Nanthawan Avishai

FGF and ERK signaling coordinately regulate mineralization-related genes and play essential roles in osteocyte differentiation

To examine the roles of FGF and ERK MAPK signaling in osteocyte differentiation and function, we performed microarray analyses using the osteocyte cell line MLO-Y4. This experiment identified a number of mineralization-related genes that were regulated by FGF2 in an ERK MAPK-dependent manner. Real-time PCR analysis indicated that FGF2 upregulates Ank, Enpp1, Mgp, Slc20a1, and Dmp1 in MLO-Y4 cells. Consistent with this observation, the selective FGF receptor inhibitor PD173074 decreased Ank, Enpp1, Slc20a1, and Dmp1 mRNA expression in mouse calvaria in organ culture. Since Dmp1 plays a central role in osteocyte differentiation and mineral homeostasis, we further analyzed FGF regulation of Dmp1. Similar to FGF2, FGF23 upregulated Dmp1 expression in MLO-Y4 cells in the presence of Klotho. Furthermore, increased extracellular phosphate levels partially inhibited FGF2-induced upregulation of Dmp1 mRNA expression, suggesting a coordinated regulation of Dmp1 expression by FGF signaling and extracellular phosphate. In MLO-Y4 osteocytes and in MC3T3E1 and primary calvaria osteoblasts, U0126 strongly inhibited both basal expression of Dmp1 mRNA and FGF2-induced upregulation. Consistent with the in vitro observations, real-time PCR and immunohistochemical analysis showed a strong decrease in Dmp1 expression in the skeletal elements of ERK1−/−; ERK2flox/flox; Prx1-Cre mice. Furthermore, scanning electron microscopic analysis revealed that no osteocytes with characteristic dendritic processes develop in the limbs of ERK1−/−; ERK2flox/flox; Prx1-Cre mice. Collectively, our observations indicate that FGF signaling coordinately regulates mineralization-related genes in the osteoblast lineage and that ERK signaling is essential for Dmp1 expression and osteocyte differentiation.

Distinct iron architecture in SF3B1 -mutant myelodysplastic syndrome patients is linked to an SLC25A37 splice variant with a retained intron

Perturbation in iron homeostasis is a hallmark of some hematologic diseases. Abnormal sideroblasts with accumulation of iron in the mitochondria are named ring sideroblasts (RS). RS is a cardinal feature of refractory anemia with RS (RARS) and RARS with marked thrombocytosis (RARS/-T). Mutations in SF3B1, a member of the RNA splicing machinery are frequent in RARS/-T and defects of this gene were linked to RS formation. Here we showcase the differences in iron architecture of SF3B1-mutant and wild-type (WT) RARS/-T and provide new mechanistic insights by which SF3B1 mutations lead to differences in iron. We found higher iron levels in SF3B1 mutant vs WT RARS/-T by transmission electron microscopy/spectroscopy/flow cytometry. SF3B1 mutations led to increased iron without changing the valence as shown by the presence of Fe2+ in mutant and WT. Reactive oxygen species and DNA damage were not increased in SF3B1-mutant patients. RNA-sequencing and Reverse transcriptase PCR showed higher expression of a specific isoform of SLC25A37 in SF3B1-mutant patients, a crucial importer of Fe2+ into the mitochondria. Our studies suggest that SF3B1 mutations contribute to cellular iron overload in RARS/-T by deregulating SLC25A37.

Serial sectioning for examination of photoreceptor cell architecture by focused ion beam technology

Structurally deciphering complex neural networks requires technology with sufficient resolution to allow visualization of single cells and their intimate surrounding connections. Scanning electron microscopy (SEM), coupled with serial ion ablation (SIA) technology, presents a new avenue to study these networks. SIA allows ion ablation to remove nanometer sections of tissue for SEM imaging, resulting in serial section data collection for three-dimensional reconstruction. Here we highlight a method for preparing retinal tissues for imaging of photoreceptors by SIA-SEM technology. We show that this technique can be used to visualize whole rod photoreceptors and the internal disc elements from wild-type (wt) mice. The distance parameters of the discs and photoreceptors are in good agreement with previous work with other methods. Moreover, we show that large planes of retinal tissue can be imaged at high resolution to display the packing of normal rods. Finally, SIA-SEM imaging of retinal tissue from a mouse model (Nrl⁻/⁻) with phenotypic changes akin to the human disease enhanced S-cone syndrome (ESCS) revealed a structural profile of overall photoreceptor ultrastructure and internal elements that accompany this disease. Overall, this work presents a new method to study photoreceptor cells at high structural resolution that has a broad applicability to the visual neuroscience field.

Tackling Characterization Challenges in High Deformation/Stress Steel Alloys Using Transmission Kikuchi Diffraction (TKD)

Although steels have been extensively studied, the application of traditional characterization methods to investigate the microstructure still poses significant challenges. One example is White Etched Areas (WEA) that are microstructural alternations in bearings induced by dynamic loading conditions [1]. Another example is ‘white layers’ at machined steel surfaces, which are generated by hard turning processes [2]. Both involve formation of nanostructured features at the surface that may lead to significant influence on surface-initiated damage, such as corrosion, fatigue and wear surface deformation. These highly deformed regions have grains that range in size from a few nanometers to 100nm and may consist of small pockets of retained austenite. In some cases preexisting carbides are no longer present in the deformed regions. In other processes such as low temperature carburization/nitridation, the challenges are not as much the structural refinement but primarily the very high level of lattice deformation and formation of nanometer size nitrides [3]. Here as well, the large stresses and very high level of interstitial alloying can result in local phase transformation that is not easily identified by scanning electron microscopy (SEM) or conventional transmission electron microscopy (TEM) without extensive effort. In these materials, sample preparation adds to the characterization challenge. Preserving the original microstructure without introducing any mechanical damage during preparation is critical. At the same time, producing adequate samples for investigating these nanometer scale features demands sample thickness and quality similar to high resolution TEM.

Promoting Undergraduate Student Experiential Learning, using Advanced Microscopy and Spectroscopy Instrumentation on Common Materials

The Swagelok Center for Surface Analysis of Materials (SCSAM) at Case Western Reserve University has initiated a program to expand undergraduate student exposure to materials characterization methods outside their curriculum. A full day short course covering the theory behind the analytical equipment and demos are offered a few times each year. However, SCSAM has attempted to find a better to way to motivate undergraduate students to be more aware of material sciences and to engage in critical thinking. Using everyday objects is an effective strategy for promoting student engagement, fostering self-directed research, and encouraging the transition from passive learning to proactive inquiry.

Analysis of Polymer-Biomacromolecule Composites in the Solid-State via Energy Dispersive Spectroscopy-Scanning Electron Microscopy

The analysis and mapping of the dispersion of biomacromolecules within solid-state polymeric materials prepared via melt-processing is important in understanding the aggregation behavior in response to different loading levels, additives, and biochemical properties of the biomacromolecules. The presence of biomacromolecules can impart new properties to the material, such as flame retardant properties, or therapeutic value through controlled release from the polymer matrix [1]. A major issue in the meltprocessing of biomacromolecules is the high temperatures necessary, typically within the range of 95 – 200 C. These elevated temperatures can cause the biomacromolecules to aggregate and denature within the polymer melt and remain once cooled [2]. The aggregation can result in loss of the desired activity of the biomacromolecule once released, irregular release profiles, or segregation of the biomacromolecule within the matrix resulting in diminished overall composite mechanical properties.

What is the effective geometrical collection efficiency of your XEDS detector? A routine procedure applied in a SEM laboratory.

In this contribution, two large-area EDS detectors were tested according to the procedure proposed recently by Procop et al. In a first step, the optimal working distance (WD) in the two different SEM chambers was determined by moving the sample stage in the Z direction and monitoring the count rates from a field of view of 25.6 μm. The WD at which the highest intensity was measured was selected as the optimal position. Next the Cu Kα peak was measured at different relative EDS positions while it was partially removed from the fully inserted position. The spectrum at each location was collected for 10 sec using the highest pulse rate and intermediate current (2.3 nA) to minimize pile up effects (13% dead time). The ‘inverse squared normalized intensities vs. relative EDS position’ used to extract the true detector – specimen distance shows a non-linear relationship even at the minimal relative positions, which indicates shadowing due to obstruction or use of an unsuitable and/or off-centered collimator. The normalized count rates measured as a function of the EDS distances, results in a too low GCE (too low true solid angles) for both tested detectors. The search for sources of losses of signal due to possible shadowing effects is in progress.

The OTO Specimen Preparation Method for Optimal Scanning Electron Microscopy Imaging of Pseudomonas aeruginosa

Pseudomonas aeruginosa, a ubiquitous gram-negative rod-shaped bacterium, has been intensively studied as an opportunistic human pathogen. It is one of the most common pathogens for nosocomial infection in immunocompromised individuals, e.g. cystic fibrosis patients [1]. Scanning electron microscopy (SEM) is a useful tool for obtaining detailed surface topography of microorganisms. For instance, it has been used for studying the ultrastructural basis of the resistance of P. aeruginosa to antiseptics, disinfectants and antibiotics [2]. Several methods for SEM sample preparation have been developed in order to enhance contrast, reduce structural damage and preserve cell structure in the native state. These techniques include glutaraldehyde fixation, negative staining, cryo-techniques, critical point drying, coating specimens with gold or osmium, and OTO staining. OTO staining has mostly been used in the preparation of biological tissues to provide bulk conductivity, which enables enhanced contrast [3]. Here, we report a specimen preparation protocol for optimal SEM imaging of P. aeruginosa using the OTO staining method.

Are Axonal Mitochondria Really Enriched at Nodes of Ranvier? A Three Dimensional Serial Ion Ablation SEM (FIB/SEM) Study.

To meet the energy demands of nerve conduction, small mitochondria are transported from the neuronal cell body and enriched at mitochondrial stationary sites located throughout the axon. Determining whether and how myelination locally regulates mitochondrial structure and function in axons is fundamental to understanding axonal biology and pathology [1]. Nodes of Ranvier are the sites of action potential propagation, and previous EM studies of myelinated axons have suggested that axons are enriched in nodal axonal cytoplasm (axoplasm). Paradoxically, several studies noted that mitochondria were frequently absent at nodes of Ranvier [2,3]. Numbers of nodes with and without mitochondria had not been quantified, presumably to difficulties in obtaining extensive 3D EM datasets including entire nodes.

Serial Sectioning in SEM: Challenges and Opportunities

*Dept. of Mat. Sci. and Eng., Case Western Reserve University, Cleveland, OH, 44106. **Dept. of Neurosciences, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44120. ***Dept. of Molecular Bio. & Microbiology, CWRU School of Medicine, Cleveland, OH, 44106. ****Dept. of Restorative Sciences, Herman Ostrow School of Dentistry, University of Southern California, Los Angeles, CA, 90089. *****Dept. of Pharmacology, Case Western Reserve University, Cleveland, OH, 44106. ******Cell and Tissue Imaging Center, St. Jude Children’s Research Hospital, Memphis, TN 38105.