Leptin‐like immunoreactivity in the muscle of juvenile sea bass (Dicentrarchus labrax)

The mammalian hormone, leptin, is mainly synthesized in adipose tissue along with other tissues. Leptin plays a role in numerous processes such as in the control of food intake, homeostasis, immune function and reproduction. In this study, we detected and localized leptin immunoreactivity to the muscle of early juvenile sea bass (Dicentrarchus labrax) by Western blot analysis and immunohistochemistry. A leptin immunopositive band with a molecular weight of ～16 kDa, corresponding to mammalian leptin, was identified in trunk skeletal muscle homogenate. Furthermore, leptin immunopositive cells were detected in the endomysium of skeletal muscular fibers. These cells showed immunostained cytoplasmic granules and roundish and oval nuclei. The most intense immunostaining was observed in the endomysial space among the superficial red muscular fibers of the trunk. These findings suggest that in early juvenile sea bass, leptin is mostly produced by skeletal muscles. Therefore, during the developmental stage lacking adipose tissue, skeletal muscles can be considered an important source of leptin. As already suggested in mammals, we can hypothesize the potential roles of leptin not only in energy expenditure for muscle contraction but also during muscle differentiation and growth. Microsc. Res. Tech. 73:797–802, 2010. © 2010 Wiley‐Liss, Inc.     

Establishing the archaeo‐metallurgic ornamentation process of an axe from the bronze age by OM,SEM‐EDX,and Micro‐FTIR

Our article presents the results of the analyses we performed by corroborating the Optical Microscopy, Scanning Electron Microscopy, Energy Dispersive Xray Analysis and micro Fourier Transformed InfraRed Analysis techniques to identify the archaeo‐metallurgic casting and ornamentation procedure of a decorated disk‐butted axe, which was discovered recently east of the Carpathian mountains, in the Moldavian Plateau. There are few known axes of that type found (A1, according to the usual typologies), as they are specific to the Middle Bronze Age period west of the Carpathians—the Wietenberg, Suciu de Sus, and Otomani‐Füzesabony cultures. The experimental data on the item under study revealed the fact that after casting it in molds made from porous silicone‐based stone, the object was coated with a thin layer, by immersing it in a lightly fusible tin alloy, whose main alloy component was copper and arsenic and iron as secondary components. After refining the shiny white layer, they applied a beautiful decoration pattern made by incision and engraving. This battle axe was an indication of higher status, such items usually being owned by community leaders. This important fact proves that the ancient metallurgic craftsmen were able to elaborate and manufacture various alloys from which they made beautiful objects and it also offers a new insight into the social and symbolic function of certain antique bronze items. Microsc. Res. Tech. 77:918–927, 2014. © 2014 Wiley Periodicals, Inc.     

Automated image segmentation of haematoxylin and eosin stained skeletal muscle cross‐sections

The ability to accurately and efficiently quantify muscle morphology is essential to determine the physiological relevance of a variety of muscle conditions including growth, atrophy and repair. There is agreement across the muscle biology community that important morphological characteristics of muscle fibres, such as cross‐sectional area, are critical factors that determine the health and function (e.g. quality) of the muscle. However, at this time, quantification of muscle characteristics, especially from haematoxylin and eosin stained slides, is still a manual or semi‐automatic process. This procedure is labour‐intensive and time‐consuming. In this paper, we have developed and validated an automatic image segmentation algorithm that is not only efficient but also accurate. Our proposed automatic segmentation algorithm for haematoxylin and eosin stained skeletal muscle cross‐sections consists of two major steps: (1) A learning‐based seed detection method to find the geometric centres of the muscle fibres, and (2) a colour gradient repulsive balloon snake deformable model that adopts colour gradient in colour space. Automatic quantification of muscle fibre cross‐sectional areas using the proposed method is accurate and efficient, providing a powerful automatic quantification tool that can increase sensitivity, objectivity and efficiency in measuring the morphometric features of the haematoxylin and eosin stained muscle cross‐sections.     

Applicability of ring‐on‐disc and pin‐on‐plate test methods for Cu–steel and Al–steel systems for large area conformal contacts

This study compares two methods for model scale testing of ferrous/non‐ferrous tribocouples under large area conformal contact condition, the kind existing in engine components such as journal bearings. Results show that the ring‐on‐disc method is better suited to visualise the performance of such tribosystems compared with the pin‐on‐plate method. The former offers greater sensitivity to minor changes in coefficient of friction and contact potential and is able to determine the thermal stability of the tribosystem under given conditions. Post‐test surface characterisation revealed protective phosphorus‐rich tribofilms on the surface of the steel counterparts from both test methods. Copyright © 2012 John Wiley & Sons, Ltd.     

Usefulness of hard X‐ray microscope using synchrotron radiation for the structure analysis of insects

Three‐dimensional (3D) printing technology has the advantage of enabling specific visualization of creative ideas. Since synchrotron based images can provide high sensitivity and high resolution, they are a very useful technology to identify the 3D anatomy of microscale samples. X‐ray images using such synchrotron radiation are grafted to 3D printing technology. We can be obtained 3D images and modeling data of an ant using synchrotron radiation, and then, it were outputted with the 3D printer. A new way to identify the usefulness of the structure analysis is then found by visualizing the micro‐internal structure of diverse biomedical samples and creating an enlarged model. This study suggests methods of utilizing a 3D printed model produced through synchrotron radiation imaging in various fields such as bioengineering, medical, and education.     

Iodine potassium iodide improves the contrast‐to‐noise ratio of micro‐computed tomography images of the human middle ear

High‐resolution imaging of middle‐ear geometry is necessary for finite‐element modeling. Although micro‐computed tomography (microCT) is widely used because of its ability to image bony structures of the middle ear, it is difficult to visualize soft tissues – including the tympanic membrane and the suspensory ligaments/tendons – because of lack of contrast. The objective of this research is to quantitatively evaluate the efficacy of iodine potassium iodide (IKI) solution as a contrast agent. Six human temporal bones were used in this experiment, which were obtained in right‐left pairs, from three cadaveric heads. All bones were fixed using formaldehyde. Three bones (one from each pair) were stained in IKI solution for 2 days, whereas the other three were not stained. Samples were scanned using a microCT system at a resolution of 20 μm. Eight soft tissues in the middle ear were segmented: anterior mallear ligament, incudomallear joint, lateral mallear ligament, posterior incudal ligament, stapedial annular ligament, stapedius muscle, tympanic membrane and tensor tympani muscle. Contrast‐to‐noise ratios (CNRs) of each soft tissue were calculated for each temporal bone. Combined CNRs of the soft tissues in unstained samples were 6.1 ± 3.0, whereas they were 8.1 ± 2.7 in stained samples. Results from Welch's t‐test indicate significant difference between the two groups at a 95% confidence interval. Results for paired t‐tests for each of the individual soft tissues also indicated significant improvement of contrast in all tissues after staining. Relatively large soft tissues in the middle ear such as the tympanic membrane and the tensor tympani muscle were impacted by staining more than smaller tissues such as the stapedial annular ligament. The increase in contrast with IKI solution confirms its potential application in automatic segmentation of the middle‐ear soft tissues.     

Assessment of polymer composites for hydrodynamic journal‐bearing applications

Polymer composites are used as facing materials in hydrodynamic bearings for their low friction and ‘compliant’ properties, which play an important role during machinery operation. In journal bearings, this low friction property can be of significant importance during start and stop cycles when insufficient oil is available to fully separate the surfaces in relative motion. Current work is aimed at determining a suitable material for use in hydrodynamic journal bearings for applications in hydroelectric power plants. This study investigates friction and wear encountered during the transition from the stationary state to operational speed (acceleration) during initial start‐up. This is examined for virgin poly‐tetra‐fluoro‐ethylene (PTFE) together with a series of commercially available PTFE‐based composites and a babbitt material in boundary/mixed lubrication conditions. Tests are performed using standard laboratory block‐on‐ring test apparatus with a VG32 mineral oil. Copyright © 2009 John Wiley & Sons, Ltd.     

Some comments on micro‐abrasion interactions of pure metals in bio‐oils

An attempt has been made to study the effect of bio‐oils on the two‐ and three‐body abrasion process of pure metals and steel. The equipment used was a micro‐abrasion tester (TE‐66, Phoenix Tribology (Plint)). The materials considered were mild steel, copper, and aluminium, being the most usable and basic materials for industrial applications such as bearings and gear assemblies. The results were used to identify transitions between wear regimes as a function of sliding speed and load. The effect on the abrasion process in the presence of various lubricant oils was also assessed. The mechanisms of abrasion with and without particles were characterised.     

Entropy‐assisted image segmentation for nano‐ and micro‐sized networks

Image analysis is an important tool for characterizing nano/micro network structures. To understand the connection, organization and proper alignment of network structures, the knowledge of the segments that represent the materials inside the image is very necessary. Image segmentation is generally carried out using statistical methods. In this study, we developed a simple and reliable masking method that improves the performance of the indicator kriging method by using entropy. This method selectively chooses important pixels in an image (optical or electron microscopy image) depending on the degree of information required to assist the thresholding step. Reasonable threshold values can be obtained by selectively choosing important pixels in a complex network image composed of extremely large numbers of thin and narrow objects. Thus, the overall image segmentation can be improved as the number of disconnected objects in the network is minimized. Moreover, we also proposed a new method for analyzing high‐pixel resolution images on a large scale and optimized the time‐consuming steps such as covariance estimation of low‐pixel resolution image, which is rescaled by performing the affine transformation on high‐pixel resolution images. Herein, image segmentation is executed in the original high‐pixel resolution image. This entropy‐based masking method of low‐pixel resolution significantly decreases the analysis time without sacrificing accuracy.     

Distribution and three‐dimensional appearance of the interstitial cells of Cajal in the rat stomach and duodenum

The relationship between the interstitial cells of Cajal (ICC) and enteric nerves or smooth muscles cells is not fully defined. Presently, distribution and appearance of ICC in the rat stomach and duodenum was studied by immunohistochemistry, electron microscopy, and three‐dimensional reconstruction. c‐kit expressing ICC were regularly observed in the Auerbach's myenteric plexus (AP) of the stomach and duodenum. ICC in stomach and duodenum muscle layers was dissimilarly distributed. c‐kit immunoreactive cells were sparsely distributed in the stomach circular muscle layer but were abundant in the duodenum deep muscular plexus (DMP). Electron microscopy revealed that stomach ICC‐AP were irregular ovals with few cytoplasmic processes, and possessed an electron‐dense cytoplasm, numerous mitochondria, intermediate filaments, and caveolae. Duodenum and stomach ICC‐AP were similar in appearance. Ultrastructure observations and three‐dimensional reconstructions revealed ICC‐AP processes wrapping the nerve fibers and projecting into the space between smooth muscle cells. While ICC‐AP was occasionally close to enteric nerves or smooth muscle cells, no connections were observed. ICC‐DMP in duodenum was elongated and adopted the same cell axis orientation as the circular muscle cells. Unlike ICC‐AP, ICC‐DMP formed gap junctions with smooth muscle cells and had close contact with nerves. These results indicate that ICC‐AP is regularly distributed in stomach and duodenum, while ICC‐DMP is exclusively located in the duodenum. ICC‐DMP, which possess gap junctions and closely contacts nerves, may participate in neuromuscular transmission. Microsc. Res. Tech. 2009. © 2009 Wiley‐Liss, Inc.     

Super‐resolution optical microscopy based on scannable cantilever‐combined microsphere

We report an ingenious method of super‐resolution optical microscopy utilizing scannable cantilever‐combined microsphere. By scanning the microsphere over the sample surface in a cantilever‐combined microsphere‐sample contact state, super‐resolution images can be acquired at arbitrary sample regions through near‐field information collection by the microsphere. In addition, such a state can effectively reduce the possibility of breaking the cantilever and damaging the microsphere or sample surface. This work has developed a new method and technique of sub‐diffraction‐limit optical microscopy, and can be practically applied in various fields of micro/nanoscopy. Microsc. Res. Tech. 78:1128–1132, 2015. © 2015 Wiley Periodicals, Inc.     

Morphological and molecular pathology of CCL4‐induced hepatic fibrosis in connexin43‐deficient mice

Gap junction channels, formed by connexins (Cx), are involved in the maintenance of tissue homeostasis, cell growth, differentiation, and development. Several studies have shown that Cx43 is involved in the control of wound healing in dermal tissue. However, it remains unknown whether Cx43 plays a role in the control of liver fibrogenesis. Our study investigated the roles of Cx43 heterologous deletion on carbon tetrachloride (CCl₄)‐induced hepatic fibrosis in mice. We administered CCl₄ to both Cx43‐deficient (Cx43^+/?) and wild‐type mice and examined hepatocellular injury and collagen deposition by histological and ultrastructural analyses. Serum biochemical analysis was performed to quantify liver injury. Hepatocyte proliferation was analyzed immunohistochemically. Protein and messenger RNA (mRNA) expression of liver connexins were evaluated using immunohistochemistry as well as immunoblotting analysis and quantitative real‐time PCR. We demonstrated that Cx43^+/? mice developed excessive liver fibrosis compared with wild‐type mice after CCl₄‐induced chronic hepatic injury, with thick and irregular collagen fibers. Histopathological evaluation showed that Cx43^+/? mice present less necroinflammatory lesions in liver parenchyma and consequent reduction of serum aminotransferase activity. Hepatocyte cell proliferation was reduced in Cx43^+/? mice. There was no difference in Cx32 and Cx26 protein or mRNA expression in fibrotic mice. Protein expression of Cx43 increased in CCl₄‐treated mice, although with aberrant protein location on cytoplasm of perisinusoidal cells. Our results demonstrate that Cx43 plays an important role in the control and regulation of hepatic fibrogenesis. Microsc. Res. Tech., 2011. © 2010 Wiley‐Liss, Inc.     

Application of Fourier transform‐second‐harmonic generation imaging to the rat cervix

We present the application of Fourier transform‐second‐harmonic generation (FT‐SHG) imaging to evaluate the arrangement of collagen fibers in five nonpregnant rat cervices. Tissue slices from the mid‐cervix and near the external orifice of the cervix were analyzed in both two‐dimensions (2D) and three‐dimensions (3D). We validate that the cervical microstructure can be quantitatively assessed in three dimensions using FT‐SHG imaging and observe collagen fibers oriented both in and out‐of‐plane in the outermost and the innermost layers, which cannot be observed using 2D FT‐SHG analysis alone. This approach has the potential to be a clinically applicable method for measuring progressive changes in collagen organization during cervical remodeling in humans.     

LABEL‐FREE BIO‐AFFINITY MASS SPECTROMETRY FOR SCREENING AND LOCATING BIOACTIVE MOLECULES

Despite the recent increase in the development of bioactive molecules in the drug industry, the enormous chemical space and lack of productivity are still important issues. Additional alternative approaches to screen and locate bioactive molecules are urgently needed. Label‐free bio‐affinity mass spectrometry (BA‐MS) provides opportunities for the discovery and development of innovative drugs. This review provides a comprehensive portrayal of BA‐MS techniques and of their applications in screening and locating bioactive molecules. After introducing the basic principles, alongside some application notes, the current state‐of‐the‐art of BA‐MS‐assisted drug discovery is discussed, including native MS, size‐exclusion chromatography‐MS, ultrafiltration‐MS, solid‐phase micro‐extraction‐MS, and cell membrane chromatography‐MS. Finally, several challenges and limitations of the current methods are summarized, with a view to potential future directions for BA‐MS‐assisted drug discovery. © 2019 John Wiley & Sons Ltd. Mass Spec Rev     

Indoxyl sulfate‐induced calcification of vascular smooth muscle cells via the PI3K/Akt/NF‐κB signaling pathway

Vascular calcification (VC) is highly prevalent in patients with chronic kidney disease (CKD) and contributes to their high rate of cardiovascular mortality. Indoxyl sulfate (IS) is a representative protein‐bound uremic toxin in CKD patients, which has been recognized as a major risk factor for VC. Recent studies have demonstrated that nuclear factor‐kappa B (NK‐κB) is highly activated in the chronic inflammation conditions of CKD patients and participated in the pathogenesis of VC. However, whether NK‐κB is involved in the progression of IS‐induced VC remains without elucidation. Here, we showed that NK‐κB activity was increased in the IS‐induced calcification of human aortic smooth muscle cells (HASMCs). Blocking the NK‐κB with a selective inhibitor (Bay‐11‐7082) significantly relieved the osteogenic transdifferentiation of HASMCs, characterized by the downregulation of early osteogenic‐specific marker, core‐binding factor alpha subunit 1 (Cbfα1), and upregulation of smooth muscle α‐actin (α‐SMA), a specific vascular smooth muscle cell marker. Besides, IS stimulated the activation of PI3K/Akt signaling. Furthermore, LY294002, a specific inhibitor of PI3K/Akt pathway, attenuated the activation of NK‐κB and osteogenic differentiation of HASMCs. Together, these results suggest that PI3K/Akt/NK‐κB signaling plays an important role in the pathogenesis of osteogenic transdifferentiation induced by IS.     

Finite‐element heat‐transfer analysis of a PEEK‐steel sliding pair in a pin‐on‐disc configuration

Finite‐element (FE) thermal models have been developed in order to study the temperature distribution in a sliding pair comprising a poly(ether ether ketone) (PEEK) pin and a steel disc in a pin‐on‐disc configuration. First, a moving heat source model for the disc was created. An alternative distributed heat source model was also produced in order to reduce computing time for the evaluation of the moving heat source model by some orders of magnitude. This latter model gave the same results as the moving heat source model, except for a small region just below the moving heat source. On the basis of the distributed heat source approach, a complete axisymmetric FE model for the disc side (taking the effect of thermal resistance between the assembled components into consideration) and a steady‐state quarter model for the pin were developed. Water cooling and air cooling of the steel shaft were also compared. It was found that air cooling allowed a higher temperature in the contact region of the two sliding partners. The experimental results obtained with thermocouples and a thermal camera showed good agreement with the model predictions.     

Metabolic labeling of Escherichia coli genomic DNA with erythrosine‐11‐dUTP for functional imaging via correlative microscopy

The fluorescent metabolic labeling of microorganisms genome is an advanced imaging technique to observe and study the native shapes, structural changes, functions, and tracking of nucleic acids in single cells or tissues. We have attempted to visualize the newly synthesized DNA within the intact nucleoid of ice‐embedded proliferating cells of Escherichia coli K‐12 (thymidine‐requiring mutant, strain N4316) via correlative light‐electron microscopy. For that purpose, erythrosine‐11‐dUTP was synthesized and used as a modified analog of the exogenous thymidine substrate for metabolic incorporation into the bacterial chromosome. The formed fluorescent genomic DNA during in cellulo polymerase reaction caused a minimal cellular arrest and cytotoxicity of E. coli at certain controlled conditions. The stained cells were visualized in typical red emission color via an epifluorescence microscope. They were further ice‐embedded and examined with a Hilbert differential contrast transmission electron microscopy. At high‐resolution, the ultrastructure of tagged nucleoid appeared with significantly higher electron dense in comparison to the unlabeled one. The enhanced contrast areas in the chromosome were ascribed to the presence of iodine contents from erythrosine dye. The presented labeling approach might be a powerful strategy to reveal the structural and dynamic changes in natural DNA replication including the relationship between newly synthesized in vivo nucleic acid and the physiological state of the cell.     

Ex vivo characterization of human atherosclerotic iliac plaque components using cryo‐imaging

We characterized atherosclerotic plaque components with a novel cryo‐imaging system in lieu of standard histological methods commonly used for imaging validation and research endpoints. We aim to accurately identify plaque tissue types from fresh cadaver specimens rapidly (less than 5 h) in three dimensions for large specimens (up to 4 cm vessel segments). A single‐blind validation study was designed to determine sensitivity, specificity and inter‐rater agreement (Fleiss' Kappa) of cryo‐imaging tissue types with histology as the gold standard. Six naïve human raters identified 344 tissue type samples in 36 cryo‐image sets after being trained. Tissue type sensitivities are as follows: greater than 90% for adventitia, media‐related, smooth muscle cell ingrowth, external elastic lamina, internal elastic lamina, fibrosis, dense calcification and haemorrhage; greater than 80% for lipid and light calcification; and greater than 50% for cholesterol clefts. Specificities were greater than 95% for all tissue types. The results demonstrate convincingly that cryo‐imaging can be used to accurately identify most tissue types. If the cryo‐imaging data are entered into visualization software, three‐dimensional renderings of the plaque can be generated to visualize and quantify plaque components.     

Preserving elemental content in adherent mammalian cells for analysis by synchrotron‐based x‐ray fluorescence microscopy

Trace metals play important roles in biological function, and x‐ray fluorescence microscopy (XFM) provides a way to quantitatively image their distribution within cells. The faithfulness of these measurements is dependent on proper sample preparation. Using mouse embryonic fibroblast NIH/3T3 cells as an example, we compare various approaches to the preparation of adherent mammalian cells for XFM imaging under ambient temperature. Direct side‐by‐side comparison shows that plunge‐freezing‐based cryoimmobilization provides more faithful preservation than conventional chemical fixation for most biologically important elements including P, S, Cl, K, Fe, Cu, Zn and possibly Ca in adherent mammalian cells. Although cells rinsed with fresh media had a great deal of extracellular background signal for Cl and Ca, this approach maintained cells at the best possible physiological status before rapid freezing and it does not interfere with XFM analysis of other elements. If chemical fixation has to be chosen, the combination of 3% paraformaldehyde and 1.5 % glutaraldehyde preserves S, Fe, Cu and Zn better than either fixative alone. When chemically fixed cells were subjected to a variety of dehydration processes, air drying was proved to be more suitable than other drying methods such as graded ethanol dehydration and freeze drying. This first detailed comparison for x‐ray fluorescence microscopy shows how detailed quantitative conclusions can be affected by the choice of cell preparation method.     

Top‐down mass spectrometry for the analysis of combinatorial post‐translational modifications

Protein post‐translational modifications (PTMs) are critically important in regulating both protein structure and function, often in a rapid and reversible manner. Due to its sensitivity and vast applicability, mass spectrometry (MS) has become the technique of choice for analyzing PTMs. Whilst the “bottom‐up' analytical approach, in which proteins are proteolyzed generating peptides for analysis by MS, is routinely applied and offers some advantages in terms of ease of analysis and lower limit of detection, “top‐down” MS, describing the analysis of intact proteins, yields unique and highly valuable information on the connectivity and therefore combinatorial effect of multiple PTMs in the same polypeptide chain. In this review, the state of the art in top‐down MS will be discussed, covering the main instrumental platforms and ion activation techniques. Moreover, the way that this approach can be used to gain insights on the combinatorial effect of multiple post‐translational modifications and how this information can assist in studying physiologically relevant systems at the molecular level will also be addressed. © 2012 Wiley Periodicals, Inc., Mass Spec Rev 32:27–42, 2013