Surface Topography Description after Turning Inconel 718 with a Conventional, Wiper and Special Insert Made by the SPS Technique.

This paper presents a comparison of surface morphology obtained after machining Inconel 718 by the conventional insert, by Wiper insert and by using the cutting insert made by Spark Plasma Sintering (SPS). The shape of the special insert was obtained by employing Wire Electrical Discharge Machining (WEDM). The paper focuses on the description of surface topography after turning in dry and wet conditions. The performed investigation included longitudinal turning tests of Inconel 718 performed in a range of variable feeds. Surface topography measurements have been performed with the application of Nanoscan 855. The performed analysis includes a parametric evaluation of the obtained surfaces. With the Wiper insert, the Sa surface roughness parameter was obtained below 0.6 µm in the whole range of used feed rates. The surface roughness parameter Sa measured on the surface after machining by special insert depends on the cutting conditions (wet and dry machining). After, the dry machining parameter Sa, similar to the Wiper insert, was below 0.6 µm in the whole range of used feed rates. Unfortunately, cutting Inconel 718 using special insert with feed rate f = 0.25 mm/rev and cooling generated a surface with Sa parameter over 2 times higher than for the same feed rate without cooling, while this parameter, after turning by conventional insert, increases over 4 times using feed rate f = 0.25 mm/rev compared to feed rate f = 0.05 mm/rev during machining with cooling. This ratio is lower for conventional insert in dry machining because of sticking, which arises at the smallest feed rate according to previous research.

Investigation of the Influence of Anti-Wear Coatings on the Surface Quality and Dimensional Accuracy during Finish Turning of the Inconel 718 Alloy.

This piece of work deals with the influence assessment of the kind of coating of the cutting inserts and their wear on the dimensional accuracy and the top layer microstructure and roughness of the surface machined with constant cutting parameters vc = 85 m/min, f = 0.14 mm/obr and ap = 0.2 mm. The tests were performed on shafts made of Inconel 718 material under the conditions of finish turning, requiring a tool life of more than 20 min. The cutting inserts of identical geometry made of fine-grained carbide covered with coatings were applied by the PVD and CVD method. The values of the obtained diameter dimensions were assessed in reference to the assumed ones, as well as the values of the surface roughness and stereometry and the microstructure of the top layer. The nature and mechanisms of edge wear and its value expressed by the VBC parameter were also assessed. It was determined in the tests that the machined surface quality defined by the Ra and Sa roughness parameters and the dimensional accuracy were influenced not only by the coating microhardness but also by the method of applying the given coating. The lowest values of the tested roughness parameters were observed for the surface machined with an edge, with the S205 coating applied by the CVD method, which was characterized by the lowest microhardness. The edge with this coating also showed the lowest wear, defined by the VBC parameter, which translated into dimensional accuracy. Furthermore, the edge with the S205 coating also provided the best results with regard to the surface layer microstructure. The least favorable results, both in terms of dimensional accuracy and surface roughness, were obtained for the surface machined with a 1115-PVD-coated edge. The highest wear value was also recorded for this edge.

Identification of Parameters and Fatigue Life Assessment of the Road Pavement Lower Construction Layers under Heavy Construction Traffic.

This article analyzes the results of testing the subgrade and the lower layers of the pavement structure with the light weight deflectometer at a load of 0.1 and 0.15 MPa. It is shown that, with layer systems with an equivalent layer modulus lower than 80 MPa, significant nonlinear phenomena occur at a load of 0.15 MPa. In this situation, the identification of a reliable replacement module, a commonly used test method, at a load of 0.1 MPa, is not appropriate-it significantly overestimates the value of the modules (even by 34%), which in turn translates into a significant overestimation of the fatigue life of the structure. In a situation where intensive exploitation of the lower layers of the pavement structure is planned before the final layer arrangement is made, it is required to apply test loads corresponding to the stress conditions occurring in these layers of the structure. Such a situation takes place under the influence of technological (construction) or temporary traffic (substitute, e.g., by-pass) during construction. In order to verify the above assumptions, numerical calculations (FEM) were carried out in the elastic model for layered structures with replacement modules determined in field tests. It was found that, especially in the case of low-bearing layer systems, it is necessary to use correction factors for modules determined with a dynamic plate at a load of 0.1 MPa. Taking into account the corrected values of the modules will allow to correctly determine the change in the durability of layers at the construction stage and in the subsequent operation of the final pavement structure.

Mapping the metabolomic and lipidomic changes in the bleomycin model of pulmonary fibrosis in young and aged mice.

Alterations in metabolic pathways were recently recognized as potential underlying drivers of idiopathic pulmonary fibrosis (IPF), translating into novel therapeutic targets. However, knowledge of metabolic and lipid regulation in fibrotic lungs is limited. To comprehensively characterize metabolic perturbations in the bleomycin mouse model of IPF, we analyzed the metabolome and lipidome by mass spectrometry. We identified increased tissue turnover and repair, evident by enhanced breakdown of proteins, nucleic acids and lipids and extracellular matrix turnover. Energy production was upregulated, including glycolysis, the tricarboxylic acid cycle, glutaminolysis, lactate production and fatty acid oxidation. Higher eicosanoid synthesis indicated inflammatory processes. Because the risk of IPF increases with age, we investigated how age influences metabolomic and lipidomic changes in the bleomycin-induced pulmonary fibrosis model. Surprisingly, except for cytidine, we did not detect any significantly differential metabolites or lipids between old and young bleomycin-treated lungs. Together, we identified metabolomic and lipidomic changes in fibrosis that reflect higher energy demand, proliferation, tissue remodeling, collagen deposition and inflammation, which might serve to improve diagnostic and therapeutic options for fibrotic lung diseases in the future.

Surface Topography Description of Threads Made with Turning on Inconel 718 Shafts.

The technology of producing threads, especially in materials that are difficult to cut, is a rare subject of research and scientific publications. The requirements for the production of these elements apply not only to the geometry, but also to the quality of the surface obtained. This is particularly important in the aviation industry, where the durability of the threaded connection affects passenger safety. Due to the design of the thread, the quality of its surface is assessed visually in industrial practice. The authors of this study decided to examine the surface topography of external threads made by turning on Inconel 718 shafts in order to confirm the visual evaluation, as well as to investigate the influence of such factors as cutting speed, turning direction and type of profile. Three types of contours were cut for the research: triangular, trapezoidal symmetrical and trapezoidal asymmetrical. Turning of each was carried out twice at cutting speeds vc = 17 m/min and vc = 30 m/min. On each of the threads, the side surface of the profile made in the direction of the insert feed and the opposite surface were examined. The surface texture parameters Sa, Sq, Sp, Sv, Sz, Ssk and Sku were determined and compared. It was noticed that the thread surfaces show a tendency to irregular roughness, which was confirmed by the analysis of the Sku and Ssk coefficients. The sides of the contours made in the direction of the insert feed are characterized by a higher roughness in relation to the opposite sides, which may result from high cutting forces and difficulties with chip evacuation. With the cutting speed being considered, lower values of Sa and Sq were obtained for vc = 17 m/min, which differed from the visual assessment, proving its high subjectivity.

Anomalies in the Geometric Surface Structure of Shaped Elements Composed of Inconel 718 Alloy.

This paper presents the results of investigation that was performed on shafts composed of Inconel 718. Tests were performed in dry and wet conditions. Cutting parameters, such as feed and depth of cut, were constant. The cutting speed was changed. The investigation was performed for various shaft shapes: cylindrical, taper 30°, taper 45°, and sphere. For that reason, the value of the angle between the machined surface and the cutting edge changed. The lowest values of the roughness parameters, Ra and Rz, were obtained for a larger value of the angle between the machined surface and cutting edge. The investigation showed that cutting speed, machining conditions (dry and wet machining), and the variable angle between the machined surface and the cutting edge influenced the surface roughness. Application of a higher cutting speed resulted in lower roughness values. Lower values of roughness parameters were obtained by wet machining.

Biomarker discovery for chronic liver diseases by multi-omics - a preclinical case study.

Nonalcoholic steatohepatitis (NASH) is a major cause of liver fibrosis with increasing prevalence worldwide. Currently there are no approved drugs available. The development of new therapies is difficult as diagnosis and staging requires biopsies. Consequently, predictive plasma biomarkers would be useful for drug development. Here we present a multi-omics approach to characterize the molecular pathophysiology and to identify new plasma biomarkers in a choline-deficient L-amino acid-defined diet rat NASH model. We analyzed liver samples by RNA-Seq and proteomics, revealing disease relevant signatures and a high correlation between mRNA and protein changes. Comparison to human data showed an overlap of inflammatory, metabolic, and developmental pathways. Using proteomics analysis of plasma we identified mainly secreted proteins that correlate with liver RNA and protein levels. We developed a multi-dimensional attribute ranking approach integrating multi-omics data with liver histology and prior knowledge uncovering known human markers, but also novel candidates. Using regression analysis, we show that the top-ranked markers were highly predictive for fibrosis in our model and hence can serve as preclinical plasma biomarkers. Our approach presented here illustrates the power of multi-omics analyses combined with plasma proteomics and is readily applicable to human biomarker discovery.

A Self-Sacrificing N-Methyltransferase Is the Precursor of the Fungal Natural Product Omphalotin.

Research on ribosomally synthesized and posttranslationally modified peptides (RiPPs) has led to an increasing understanding of biosynthetic mechanisms, mostly drawn from bacterial examples. In contrast, reports on RiPPs from fungal producers, apart from the amanitins and phalloidins, are still scarce. The fungal cyclopeptide omphalotin A carries multiple N-methylations on the peptide backbone, a modification previously known only from nonribosomal peptides. Mining the genome of the omphalotin-producing fungus for a precursor peptide led to the identification of two biosynthesis genes, one encoding a methyltransferase OphMA that catalyzes the automethylation of its C-terminus, which is then released and cyclized by the protease OphP. Our findings suggest a novel biosynthesis mechanism for a RiPP in which a modifying enzyme bears its own precursor peptide.

Dissecting reactions of nonlinear precursor peptide processing of the class III lanthipeptide curvopeptin.

Lanthipeptides are ribosomally synthesized peptides which undergo extensive post-translational modifications. In addition to novel structural features and bioactivities, the in vitro study on the biosynthesis of the class III lanthipeptide labyrinthopeptin revealed a unique C- to N-terminal directionality of biosynthetic processing. The recently described class III lanthipeptide curvopeptin allowed investigating the directionality aspect in much greater detail: Structural characterization of nine curvopeptin biosynthesis intermediates by high-resolution mass spectrometry combined with a deuterium-labeling approach enabled for the first time building a comprehensive biosynthesis model featuring all three post-translational modification reactions: phosphorylation, elimination, and cyclization. These results point to a nonlinear processing scheme with a predominant C → N-terminal directionality. Our data give important mechanistic insights into the concerted processing and directionality of the multifunctional class III modifying enzymes. The data are of significance in the light of obtaining a mechanistic understanding of the post-translational biosynthesis machinery of the growing variety of ribosomally synthesized and post-translationally modified peptides.

Involvement and unusual substrate specificity of a prolyl oligopeptidase in class III lanthipeptide maturation.

Lanthipeptides represent an important group of ribosomally synthesized and post-translationally modified peptides (RiPPs). Commonly, in the last steps of their maturation, a part of the peptide, termed the leader, is removed, providing the active compound. This contribution describes for the first time the identification of a protease involved in the removal of the leader peptide of a class III lanthipeptide. Four putative class III biosynthetic gene clusters were identified in bacterial genomes, each containing a gene encoding a prolyl oligopeptidase (POP). Further in vitro investigations of the gene cluster from Kribbella flavida , involving reconstitution of the biosynthesis of the new lanthipeptide flavipeptin, proved that a POP-type FlaP protease is responsible for leader removal. Interestingly, detailed in vitro studies of the substrate specificity revealed that FlaP is specific to the post-translationally modified peptide and can discriminate between N- and C-terminal rings. Therefore, it has been shown for the first time that factors other than size and amino acid sequence might be involved in substrate recognition by POPs.

Heterologous expression and engineering studies of labyrinthopeptins, class III lantibiotics from Actinomadura namibiensis.

Labyrinthopeptins are class III lantibiotics produced by the actinomycete Actinomadura namibiensis. The most characteristic structural feature is the posttranslationally installed triamino triacid labionin with a quaternary α-carbon. In addition to the unique structure, labyrinthopeptin A2 possess remarkable antiviral and antiallodynic biological activities. To harness the substrate tolerance of the biosynthetic machinery, we developed an efficient system for the generation of labyrinthopeptin analogs. Streptomyces lividans was used as a heterologous host since the natural producer Actinomadura namibiensis remained genetically intractable. Generation of a library of 39 mutants allowed identification of variable and invariable regions in the labyrinthopeptin structures. Additional data on the flexibility of the biosynthetic machinery were provided by in vitro experiments. This study is detailed investigation on the potential to generate analogs of class III lantibiotics by genetic engineering.

Curvopeptin: a new lanthionine-containing class III lantibiotic and its co-substrate promiscuous synthetase.

Lantibiotics are ribosomally synthesized peptides containing post-translationally installed lanthionine thioether bridges. Recently characterized class III lantibiotics have also revealed the occurrence of labionin, a novel carbacyclic variation of lanthionine, and highlighted the structural diversity within this group. Here we describe the discovery and characterization of curvopeptins produced by Thermomonospora curvata, the first class III lantibiotics of thermophilic origin. Furthermore, investigation of the modifying enzyme CurKC and in particular the characterization of its specificity toward phosphorylation co-substrates was performed. Remarkably, all investigated NTPs and dNTPs were accepted by the enzyme, although the purine nucleotides ATP/dATP and GTP/dGTP were the preferred co-substrates. This finding complements previous studies on the class III lantibiotic synthetases LabKC and EryKC and underlines the surprising promiscuity of the Ser/Thr-kinase domain. Enzymatic studies with a precursor peptide mutant allowed the assignment of all dehydration sites and further GC-MS analysis revealed the presence of lanthionine as the main type of intramolecular ring.

Deuterium labeled peptides give insights into the directionality of class III lantibiotic synthetase LabKC.

The biosynthesis of a considerable number of ribosomally synthesized peptide antibiotics involves the modification of Ser and Thr residues of a precursor peptide. This post-translational processing is performed by one or multiple modifying enzymes encoded in the biosynthetic gene cluster. We present a deuterium-label based enzyme assay, utilizing a series of peptide substrates with α-deuterated Ser, for the determination of the dehydration order during the biosynthesis of class III lantibiotic labyrinthopeptin A2. Remarkably, the data show that, in contrast to other modifying enzymes of class I and II lantibiotics, LabKC has a C- to N-terminal processing mode. This surprising finding, which we consider relevant for the biosyntheses of other class III lantibiotics, underlines significant differences of this class of modifying enzymes compared to other investigated systems.

Characterization of new class III lantibiotics--erythreapeptin, avermipeptin and griseopeptin from Saccharopolyspora erythraea, Streptomyces avermitilis and Streptomyces griseus demonstrates stepwise N-terminal leader processing.

Lantibiotics are a large group of ribosomally synthesized peptides post-translationally modified to incorporate the amino acid lanthionine. They are classified, according to their biosynthetic pathway and bioactivity, into three major subtypes. Of Actinomycetes type III lantibiotics, only four peptides (SapB, SapT, LabA1, and LabA2) have been described and structurally characterized, although homologous gene clusters are abundant in other Actinomycetes. All these gene clusters share a similar architecture with a characteristic Ser/Ser/Cys motif in precursor peptides, which has previously been suggested to act as a precursor for lanthionine (SapB) and labionin (LabA2) rings. Mass spectrometry screening led to the discovery and characterization of three new representatives of type III lantibiotics: Avermipeptin (Avi), Erythreapeptin (Ery), and Griseopeptin (Gri) from Streptomyces avermitilis DSM 46492, Saccharopolyspora erythraea NRRL 2338, and Streptomyces griseus DSM 40236, respectively. Apart from the assignment of these peptides to their corresponding gene clusters, additional investigations on Avi, Ery and Gri peptides indicate stepwise leader processing by putative aminopeptidase-like protease(s), thus yielding mixtures of differently N-terminal-processed lantibiotic peptides. Similar peptide processing was observed for a heterologously expressed eryth biosynthetic gene cluster expressed in a Streptomyces host system. Remarkably, all isolates of the new type III lantibiotics contain both the amino acids lanthionine and labionin, thus implying dual-mode cyclase activity of the processing lyase-kinase-cyclase enzymes. These findings have implications for the structures and maturation of other type III lantibiotics from Actinomycetes.

Leader peptide-directed processing of labyrinthopeptin A2 precursor peptide by the modifying enzyme LabKC.

Lantibiotics are peptide antibiotics, realizing their unique secondary structure by posttranslational modifications, the most important one being the formation of the characteristic amino acid lanthionine. Like other ribosomal peptide antibiotics, they are synthesized with an N-terminal leader peptide important for posttranslational processing by modifying enzymes; after peptide maturation, the leader peptide is proteolytically cleaved off. Numerous studies of the leader peptides of class I and II lantibiotics already showed their crucial role in recognition, self-immunity, and extracellular transport. The recently described labyrinthopeptins, members of the family of class III lantibiotics, exhibit the characteristic novel amino acid labionin, which was revealed by elucidation of the structure of labyrinthopeptin A2. The assembly of the labionin motif in the linear peptide chain is mediated by the lyase-kinase-cyclase-type enzyme LabKC through a serine side chain phosphorylation with GTP, elimination of the phosphate group, and a subsequent 2-fold Michael-type addition cyclization. In this work, we systematically investigated for the first time the importance of the leader peptide in the processing of class III lantibiotics using the example of the labyrinthopeptin A2 precursor peptide. In vitro studies with synthetic leader peptide analogues revealed that a conserved N-terminal hydrophobic patch on a putative helical structure is required for the proper peptide processing by the modifying enzyme LabKC. On the other hand, studies showed that the C-terminal part of the leader peptide serves as a spacer between the binding site and active sites for phosphorylation and elimination, thus restricting the number of hydroxy amino acid side chains that could undergo dehydration. Finally, a model for the peptide recognition and processing by the LabKC has been postulated.