Immunofluorescence staining indicated the acrosome of spermatozoa contained the Mrr protein, which is phosphorylated with serine/threonine and tyrosine, and also glycosylated. The Mrr is likely to be involved in acrosomal activation during fertilization of eggs. Microsc. Res. Tech., 2013. © 2012 Wiley Periodicals, Inc.     

Detection of single copy sequences using BAC-FISH and C-PRINS techniques in sunflower chromosomes

Bacterial artificial chromosome - fluorescence in situ hybridization (BAC-FISH) and cyclingprimed in situ labeling (C-PRINS) techniques were evaluated for integration of physical and genetic maps of sunflower (Helianthus annuus L.). Single-site SSR markers were selected from three linkage groups of a high-density sunflower genetic map. This selection was based on previously identified QTL associated to S. sclerotiorum. These markers were used to select BACs contaning single copy sequences for BAC-FISH aplication. Blocking of highly dispersed repetitive sunflower sequences reduced unspecific hybridization, and allowed the detection of specific signals for BACs containing SSR markers HA4222 and HA2600, anchored to LG 16 and LG 10, respectively. Single-site FISH signal detection was optimized by adjusting the relative quantity and quality of unlabelled repetitive sequences present in the blocking DNA. The SSR marker ORS1247 anchored to the LG 17 was detected by C-PRINS, which yielded fluorescence signals that were specific and intense. This progress in localizing single-copy sequences using BAC-FISH and indirect CPRINS strategies in sunflower will facilitate the integration of genetic and physical maps, allowing the identification of     

TiF4 gel effects on tubular occlusion of eroded/abraded human dentin

This in situ study evaluated the tubular occlusion caused by 4% TiF₄ gel on the surface of eroded/abraded dentin. Sixty human dentin samples were eroded in vitro and assigned into six groups (n = 10) according to the in situ surface treatment and number of cycling days: 4% TiF₄ gel applied once (TiF₄1), twice (TiF₄2), or three times (TiF₄3) followed by 2, 4, and 6 days of erosive/abrasive in situ cycling, respectively. Control groups (no treatment) were subjected to 2 (C1), 4 (C2), and 6 (C3) days of erosive/abrasive in situ cycling only. A seventh group (n = 10) was comprised by in vitro uneroded samples (UN), subjected to 6 days of in situ erosive/abrasive cycling. Each cycling day consisted on six erosive (0.5% citric acid, pH 2.6) and one abrasive events. Environmental scanning electron microscopy micrographs were taken. For all groups, blinded examiners assessed dentin tubules occlusion using visual scores (0—unoccluded, 1—partially occluded by granular deposits, 2—partially occluded by reduction in tubular lumen into diamond shape, 3—completely occluded) on images captured prior and after the in situ phase. Scheirer‐Ray‐Hare test demonstrated that treatments significantly affected tubule occlusion (p < .001). Dunn's test showed that tubule occlusion in TiF₄3 was significantly higher than in C1. Tubule occlusion in remaining groups did not differ from that observed in groups TiF₄3 and C1. Tubule occlusion was significantly higher after in situ phase. It may be suggested that TiF₄, when applied three times, was able to positively change tubule occlusion of dentin samples.     

Real-time enzymatic degradation of human dentin collagen fibrils exposed to exogenous collagenase: an AFM study in situ

Objective: This study was carried out to observe the enzymatic degradation of human dentin collagen fibrils exposed to exogenous collagenase in situ using atomic force microscopy, to understand the characteristics of the enzymatic degradation of collagen fibrils on dentin specimens. Methods: Polished dentin specimens from caries‐free third molars were etched with citric acid, and then treated with an aqueous solution of 6.5% NaOCl for 120 s. The specimen was then put into a fluid cell and treated with a mixed solution of collagenase I (MMP‐1) and collagenase II (MMP‐8) for 9 h. AFM with contact mode was performed in situ to monitor the enzymatic degradation process of the dentin collagen fibrils. The distinctly topographic changes of the dentin surface were recorded continuously during different stages of the enzymatic degradation process. Results: The mixed solution of exogenous collagenase I and collagenase II could degrade dentin organic matrix (mainly collagen) efficiently, and the structures of dentin substrate were clearly exposed. Conclusion: It is possible to carry out real‐time observations on the enzymatic biodegradation process of human dentin collagen fibrils on dentin specimens with atomic force microscopy in situ. By this means, the fine structures of the etched dentin substrate were clearly revealed, possibly contributing to the related study of human dentin in vitro.     

Mapping the chemistry of resinite,funginite and associated vitrinite in coal with micro‐FTIR

Micro‐FTIR mapping is a powerful tool for nondestructive, in situ chemical characterization of coal macerals at high resolution. In this study, the chemistry of resinite, funginite and associated vitrinite is characterized via reflectance micro‐FTIR for Cenozoic high volatile C bituminous coals from Colombia. In comparison with the micro‐FTIR spectra of vitrinite and inertinite, the corresponding spectra of liptinite macerals in the same coals are characterized by stronger aliphatic CH_x absorbance at 3000–2800 and 1460–1450 cm^?1, but less intense aromatic C=C ring stretching vibration and aromatic CH_x out‐of‐plane deformation at 700–900 cm^?1. The aliphatic components in resinite have the longest carbon chains and are least branched, bestowing the highest hydrocarbon generation potential on resinite among the three macerals studied. In contrast, funginite exhibits the strongest aromatic character, the highest aromaticity, the lowest ‘A’ factor values and the lowest C=O/C=C ratios among the three maceral groups. Vitrinite generally displays intermediate chemical characteristics. Reflectance micro‐FTIR mapping of coal samples further confirms the aliphatic character of resinite and the aromatic nature of funginite. In addition, chemical mapping of resinite and adjacent vitrinite shows that vitrinite immediately adjacent to resinite displays higher aliphatic CH_x stretching intensity than more distant vitrinite, suggesting that chemical components from resinite can diffuse over short distances into adjacent vitrinite, specifically causing hydrogen enrichment. It needs to be pointed out, however, that the region of influence is localized and limited to a narrow zone, whose extent likely depends on resinite's properties, such as its size and aliphatic material content. This way, the chemical map of resinite and associated vitrinite provides direct evidence of the intermaceral effects occurring during the peat forming stage or during later coalification. No influence of funginite (primarily fungal spores and sclerotia) on the chemistry of adjacent vitrinite has been demonstrated, which is likely due to the highly aromatic structure of this type of funginite.     

Transpiration-assisted perfusion fixation provides in situ preservation of developing ray parenchyma cells in Eucalyptus nitens

The technique of animal vascular perfusion fixation was adapted for in situ fixation of the fragile and difficult to access cells of the ray parenchyma system in stems of 10-year-old Eucalyptus nitens trees. In situ fixation enabled tissue to be safely dissected for histological processing without risk of damage to microstructure or initiation of wound response. Fixative was perfused through the active vascular system under hydrostatic and transpiration pressure directly to vessel-associated ray cells. Diffusion from vessels allowed fixative to access nonvessel-associated ray cells. Acrolein was included to aid diffusion fixation and safranin dye was included to define fixed regions within the xylem. Sections prepared for light and electron microscopy from samples cut from regions showing intense safranin staining showed good microstructural preservation and were free of artefacts caused by mechanical injury or wound response.     

The influence of tissue processing on quantitative histopathology in breast cancer

Objective grading of breast cancer by morphometry has been suggested for improving the precision of the prognostic prediction. However, the tissue components evaluated might be influenced by variations in the processing, reducing the clinical value. In the present study, the impact of the period of fixation, of the acidity of the fixative and of the embedding medium was investigated by allocating tissue samples from 27 surgical breast cancer specimens systematically randomly to different modes of processing. The volume-weighted mean volume of cancer cell nuclei, v?_V(nuc), was estimated using the method of point-sampled intercepts on vertical sections. In addition, estimates of the mean nuclear profile area, ā_H(nuc), the nuclear volume fraction, V_V(nuc), the nuclear profile density, ND, and the mitotic profile frequency, MF, were obtained. The quantitative histopathological estimates were stable with respect to the investigated variables of the tissue processing. No significant differences were found when comparing the estimates obtained in samples from five tumours fixed in formalin at pH 5·0, 6·0, 7·0, 7·4 and 8·0, respectively. Similarly, no significant correlations between the estimates and the period of formalin fixation (24 h, 3 days and 3 months) were found in samples from five other tumours. However, the v?_V(nuc) was 13% larger (2p = 0·004) and the mean ND 17% smaller (2p = 0·04) in hydroxyethyl-methacrylate-embedded samples from 17 tumours as compared to paraffin-embedded samples. Thus, the shrinkage observed in paraffin seems to affect nuclei less than tissue.     

Dual-mode reflectance and fluorescence near-video-rate confocal microscope for architectural, morphological and molecular imaging of tissue

We have developed a near‐video‐rate dual‐mode reflectance and fluorescence confocal microscope for the purpose of imaging ex vivo human specimens and in vivo animal models. The dual‐mode confocal microscope (DCM) has light sources at 488, 664 and 784 nm, a frame rate of 15 frames per second, a maximum field of view of 300 × 250 μm and a resolution limit of 0.31 μm laterally and 1.37 μm axially. The DCM can image tissue architecture and cellular morphology, as well as molecular properties of tissue, using reflective and fluorescent molecular‐specific optical contrast agents. Images acquired with the DCM demonstrate that the system has the sub‐cellular resolution needed to visualize the morphological and molecular changes associated with cancer progression and has the capability to image animal models of disease in vivo. In the hamster cheek pouch model of oral carcinogenesis, the DCM was used to image the epithelium and stroma of the cheek pouch; blood flow was visible and areas of dysplasia could be distinguished from normal epithelium using 6% acetic acid contrast. In human oral cavity tissue slices, DCM reflectance images showed an increase in the nuclear‐to‐cytoplasmic ratio and density of nuclei in neoplastic tissues as compared to normal tissue. After labelling tissue slices with fluorescent contrast agents targeting the epidermal growth factor receptor, an increase in epidermal growth factor receptor expression was detected in cancerous tissue as compared to normal tissue. The combination of reflectance and fluorescence imaging in a single system allowed imaging of two different parameters involved in neoplastic progression, providing information about both the morphological and molecular expression changes that occur with cancer progression. The dual‐mode imaging capabilities of the DCM allow investigation of both morphological changes as well as molecular changes that occur in disease processes. Analyzing both factors simultaneously may be advantageous when trying to detect and diagnose disease. The DCM's high resolution and near‐video‐rate image acquisition and the growing inventory of molecular‐specific contrast agents and disease‐specific molecular markers holds significant promise for in vivo studies of disease processes such as carcinogenesis.     

Prognostic tumor microenvironment gene and the relationship with immune infiltration characteristics in metastatic breast cancer

The aim of this study was to reveal genes associated with breast cancer metastasis, to investigate their intrinsic relationship with immune cell infiltration in the tumor microenvironment, and to screen for prognostic biomarkers. Gene expression data of breast cancer patients and their metastases were downloaded from the GEO, TCGA database. R language package was used to screen for differentially expressed genes, enrichment analysis of genes, PPI network construction, and also to elucidate key genes for diagnostic and prognostic survival. Spearman’s r correlation was used to analyze the correlation between key genes and infiltrating immune cells. We screened 25 hub genes, FN1, CLEC5A, ATP8B4, TLR7, LY86, PTGER3 and other genes were differentially expressed in cancer and paraneoplastic tissues. However, patients with higher expression of CD1C, IL-18 breast cancer had a better prognosis in the 10 years survival period, while patients with high expression of FN1, EIF4EBP1 tumors had a worse prognosis. In addition, TP53 and HIF1 genes are closely related to the signaling pathway of breast cancer metastasis. In this study, gene expression of ATP8B4 and CD1C were correlated with cancer tissue infiltration of CD8⁺ T lymphocytes, while GSE43816, GSE62327 and TCGA databases showed that CD8⁺ T lymphocytes were closely associated with breast cancer progression. Functional enrichment analysis of genes based on expression differences yielded key genes of prognostic value in the breast cancer microenvironment.     

Cetyltrimethylammonium bromide inhibits the metastasis of breast cancer to the lungs by inhibiting epithelial–mesenchymal transition

Breast cancer is a highly aggressive cancer in females. Metastasis is a major obstacle to the efficient and successful treatment of breast cancer. Cetyltrimethylammonium bromide (CTAB) has anti-tumor effects on a variety of tumors. We showed that CTAB inhibits the metastasis of breast cancer to the lungs both in vitro and in vivo. Epithelial-mesenchymal transition (EMT) is thought to be one of the major processes mediating breast cancer metastasis. We found that CTAB suppressed EMT and regulated the levels of the classical EMT markers E-cadherin, N-cadherin, vimentin, Snail and Twist1. Moreover, as a candidate anti-tumor agent, CTAB showed primary safety in vivo. Taken together, our results suggest that CTAB inhibits the migration of primary breast cancer to the lungs. Our findings confirm the clinical potential of CTAB for the treatment of breast cancer by targeting EMT. CTAB may thus be a promising novel anti-tumor drug for the treatment of breast cancer metastasis.     

A MEMS‐based heating holder for the direct imaging of simultaneous in‐situ heating and biasing experiments in scanning/transmission electron microscopes

The introduction of scanning/transmission electron microscopes (S/TEM) with sub‐Angstrom resolution as well as fast and sensitive detection solutions support direct observation of dynamic phenomena in‐situ at the atomic scale. Thereby, in‐situ specimen holders play a crucial role: accurate control of the applied in‐situ stimulus on the nanostructure combined with the overall system stability to assure atomic resolution are paramount for a successful in‐situ S/TEM experiment. For those reasons, MEMS‐based TEM sample holders are becoming one of the preferred choices, also enabling a high precision in measurements of the in‐situ parameter for more reproducible data. A newly developed MEMS‐based microheater is presented in combination with the new NanoEx?‐i/v TEM sample holder. The concept is built on a four‐point probe temperature measurement approach allowing active, accurate local temperature control as well as calorimetry. In this paper, it is shown that it provides high temperature stability up to 1,300°C with a peak temperature of 1,500°C (also working accurately in gaseous environments), high temperature measurement accuracy (<4%) and uniform temperature distribution over the heated specimen area (<1%), enabling not only in‐situ S/TEM imaging experiments, but also elemental mapping at elevated temperatures using energy‐dispersive X‐ray spectroscopy (EDS). Moreover, it has the unique capability to enable simultaneous heating and biasing experiments. Microsc. Res. Tech. 79:239–250, 2016. © 2016 Wiley Periodicals, Inc.     

Near real time in vivo fibre optic confocal microscopy: sub-cellular structure resolved

We have built a fibre optic confocal reflectance microscope capable of imaging biological tissue in near real time. The measured lateral resolution is 3 µm and axial resolution is 6 µm. Images of epithelial cells, excised tissue biopsies, and the human lip in vivo have been obtained at 15 frames s^?1. Both cell morphology and tissue architecture can be appreciated from images obtained with this microscope. This device has the potential to enable reflected light confocal imaging of internal organs for in situ detection of pathology.     

Nucleotide and protein distribution in BrdU-labelled polytene chromosomes revealed by ion probe mass spectrometry

Detailed chemical maps of BrdU-labelled polytene chromosomes of Drosophila melanogaster, obtained by imaging secondary ion mass spectrometry, reveal separately the distribution of DNA and proteins in the chromosomes. The thymidine-analogue BrdU within the chromosomal DNA is localized by detecting the Br^? secondary ion signal, while both nucleic acid and protein content are mapped through the abundantly emitted CN^? signal. This novel approach supercedes, and helps explain the origin of, the banding patterns that are observed by conventional staining techniques. The high spatial resolution and chemical and isotopic sensitivity of this technique should enhance the localization of specific genes by in situ hybridization in mitotic chromosomes.