Advanced Spectroscopy and APBS Modeling for Determination of the Role of His190 and Trp103 in Mouse Thymidylate Synthase Interaction with Selected dUMP Analogues

A homo-dimeric enzyme, thymidylate synthase (TS), has been a long-standing molecular target in chemotherapy. To further elucidate properties and interactions with ligands of wild-type mouse thymidylate synthase (mTS) and its two single mutants, H190A and W103G, spectroscopic and theoretical investigations have been employed. In these mutants, histidine at position 190 and tryptophan at position 103 are substituted with alanine and glycine, respectively. Several emission-based spectroscopy methods used in the paper demonstrate an especially important role for Trp 103 in TS ligands binding. In addition, the Advanced Poisson–Boltzmann Solver (APBS) results show considerable differences in the distribution of electrostatic potential around Trp 103, as compared to distributions observed for all remaining Trp residues in the mTS family of structures. Together, spectroscopic and APBS results reveal a possible interplay between Trp 103 and His190, which contributes to a reduction in enzymatic activity in the case of H190A mutation. Comparison of electrostatic potential for mTS complexes, and their mutants, with the substrate, dUMP, and inhibitors, FdUMP and N4-OH-dCMP, suggests its weaker influence on the enzyme–ligand interactions in N4OH-dCMP-mTS compared to dUMP-mTS and FdUMP-mTS complexes. This difference may be crucial for the explanation of the ”abortive reaction” inhibitory mechanism of N4OH-dCMP towards TS. In addition, based on structural analyses and the H190A mutant capacity to form a denaturation-resistant complex with N4-OH-dCMP in the mTHF-dependent reaction, His190 is apparently responsible for a strong preference of the enzyme active center for the anti rotamer of the imino inhibitor form.     

Visualization of particles arrangement during filling stage of polyamide 6 – metal insert injection molding

An isotactic polypropylene filled with talc and mica particles was used for visualization of molten polymer flow lines during filling of mold cavity with metal insert. The talc particles orientation in polypropylene matrix was followed by scanning electron microscopy. For the quantification of platelet particles orientation in molten polymer flow localized near metal wall, the Herman's function was applied. An existence of regions with well oriented, randomly organized talc particles depending on the shape and cross‐section of the channels, was found and reported. The domain with the most disorganized platelet particles was detected in the region where polymer streamed around the sharp corner of the metal insert. Such a kind of disarranged region could be the weakest part of the whole injection molded polymeric element. POLYM. ENG. SCI., 59:E271–E278, 2019. © 2019 Society of Plastics Engineers     

Central Apparatus,the Molecular Kickstarter of Ciliary and Flagellar Nanomachines

Motile cilia and homologous organelles, the flagella, are an early evolutionarily invention, enabling primitive eukaryotic cells to survive and reproduce. In animals, cilia have undergone functional and structural speciation giving raise to typical motile cilia, motile nodal cilia, and sensory immotile cilia. In contrast to other cilia types, typical motile cilia are able to beat in complex, two-phase movements. Moreover, they contain many additional structures, including central apparatus, composed of two single microtubules connected by a bridge-like structure and assembling numerous complexes called projections. A growing body of evidence supports the important role of the central apparatus in the generation and regulation of the motile cilia movement. Here we review data concerning the central apparatus structure, protein composition, and the significance of its components in ciliary beating regulation.     

3D characterization of porosity in an air plasma‐sprayed thermal barrier coating and its effect on thermal conductivity

Air plasma spray (APS) thermal barrier coatings (TBCs) contain pores, cracks and splat interfaces that are preferentially aligned normal to the heat flux direction. These significantly reduce (by as much as 50%) the thermal conductivity over a fully dense coating. Here the microstructures of APS thermal barrier coatings (TBCs) have been characterized in 3D using X‐ray microcomputed tomography (μ‐CT). Pores larger than 2.8 μm³ are resolved and their contribution to thermal conductivity reduction is evaluated using image‐based microstructurally realistic numerical models. However, the models overestimate (by almost 50%) the measured thermal conductivity of the as‐deposited TBC sample. It is shown that this discrepancy is due to very fine (micrometer and submicrometer) cracks which readily sinter after short‐term exposure to temperatures representative of TBC operating conditions causing the measured thermal conductivity to rise to within 15% of that predicted. This suggests that under the realistic service conditions, the 3D image‐based models based on CT images provide a good indicator of the likely long‐term TBC performance. Virtual experiments showed that for the retained pores, the larger pores which are flatter and more oriented in the plane of the APS splats, contribute disproportionately to the beneficial reduction in thermal conductivity. Our results demonstrate that X‐ray imaging is a useful tool in establishing APS process conditions that give rise to a beneficial distribution of such pores.     

Integrated process planning and scheduling in networked manufacturing systems for I4.0: a review and framework proposal

Wireless Networks - Integrated process planning and scheduling in networked manufacturing systems plays a crucial role nowadays and in the forthcoming context of Industry 4.0 to enable effective...     

Novel Polymorph of GaSe

2D GaSe is a semiconductor belonging to the group of post-transition metal chalcogenides with great potential for advanced optoelectronic applications. The weak interlayer interaction in multilayer 2D materials allows the formation of several polymorphs. Here, the first structural observation of a new GaSe polymorph is reported, characterized by a distinct atomic configuration with a centrosymmetric monolayer (D_3d point group). The atomic structure of this new GaSe polymorph is determined by aberration-corrected scanning transmission electron microscopy. Density-functional theory calculations verify the structural stability of this polymorph. Furthermore, the band structure and Raman intensities are calculated, predicting slight differences to the currently known polymorphs. In addition, the occurrence of layer rotations, interlayer relative orientations, as well as translation shear faults is discussed. The experimental confirmation of the new GaSe polymorph indicates the importance of investigating changes in the crystal structure, which can further impact the properties of this family of compounds.     

Protein‐Corona‐by‐Design in 2D: A Reliable Platform to Decode Bio–Nano Interactions for the Next‐Generation Quality‐by‐Design Nanomedicines

Hard corona (HC) protein, i.e., the environmental proteins of the biological medium that are bound to a nanosurface, is known to affect the biological fate of a nanomedicine. Due to the size, curvature, and specific surface area (SSA) 3‐factor interactions inherited in the traditional 3D nanoparticle, HC‐dependent bio–nano interactions are often poorly probed and interpreted. Here, the first HC‐by‐design case study in 2D is demonstrated that sequentially and linearly changes the HC quantity using functionalized graphene oxide (GO) nanosheets. The HC quantity and HC quality are analyzed using NanoDrop and label‐free liquid chromatography–mass spectrometry (LC‐MS) followed by principal component analysis (PCA). Cellular responses (uptake and cytotoxicity in J774 cell model) are compared using imaging cytometry and the modified lactate dehydrogenase assays, respectively. Cellular uptake linearly and solely correlates with HC quantity (R² = 0.99634). The nanotoxicity, analyzed by retrospective design of experiment (DoE), is found to be dependent on the nanomaterial uptake (primary), HC composition (secondary), and nanomaterial exposure dose (tertiary). This unique 2D design eliminates the size–curvature–SSA multifactor interactions and can serve as a reliable screening platform to uncover HC‐dependent bio–nano interactions to enable the next‐generation quality‐by‐design (QbD) nanomedicines for better clinical translation.     

Transfer of fibroblast sheets cultured on thermoresponsive dishes with membranes

In cell or tissue engineering, it is essential to develop a support for cell-to-cell adhesion, which leads to the generation of cell sheets connected by extracellular matrix. Such supports must be hydrophobic and should result in a detachable cell sheet. A thermoresponsive support that enables the cultured cell sheet to detach using only a change in temperature could be an interesting alternative in regenerative medicine. The aim of this study was to evaluate plates covered with thermoresponsive polymers as supports for the formation of fibroblast sheets and to develop a damage-free procedure for cell sheet transfer with the use of membranes as transfer tools. Human skin fibroblasts were seeded on supports coated with a thermoresponsive polymer: commercial UpCell? dishes (NUNC?) coated with thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) and dishes coated with thermoresponsive poly(tri(ethylene glycol) monoethyl ether methacrylate) (P(TEGMA-EE)). Confluent fibroblast sheets were effectively cultured and harvested from both commercial PNIPAM-coated dishes and laboratory P(TEGMA-EE)-coated dishes. To transfer a detached cell sheet, two membranes, Immobilon-P^® and SUPRATHEL^®, were examined. The use of SUPRATHEL for relocating the cell sheets opens a new possibility for the clinical treatment of wounds. This study established the background for implementing thermoresponsive supports for transplanting in vitro cultured fibroblasts.     

Cyber-physical systems challenges: a needs analysis for collaborating embedded software systems

Embedding computing power in a physical environment has provided the functional flexibility and performance necessary in modern products such as automobiles, aircraft, smartphones, and more. As product features came to increasingly rely on software, a network infrastructure helped factor out common hardware and offered sharing functionality for further innovation. A logical consequence was the need for system integration. Even in the case of a single original end manufacturer who is responsible for the final product, system integration is quite a challenge. More recently, there have been systems coming online that must perform system integration even after deployment—that is, during operation. This has given rise to the cyber-physical systems (CPS) paradigm. In this paper, select key enablers for a new type of system integration are discussed. The needs and challenges for designing and operating CPS are identified along with corresponding technologies to address the challenges and their potential impact. The intent is to contribute to a model-based research agenda in terms of design methods, implementation technologies, and organization challenges necessary to bring the next-generation systems online.     

Proteomics of citrullination in cardiovascular disease

Citrullination is an enzymatic posttranslational modification (PTM), which has become a topic of recent research due to its involvement in various physiologic and pathologic processes. This review will focus primarily on the cardiovascular pathology associated to date with citrullination, including myocardial citrullination as well as the potential role of citrullination in atherosclerosis as a driver inflammation, especially in patients with rheumatoid arthritis (RA). There is extensive citrullination within normal and RA myocardium as well as the atherosclerotic plaque; and increased levels of antibodies to citrullinated proteins have been associated with increased cardiovascular burden in patients with RA. Given robust citrullination in both RA as well as non-RA patients, there also exists the potential for protein citrullination to contribute to cardiovascular pathology in the general population. However, to investigate this possibility will require development of improved biochemical and proteomic tools for the study of protein citrullination. The remainder of this review will discuss current and developing methodologies to study protein citrullination and discuss their applicability for the analysis of complex samples. The ability to identify and quantify citrullinated protein is a key to understanding the role of this PTM. Methodologies and limitations of current technology for the identification of citrullination are discussed.