Microfluidics-based super-resolution microscopy enables nanoscopic characterization of blood stem cell rolling.

Hematopoietic stem/progenitor cell (HSPC) homing occurs via cell adhesion mediated by spatiotemporally organized ligand-receptor interactions. Although molecules and biological processes involved in this multistep cellular interaction with endothelium have been studied extensively, molecular mechanisms of this process, in particular the nanoscale spatiotemporal behavior of ligand-receptor interactions and their role in the cellular interaction, remain elusive. We introduce a microfluidics-based super-resolution fluorescence imaging platform and apply the method to investigate the initial essential step in the homing, tethering, and rolling of HSPCs under external shear stress that is mediated by selectins, expressed on endothelium, with selectin ligands (that is, CD44) expressed on HSPCs. Our new method reveals transient nanoscale reorganization of CD44 clusters during cell rolling on E-selectin. We demonstrate that this mechanical force-induced reorganization is accompanied by a large structural reorganization of actin cytoskeleton. The CD44 clusters were partly disrupted by disrupting lipid rafts. The spatial reorganization of CD44 and actin cytoskeleton was not observed for the lipid raft-disrupted cells, demonstrating the essential role of the spatial clustering of CD44 on its reorganization during cell rolling. The lipid raft disruption causes faster and unstable cell rolling on E-selectin compared with the intact cells. Together, our results demonstrate that the spatial reorganization of CD44 and actin cytoskeleton is the result of concerted effect of E-selectin-ligand interactions, external shear stress, and spatial clustering of the selectin ligands, and has significant effect on the tethering/rolling step in HSPC homing. Our new experimental platform provides a foundation for characterizing complicated HSPC homing

Review of In vitro Toxicity of Nanoparticles and Nanorods: Part 1

The specific use of engineered nanostructures in biomedical applications has become very attractive, due to their ability to interface and target specific cells and tissues to execute their functions. Additionally, there is continuous progress in research on new nanostructures with unique optical, magnetic, catalytic, and electrochemical properties that can be exploited for therapeutic or diagnostic methods. On the other hand, as nanostructures become widely used in many different applications, the unspecific exposure of humans to them is also unavoidable. Therefore, studying and understanding the toxicity of such materials is of increasing importance. Previously published reviews regarding the toxicological effects of nanostructures focuses mostly on the cytotoxicity of nanoparticles and their internalization, activated signaling pathways, and cellular response. Here, the most recent studies on the in vitro cytotoxicity of NPs, nanowires, and nanorods for biomedical applications are reviewed and divided into two parts. The first part considers nonmagnetic metallic and magnetic nanostructures. While part 2 covers carbon structures and semiconductors. The factors influencing the toxicity of these nanostructures are elaborated, to help elucidating the effects of these nanomaterials on cells, which is a prerequisite for their save clinical use

Dynamic structure mediates halophilic adaptation of a DNA polymerase from the deep-sea brines of the Red Sea.

The deep-sea brines of the Red Sea are remote and unexplored environments characterized by high temperatures, anoxic water, and elevated concentrations of salt and heavy metals. This environment provides a rare system to study the interplay between halophilic and thermophilic adaptation in biologic macromolecules. The present article reports the first DNA polymerase with halophilic and thermophilic features. Biochemical and structural analysis by Raman and circular dichroism spectroscopy showed that the charge distribution on the protein's surface mediates the structural balance between stability for thermal adaptation and flexibility for counteracting the salt-induced rigid and nonfunctional hydrophobic packing. Salt bridge interactions via increased negative and positive charges contribute to structural stability. Salt tolerance, conversely, is mediated by a dynamic structure that becomes more fixed and functional with increasing salt concentration. We propose that repulsive forces among excess negative charges, in addition to a high percentage of negatively charged random coils, mediate this structural dynamism. This knowledge enabled us to engineer a halophilic version of Thermococcus kodakarensis DNA polymerase.-Takahashi, M., Takahashi, E., Joudeh, L. I., Marini, M., Das, G., Elshenawy, M. M., Akal, A., Sakashita, K., Alam, I., Tehseen, M., Sobhy, M. A., Stingl, U., Merzaban, J. S., Di Fabrizio, E., Hamdan, S. M. Dynamic structure mediates halophilic adaptation of a DNA polymerase from the deep-sea brines of the Red Sea

Formation of functional selectin ligands on activated T cells and thymic progenitors : the role of core 2 β1,6-N-acetylglucosaminyltransferases in the control of lymphocyte trafficking and thymic progenitor homing

The core 2 β 1,6-N-acetylglucosaminyltransferase (C2GlcNAcT) family of enzymes (C2GlcNAcT-I,-II,-III) synthesize branched O-glycans. A significant body of work highlights the importance of C2GlcNAcT-I in controlling selectin-ligand-mediated cell trafficking while little is known about the role of the two other C2GlcNAcT isoenzymes. 1) The first objective of this thesis is to determine in vitro and in vivo T cell stimulation conditions that guide P-selectin ligand expression in absence of C2GlcNAcT-I. Mitogen stimulation of splenocytes maintained under very-high-density culture conditions uncovers C2GlcNAcT-I-independent P-selectin ligand formation in CD8⁺ T cells, but not CD4⁺ T cells. CD8⁺ T cells of C2GlcNAcT-I[sup null] mice also roll under shear flow on immobilized Pselectin in a PSGL-l(P-selectin-glycoprotein-ligand- l)-specific manner. Using RT-PCR analysis, we identify C2GlcNAcT-III as the likely source of core 2 activity. Up regulation of P-selectin ligand in C2GlcNAcT-I[sup null] CD8⁺ T cells correlates with higher core 2 enzyme activity, as measured by a standard enzymatic assay and cell-surface binding of the core 2- sensitive mAb 1B11. This reveals the well-established C2GlcNAcT-I substrates - CD43 and CD45 - as additional physiological targets of C2GlcNAcT-III. Adoptive transfer of C2GlcNAcT-I[sup null] T cells from mice transgenic for the male antigen (HY) T cell receptor shows that C2GlcNAcT-I-independent P-selectin ligand formation occurs on CD8⁺ T cells under in vivo stimulation conditions. In consequence, C2GlcNAcT-III emerges as a contributor to P-selectin ligand formation that may co-operate with C2GlcNAcT-I to control CD8+ T cell trafficking. 2) A second objective of this thesis is to explore whether C2GlcNAcT-I plays a role in controlling early T cell progenitor migration to the thymus. PSGL-1 is expressed by these thymic progenitors. In order for PSGL-1 to be recognized by P-selectin which is constitutively expressed at low levels on the thymic endothelium, it must be modified by C2GlcNAcT-I. Using mice with deficiencies in PSGL-1, C2GlcNAcT-I and P-selectin, we employ parabiosis and competitive repopulation to show that C2GlcNAcT-I modification of PSGL-1 expressed on thymic progenitors is a functionally imperative component of the thymic homing process.Medicine, Faculty ofMedicine, Department ofExperimental Medicine, Division ofGraduat

Scripting Inclusion

Efforts to bring underrepresented modernist women artists of Arabic speaking countries into the scope of Western art exhibitions has been on the rise, particularly since the early 2000s. Who decides what artists get shown and how their stories are told? What are the power structures guiding their inclusion? I inspect the consequences of the prevailing power dynamic through a feminist lens. This thesis is meant to offer a way of reviewing these systems of power so it can be more explicitly analyzed and discussed in tandem with how art is inscribed into Western discourse.S.M

Theory and applications of NMR-based metabolomics in human disease diagnosis

Metabolomics is a dynamic and emerging research field, joining proteomics, transcriptomics and genomics in affording a comprehensive understanding of biological systems and how these systems are affected by environmental stimuli and/or genetic modification. Metabolomics is particularly helpful for identifying biomarkers of disease processes such as the effects of a high fat diet on cardiovascular disease, providing insight into the interaction between genes and diet. Nuclear Magnetic Resonance (NMR) and Mass spectrometry (MS) are the most common analytical tools in metabolomics research. The high reproducibility of NMR-based techniques makes it superior to other analytical techniques especially in terms of searching for new and novel biomarkers in human diseases. Recently, NMR-based metabolomics approaches have been proposed as a promising and powerful technique for diagnosis of several human diseases. They have been used to investigate a wide range of diseases, through the examination of different kinds of human samples, including urine, blood plasma/serum, blister fluid, saliva, as well as intact tissue biopsies and tissue extracts. However, several factors can influence the metabolic balance within the human body and therefore in samples drawn from the body, including gender, age, fasting, diet, emotional stress, drug administration, physical activity and life style, thus complicating the use of NMR-based metabolomics approaches in diagnosing specific human disease. This chapter highlights the potential applications of NMR-based metabolomics approaches as a promising technique for diagnosis of human diseases