A Note on Interpreting Tool Temperature Measurements from Thermography

Thermography (thermal imaging) is a well-established experimental method for studying cutting tool temperature distributions. In one form, cutting edge temperatures within the chip / tool contact area are deduced from thermal images of tool faces normal to the cutting edge but offset from the contact region. In general practice, the offset is made as small as possible (<< 1 mm) and it is assumed that the observed temperature is the same as that within the contact. In this short communication an approximate analytical model is developed for the influence of the offset on the observed temperature. The predictions from the model are compared with previously unpublished existing results on the machining of Ti alloys (Ti6Al4V and Ti5Al4V) and on steel (AISI 4140). It is shown that ignoring the offset may introduce underestimates of cutting edge temperature of ≈ 30% or more. This is large compared to the usually considered uncertainties of ± 5% from camera and tool emissivity calibration. There is a need for a dedicated study of this effect.     

Quantification of tool wear using white light interferometry and three-dimensional computational metrology

Although several wear modes can result from machining, the most common tend to be what are referred to as flank wear and crater wear. Flank wear can be easily measured directly from images of a worn cutting tool, and this is the typical method used to quantify the condition of a tool. On the other hand, crater wear is difficult to quantify, and thus has typically been treated in a qualitative manner. The inability to characterize and compare the two wear modes in a quantitative way is an increasingly important problem as the precision of machining operations improves and cutting moves almost exclusively to the nose radius of cutting tools. This paper introduces a new approach to this problem by proposing a technique to quantify both wear modes for direct comparison. The technique measures the volumetric wear loss in the two regions by comparing three-dimensional wear data obtained by white light interferometry with ideal representations of unworn cutting tools. The resulting wear measurements are compared and related to changes in the cutting process, specifically increases in cutting forces and changes in the topography of machined surfaces.     

Microstructures and Mechanical Properties of Ti6Al4V Parts Fabricated by Selective Laser Melting and Electron Beam Melting

This work compares two metal additive manufacturing processes, selective laser melting (SLM) and electron beam melting (EBM), based on microstructural and mechanical property evaluation of Ti6Al4V parts produced by these two processes. Tensile and fatigue bars conforming to ASTM standards were fabricated using Ti6Al4V ELI grade material. Microstructural evolution was studied using optical and scanning electron microscopy. Tensile and fatigue tests were carried out to understand mechanical properties and to correlate them with the corresponding microstructure. The results show differences in microstructural evolution between SLM and EBM processed Ti6Al4V and their influence on mechanical properties. The microstructure of SLM processed parts were composed of an α′ martensitic phase, whereas the EBM processed parts contain primarily α and a small amount of β phase. Consequently, there are differences in tensile and fatigue properties between SLM- and EBM-produced Ti6Al4V parts. The differences are related to the cooling rates experienced as a consequence of the processing conditions associated with SLM and EBM processes.     

Evaluating and monitoring nucleation and growth in copper foil

The electrodeposition of copper foil for use in electronic materials applications is a complex and demanding process. The specific aspects of producing and controlling the structure-property-performance requirements of the foil are important because of the stringent demands placed on their use in printed circuit boards and similar products. In this paper, a brief review of the electrodeposition process for raw copper foil is presented. Since electrolyte additives play such a significant role in the copper-depositionprocess, the effects of two essential additives, chloride ion and an organic (e.g., glue or gelatine), on the foil are described. Also, the influence of other operating parameters on the initial nucleation, growth, and subsequent electrocrystallization are discussed. Selected characterization methods, such as polarization and scanning electron micrography techniques, are described as a means of monitoring the process, but universally accepted methods of evaluating and controlling the additives and foil quality during electrolysis are still being sought.     

The GAUGAA Motif Is Responsible for the Binding between circSMARCA5 and SRSF1 and Related Downstream Effects on Glioblastoma Multiforme Cell Migration and Angiogenic Potential

Circular RNAs (circRNAs) are a large class of RNAs with regulatory functions within cells. We recently showed that circSMARCA5 is a tumor suppressor in glioblastoma multiforme (GBM) and acts as a decoy for Serine and Arginine Rich Splicing Factor 1 (SRSF1) through six predicted binding sites (BSs). Here we characterized RNA motifs functionally involved in the interaction between circSMARCA5 and SRSF1. Three different circSMARCA5 molecules (Mut1, Mut2, Mut3), each mutated in two predicted SRSF1 BSs at once, were obtained through PCR-based replacement of wild-type (WT) BS sequences and cloned in three independent pcDNA3 vectors. Mut1 significantly decreased its capability to interact with SRSF1 as compared to WT, based on the RNA immunoprecipitation assay. In silico analysis through the “Find Individual Motif Occurrences” (FIMO) algorithm showed GAUGAA as an experimentally validated SRSF1 binding motif significantly overrepresented within both predicted SRSF1 BSs mutated in Mut1 (q-value = 0.0011). U87MG and CAS-1, transfected with Mut1, significantly increased their migration with respect to controls transfected with WT, as revealed by the cell exclusion zone assay. Immortalized human brain microvascular endothelial cells (IM-HBMEC) exposed to conditioned medium (CM) harvested from U87MG and CAS-1 transfected with Mut1 significantly sprouted more than those treated with CM harvested from U87MG and CAS-1 transfected with WT, as shown by the tube formation assay. qRT-PCR showed that the intracellular pro- to anti-angiogenic Vascular Endothelial Growth Factor A (VEGFA) mRNA isoform ratio and the amount of total VEGFA mRNA secreted in CM significantly increased in Mut1-transfected CAS-1 as compared to controls transfected with WT. Our data suggest that GAUGAA is the RNA motif responsible for the interaction between circSMARCA5 and SRSF1 as well as for the circSMARCA5-mediated control of GBM cell migration and angiogenic potential.     

Characterization of Apo-Form Selective Inhibition of Indoleamine 2,3-Dioxygenase**

Indoleamine-2,3-dioxygenase 1 (IDO1) is a heme-containing enzyme that catalyzes the rate-limiting step in the kynurenine pathway of tryptophan (TRP) metabolism. As it is an inflammation-induced immunoregulatory enzyme, pharmacological inhibition of IDO1 activity is currently being pursued as a potential therapeutic tool for the treatment of cancer and other disease states. As such, a detailed understanding of the mechanism of action of IDO1 inhibitors with various mechanisms of inhibition is of great interest. Comparison of an apo-form-binding IDO1 inhibitor (GSK5628) to the heme-coordinating compound, epacadostat (Incyte), allows us to explore the details of the apo-binding inhibition of IDO1. Herein, we demonstrate that GSK5628 inhibits IDO1 by competing with heme for binding to a heme-free conformation of the enzyme (apo-IDO1), whereas epacadostat coordinates its binding with the iron atom of the IDO1 heme cofactor. Comparison of these two compounds in cellular systems reveals a long-lasting inhibitory effect of GSK5628, previously undescribed for other known IDO1 inhibitors. Detailed characterization of this apo-binding mechanism for IDO1 inhibition might help design superior inhibitors or could confer a unique competitive advantage over other IDO1 inhibitors vis-à-vis specificity and pharmacokinetic parameters.     

Courses Based on iGEM/BIOMOD Competitions Are the Ideal Format for Research-Based Learning of Xenobiology

Synthetic biology and especially xenobiology, as emerging new fields of science, have reached an intellectual and experimental maturity that makes them suitable for integration into the university curricula of chemical and biological disciplines. Novel scientific fields that include laboratory work are perfect playgrounds for developing highly motivating research-based teaching modules. We believe that research-based learning enriched by digital tools is the best approach for teaching new emerging essentials of academic education. This is especially true when the scientific field as such is still not canonized with text books and best-practice examples. Our experience shows that iGEM/BIOMOD competitions represent an excellent basis for designing research-based courses in xenobiology. Therefore, we present a report on “iGEM–Synthetic Biology” offered at the Technische Universität Berlin as an example.     

Simplified Monopalmitoyl Toll-like Receptor 2 Ligand Mini-UPam for Self-Adjuvanting Neoantigen-Based Synthetic Cancer Vaccines

Synthetic vaccines, based on antigenic peptides that comprise MHC−I and MHC-II T-cell epitopes expressed by tumors, show great promise for the immunotherapy of cancer. For optimal immunogenicity, the synthetic peptides (SPs) should be adjuvanted with suitable immunostimulatory additives. Previously, we have shown that improved immunogenicity in vivo is obtained with vaccine modalities in which an SP is covalently connected to an adjuvanting moiety, typically a ligand to Toll-like receptor 2 (TLR2). SPs were covalently attached to UPam, which is a derivative of the classic TLR2 ligand Pam₃CysSK₄. A disadvantage of the triply palmitoylated UPam is its high lipophilicity, which precludes universal adoption of this adjuvant for covalent modification of various antigenic peptides as it renders the synthetic vaccine insoluble in several cases. Here, we report a novel conjugatable TLR2 ligand, mini-UPam, which contains only one palmitoyl chain, rather than three, and therefore has less impact on the solubility and other physicochemical properties of a synthetic peptide. In this study, we used SPs that contain the clinically relevant neoepitopes identified in a melanoma patient who completely recovered after T-cell therapy. Homogeneous mini-UPam-SP conjugates have been prepared in good yields by stepwise solid-phase synthesis that employed a mini-UPam building block pre-prepared in solution and the standard set of Fmoc-amino acids. The immunogenicity of the novel mini-UPam-SP conjugates was demonstrated by using the cancer patient's T-cells.     

CYP154C5 Regioselectivity in Steroid Hydroxylation Explored by Substrate Modifications and Protein Engineering**

CYP154C5 from Nocardia farcinica is a P450 monooxygenase able to hydroxylate a range of steroids with high regio- and stereoselectivity at the 16α-position. Using protein engineering and substrate modifications based on the crystal structure of CYP154C5, an altered regioselectivity of the enzyme in steroid hydroxylation had been achieved. Thus, conversion of progesterone by mutant CYP154C5 F92A resulted in formation of the corresponding 21-hydroxylated product 11-deoxycorticosterone in addition to 16α-hydroxylation. Using MD simulation, this altered regioselectivity appeared to result from an alternative binding mode of the steroid in the active site of mutant F92A. MD simulation further suggested that the entrance of water to the active site caused higher uncoupling in this mutant. Moreover, exclusive 15α-hydroxylation was observed for wild-type CYP154C5 in the conversion of 5α-androstan-3-one, lacking an oxy-functional group at C17. Overall, our data give valuable insight into the structure–function relationship of this cytochrome P450 monooxygenase for steroid hydroxylation.