Prediction of Fatigue Life of Polyetherimide/Carbon Fiber Particulate Composites at Various Maximum Stresses and Filler Contents.

The objective of this research was to predict the fatigue behavior of polyetherimide-based composites loaded with short carbon fibers 200 µm long under cyclic loads. The weight fraction of the filler was 10, 20, and 30 wt.%, while the maximum stress in a cycle was 55, 65, and 75 MPa. A modified fatigue model based on the obtained experimental results and Basquin equation was developed. The novelty of the results is related to developing a model on the structure-property relationship, which accounts for both the maximum stress in a cycle and the carbon fiber content in the composites. In addition, an "algorithm" for designing such composites according to the fatigue life criterion was proposed. The approach to determine relationships between the composition, structure, and properties of PCMs described in this study can be applied to further expand the model and to improve its versatility in the use of other thermoplastic matrices and fillers. The results of this study can be applied for the design of composites for structural applications with designated fatigue properties.

Impact Behavior and Residual Strength of PEEK/CF-Laminated Composites with Various Stacking Sequences.

Carbon fiber-reinforced composites are popular due to their high strength and light weight; thus, the structures demonstrate high performance and specific strength. However, these composites are susceptible to impact damage. The objective of this research was to study the behavior of carbon fiber-reinforced laminates based on a polyetheretherketone (PEEK) matrix with six stacking sequences under static and impact loading. Four-point bending, short-beam bending, drop weight impact, and compression after impact tests were carried out. The results were complemented with digital shearography to estimate the damaged areas. Finite element modeling served to assess the failure mechanisms, such as fiber and matrix failure, in different layers due to tension of compression. Three behavior pattern of layups under drop-weight impact were found: (i)-energy redistribution due to mostly linear behavior (like a trampoline) and thus lower kinetic energy absorption for damage initiation, (ii)-moderate absorption of energy with initiation and propagation of concentrated damage with depressed redistribution of energy in the material, (iii)-moderate energy absorption with good redistribution due to initiation of small, dispersed damage. The results can be used to predict the mechanical behavior of composites with different stacking sequences in materials for proper structural design.

Triphenylphosphonium Analogs of Short Peptide Related to Bactenecin 7 and Oncocin 112 as Antimicrobial Agents.

Antimicrobial peptides (AMPs) have recently attracted attention as promising antibacterial agents capable of acting against resistant bacterial strains. In this work, an approach was applied, consisting of the conjugation of a peptide related to the sequences of bactenecin 7 (Bac7) and oncocin (Onc112) with the alkyl(triphenyl)phosphonium (alkyl-TPP) fragment in order to improve the properties of the AMP and introduce new ones, expand the spectrum of antimicrobial activity, and reduce the inhibitory effect on the eukaryotic translation process. Triphenylphosphonium (TPP) derivatives of a decapeptide RRIRPRPPYL were synthesized. It was comprehensively studied how the modification of the AMP affected the properties of the new compounds. It was shown that while the reduction in the Bac7 length to 10 a.a. residues dramatically decreased the affinity to bacterial ribosomes, the modification of the peptide with alkyl-TPP moieties led to an increase in the affinity. New analogs with structures that combined a decapeptide related to Bac7 and Onc112-Bac(1-10, R/Y)-and TPP attached to the C-terminal amino acid residue via alkylamide linkers, inhibited translation in vitro and were found to be more selective inhibitors of bacterial translation compared with eukaryotic translation than Onc112 and Bac7. The TPP analogs of the decapeptide related to Bac7 and Onc112 suppressed the growth of both Gram-negative bacteria, similar to Onc112 and Bac7, and Gram-positive ones, similar to alkyl-TPP derivatives, and also acted against some resistant laboratory strains. Bac(1-10, R/Y)-C2-TPP, containing a short alkylamide linker between the decapeptide and TPP, was transferred into the E. coli cells via the SbmA transporter protein. TPP derivatives of the decapeptide Bac(1-10, R/Y) containing either a decylamide or ethylamide linker caused B. subtilis membrane depolarization, similar to alkyl-TPP. The Bac(1-10, R/Y)-C2-TPP analog was proven to be non-toxic for mammalian cells using the MTT test.

Multiple Non-Canonical Base-Stacking Interactions as One of the Major Determinants of RNA Tertiary Structure Organization.

Stacking interactions of heterocyclic bases of ribonucleotides are one of the most important factors in the organization of RNA secondary and tertiary structure. Most of these (canonical) interactions are formed between adjacent residues in RNA polynucleotide chains. However, with the accumulation of data on the atomic tertiary structures of various RNAs and their complexes with proteins, it has become clear that nucleotide residues that are not adjacent in the polynucleotide chains and are sometimes separated in the RNA primary structure by tens or hundreds of nucleotides can interact via (non-canonical) base stacking. This paper presents an exhaustive database of such nonadjacent base-stacking elements (NA-BSEs) and their environment in the macromolecules of natural and synthetic RNAs. Analysis of these data showed that NA-BSE-forming nucleotides, on average, account for about a quarter of all nucleotides in a particular RNA and, therefore, should be considered as bona fide motifs of the RNA tertiary structure. We also classified NA-BSEs by their location in RNA macromolecules. It was shown that the structure-forming role of NA-BSEs involves compact folding of single-stranded RNA loops, transformation of double-stranded bulges into imperfect helices, and binding of RNA regions distant in the primary and secondary RNA structure.

Ultrasonic Welding of PEEK Plates with CF Fabric Reinforcement-The Optimization of the Process by Neural Network Simulation.

The optimal mode for ultrasonic welding (USW) of the "PEEK-ED (PEEK)-prepreg (PEI impregnated CF fabric)-ED (PEEK)-PEEK" lap joint was determined by artificial neural network (ANN) simulation, based on the sample of the experimental data expanded with the expert data set. The experimental verification of the simulation results showed that mode 10 (t = 900 ms, P = 1.7 atm, τ = 2000 ms) ensured the high strength properties and preservation of the structural integrity of the carbon fiber fabric (CFF). Additionally, it showed that the "PEEK-CFF prepreg-PEEK" USW lap joint could be fabricated by the "multi-spot" USW method with the optimal mode 10, which can resist the load per cycle of 50 MPa (the bottom HCF level). The USW mode, determined by ANN simulation for the neat PEEK adherends, did not provide joining both particulate and laminated composite adherends with the CFF prepreg reinforcement. The USW lap joints could be formed when the USW durations (t) were significantly increased up to 1200 and 1600 ms, respectively. In this case, the elastic energy is transferred more efficiently to the welding zone through the upper adherend.

Effect of Polymer Matrix on Inelastic Strain Development in PI- and PEI-Based Composites Reinforced with Short Carbon Fibers under Low-Cyclic Fatigue.

Since the inelastic strain development plays an important role in the low-cycle fatigue (LCF) of High-Performance Polymers (HPPs), the goal of the research was to study the effect of an amorphous polymer matrix type on the resistance to cyclic loading for both polyimide (PI)- and polyetherimide (PEI)-based composites, identically loaded with short carbon fibers (SCFs) of various lengths, in the LCF mode. The fracture of the PI and PEI, as well as their particulate composites loaded with SCFs at an aspect ratio (AR) of 10, occurred with a significant role played by cyclic creep processes. Unlike PEI, PI was less prone to the development of creep processes, probably because of the greater rigidity of the polymer molecules. This increased the stage duration of the accumulation of scattered damage in the PI-based composites loaded with SCFs at AR = 20 and AR = 200, causing their greater cyclic durability. In the case of SCFs 2000 µm long, the length of the SCFs was comparable to the specimen thickness, causing the formation of a spatial framework of unattached SCFs at AR = 200. The higher rigidity of the PI polymer matrix provided more effective resistance to the accumulation of scattered damage with the simultaneously higher fatigue creep resistance. Under such conditions, the adhesion factor exerted a lesser effect. As shown, the fatigue life of the composites was determined both by the chemical structure of the polymer matrix and the offset yield stresses. The essential role of the cyclic damage accumulation in both neat PI and PEI, as well as their composites reinforced with SCFs, was confirmed by the results of XRD spectra analysis. The research holds the potential to solve problems related to the fatigue life monitoring of particulate polymer composites.

An Automated Optical Strain Measurement System for Estimating Polymer Degradation under Fatigue Testing.

(1) Background: this study deals with design of an automated laboratory facility based on a servo-hydraulic testing machine for estimating parameters of mechanical hysteresis loops by means of the digital image correlation (DIC) method. (2) Methods: the paper presents a description of the testing facility, describes the grounds for calculating the elastic modulus, the offset yield strength (OYS) and the parameters of the mechanical hysteresis loops by the DIC method. (3) Results: the developed hardware-software facility was tested by studying the fatigue process in neat polyimide (PI) under various amplitude tension-tension loadings. It was found that the damage accumulation was accompanied by the decrease in the loop areas, while failure occurred when it reduced by at least ~5 kJ/m3. (4) Conclusions: it was shown that lowering the loop area along with changing the secant modulus value makes it possible to estimate the level of the scattered damage accumulation (mainly at the stresses above the OYS level). It was revealed that fractography data, namely the pattern and sizes of the fatigue crack initiation and propagation zones, did not correlate well with the dependences of the parameters of the hysteresis loops.

Estimating Low- and High-Cyclic Fatigue of Polyimide-CF-PTFE Composite through Variation of Mechanical Hysteresis Loops.

The fatigue properties of neat polyimide and the "polyimide + 10 wt.% milled carbon fibers + 10 wt.% polytetrafluoroethylene" composite were investigated under various cyclic loading conditions. In contrast to most of the reported studies, constructing of hysteresis loops was performed through the strain assessment using the non-contact 2D Digital Image Correlation method. The accumulation of cyclic damage was analyzed by calculating parameters of mechanical hysteresis loops. They were: (i) the energy losses (hysteresis loop area), (ii) the dynamic modulus (proportional to the compliance/stiffness of the material) and (iii) the damping capacity (calculated through the dissipated and total mechanical energies). On average, the reduction in energy losses reached 10-18% at the onset of fracture, whereas the modulus variation did not exceed 2.5% of the nominal value. The energy losses decreased from 20 down to 18 J/m3 (10%) for the composite, whereas they reduced from 30 down to 25 J/m3 (17%) for neat PI in the low-cycle fatigue mode. For high-cycle fatigue, energy losses decreased from 10 to 9 J/m3 (10%) and from 17 to 14 J/m3 (18%) for neat PI and composite, respectively. For this reason, the changes of the energy losses due to hysteresis are of prospects for the characterization of both neat PI and the reinforced PI-based composites.

Ribosomal protein S18 acetyltransferase RimI is responsible for the acetylation of elongation factor Tu.

N-terminal acetylation is widespread in the eukaryotic proteome but in bacteria is restricted to a small number of proteins mainly involved in translation. It was long known that elongation factor Tu (EF-Tu) is N-terminally acetylated, whereas the enzyme responsible for this process was unclear. Here, we report that RimI acetyltransferase, known to modify ribosomal protein S18, is likewise responsible for N-acetylation of the EF-Tu. With the help of inducible tufA expression plasmid, we demonstrated that the acetylation does not alter the stability of EF-Tu. Binding of aminoacyl tRNA to the recombinant EF-Tu in vitro was found to be unaffected by the acetylation. At the same time, with the help of fast kinetics methods, we demonstrate that an acetylated variant of EF-Tu more efficiently accelerates A-site occupation by aminoacyl-tRNA, thus increasing the efficiency of in vitro translation. Finally, we show that a strain devoid of RimI has a reduced growth rate, expanded to an evolutionary timescale, and might potentially promote conservation of the acetylation mechanism of S18 and EF-Tu. This study increased our understanding of the modification of bacterial translation apparatus.

Conjugates of Chloramphenicol Amine and Berberine as Antimicrobial Agents.

In order to obtain antimicrobial compounds with improved properties, new conjugates comprising two different biologically active agents within a single chimeric molecule based on chloramphenicol (CHL) and a hydrophobic cation were synthesized and studied. Chloramphenicol amine (CAM), derived from the ribosome-targeting antibiotic CHL, and the plant isoquinoline alkaloid berberine (BER) are connected by alkyl linkers of different lengths in structures of these conjugates. Using competition binding, double reporter system, and toeprinting assays, we showed that synthesized CAM-Cn-BER compounds bound to the bacterial ribosome and inhibited protein synthesis like the parent CHL. The mechanism of action of CAM-C5-BER and CAM-C8-BER on the process of bacterial translations was similar to CHL. Experiments with bacteria demonstrated that CAM-Cn-BERs suppressed the growth of laboratory strains of CHL and macrolides-resistant bacteria. CAM-C8-BER acted against mycobacteria and more selectively inhibited the growth of Gram-positive bacteria than the parent CHL and the berberine derivative lacking the CAM moiety (CH3-C8-BER). Using a potential-sensitive fluorescent probe, we found that CAM-C8-BER significantly reduced the membrane potential in B. subtilis cells. Crystal violet assays were used to demonstrate the absence of induction of biofilm formation under the action of CAM-C8-BER on E. coli bacteria. Thus, we showed that CAM-C8-BER could act both on the ribosome and on the cell membrane of bacteria, with the alkylated berberine fragment of the compound making a significant contribution to the inhibitory effect on bacterial growth. Moreover, we showed that CAM-Cn-BERs did not inhibit eukaryotic translation in vitro and were non-toxic for eukaryotic cells.

Minimal PK/PD model for simultaneous description of the maximal tolerated dose and metronomic treatment outcomes in mouse tumor models.

PURPOSE: Metronomic chemotherapy (MC) is a promising approach where, in contrast to the conventional maximal tolerated dose (MTD) strategy, regular fractionated doses of the drug are used. This approach has proven its efficacy, although drug dosing and scheduling are often chosen empirically. Pharmacokinetic/pharmacodynamic (PK/PD) models provide a way to choose optimal protocols with computational methods. Existing models are usually too complicated and are valid for only a subset of drug schedules. To address this issue, we propose herein a simple model that can describe MC and MTD regimens simultaneously. METHODS: The minimal model comprises tumor suppression due to antiangiogenic drug effect together with a cell-kill term, responsible for its cytotoxicity. The model was tested on data obtained on tumor-bearing mice treated with gemcitabine in ether MTD, MC, or combined (MTD + MC) regimens. RESULTS: We conducted a number of tests in which data were divided in various ways into training and validation sets. The model successfully described different trends in the MTD and MC regimens. With parameters obtained by fitting the model to MTD data, the simulations correctly predicted trends in both the MC and combined therapy groups. CONCLUSION: Our results demonstrate that the proposed model presents a minimal yet efficient tool for modeling outcomes in different treatment regimens in mice. We hope that this model has the potential for use in clinical practice in the development of patient-specific chemotherapy scheduling protocols based on observed treatment response.

Triphenilphosphonium Analogs of Chloramphenicol as Dual-Acting Antimicrobial and Antiproliferating Agents.

In the current work, in continuation of our recent research, we synthesized and studied new chimeric compounds, including the ribosome-targeting antibiotic chloramphenicol (CHL) and the membrane-penetrating cation triphenylphosphonium (TPP), which are linked by alkyl groups of different lengths. Using various biochemical assays, we showed that these CAM-Cn-TPP compounds bind to the bacterial ribosome, inhibit protein synthesis in vitro and in vivo in a way similar to that of the parent CHL, and significantly reduce membrane potential. Similar to CAM-C4-TPP, the mode of action of CAM-C10-TPP and CAM-C14-TPP in bacterial ribosomes differs from that of CHL. By simulating the dynamics of CAM-Cn-TPP complexes with bacterial ribosomes, we proposed a possible explanation for the specificity of the action of these analogs in the translation process. CAM-C10-TPP and CAM-C14-TPP more strongly inhibit the growth of the Gram-positive bacteria, as compared to CHL, and suppress some CHL-resistant bacterial strains. Thus, we have shown that TPP derivatives of CHL are dual-acting compounds targeting both the ribosomes and cellular membranes of bacteria. The TPP fragment of CAM-Cn-TPP compounds has an inhibitory effect on bacteria. Moreover, since the mitochondria of eukaryotic cells possess qualities similar to those of their prokaryotic ancestors, we demonstrate the possibility of targeting chemoresistant cancer cells with these compounds.

Binding and Action of Triphenylphosphonium Analog of Chloramphenicol upon the Bacterial Ribosome.

Chloramphenicol (CHL) is a ribosome-targeting antibiotic that binds to the peptidyl transferase center (PTC) of the bacterial ribosome and inhibits peptide bond formation. As an approach for modifying and potentially improving the properties of this inhibitor, we explored ribosome binding and inhibitory properties of a semi-synthetic triphenylphosphonium analog of CHL-CAM-C4-TPP. Our data demonstrate that this compound exhibits a ~5-fold stronger affinity for the bacterial ribosome and higher potency as an in vitro protein synthesis inhibitor compared to CHL. The X-ray crystal structure of the Thermus thermophilus 70S ribosome in complex with CAM-C4-TPP reveals that, while its amphenicol moiety binds at the PTC in a fashion identical to CHL, the C4-TPP tail adopts an extended propeller-like conformation within the ribosome exit tunnel where it establishes multiple hydrophobic Van der Waals interactions with the rRNA. The synthesized compound represents a promising chemical scaffold for further development by medicinal chemists because it simultaneously targets the two key functional centers of the bacterial ribosome-PTC and peptide exit tunnel.

Tetracenomycin X inhibits translation by binding within the ribosomal exit tunnel.

The increase in multi-drug resistant pathogenic bacteria is making our current arsenal of clinically used antibiotics obsolete, highlighting the urgent need for new lead compounds with distinct target binding sites to avoid cross-resistance. Here we report that the aromatic polyketide antibiotic tetracenomycin (TcmX) is a potent inhibitor of protein synthesis, and does not induce DNA damage as previously thought. Despite the structural similarity to the well-known translation inhibitor tetracycline, we show that TcmX does not interact with the small ribosomal subunit, but rather binds to the large subunit, within the polypeptide exit tunnel. This previously unappreciated binding site is located adjacent to the macrolide-binding site, where TcmX stacks on the noncanonical basepair formed by U1782 and U2586 of the 23S ribosomal RNA. Although the binding site is distinct from the macrolide antibiotics, our results indicate that like macrolides, TcmX allows translation of short oligopeptides before further translation is blocked.

Translation at first sight: the influence of leading codons.

First triplets of mRNA coding region affect the yield of translation. We have applied the flowseq method to analyze >30 000 variants of the codons 2-11 of the fluorescent protein reporter to identify factors affecting the protein synthesis. While the negative influence of mRNA secondary structure on translation has been confirmed, a positive role of rare codons at the beginning of a coding sequence for gene expression has not been observed. The identity of triplets proximal to the start codon contributes more to the protein yield then more distant ones. Additional in-frame start codons enhance translation, while Shine-Dalgarno-like motifs downstream the initiation codon are inhibitory. The metabolic cost of amino acids affects the yield of protein in the poor medium. The most efficient translation was observed for variants with features resembling those of native Escherichia coli genes.

Comprehensive Functional Analysis of Escherichia coli Ribosomal RNA Methyltransferases.

Ribosomal RNAs in all organisms are methylated. The functional role of the majority of modified nucleotides is unknown. We systematically questioned the influence of rRNA methylation in Escherichia coli on a number of characteristics of bacterial cells with the help of a set of rRNA methyltransferase (MT) gene knockout strains from the Keio collection. Analysis of ribosomal subunits sedimentation profiles of the knockout strains revealed a surprisingly small number of rRNA MT that significantly affected ribosome assembly. Accumulation of the assembly intermediates was observed only for the rlmE knockout strain whose growth was retarded most significantly among other rRNA MT knockout strains. Accumulation of the 17S rRNA precursor was observed for rsmA(ksgA) knockout cells as well as for cells devoid of functional rsmB and rlmC genes. Significant differences were found among the WT and the majority of rRNA MT knockout strains in their ability to sustain exogenous protein overexpression. While the majority of the rRNA MT knockout strains supported suboptimal reporter gene expression, the strain devoid of the rsmF gene demonstrated a moderate increase in the yield of ectopic gene expression. Comparative 2D protein gel analysis of rRNA MT knockout strains revealed only minor perturbations of the proteome.

Insights into the improved macrolide inhibitory activity from the high-resolution cryo-EM structure of dirithromycin bound to the E. coli 70S ribosome.

Macrolides are one of the most successful and widely used classes of antibacterials, which kill or stop the growth of pathogenic bacteria by binding near the active site of the ribosome and interfering with protein synthesis. Dirithromycin is a derivative of the prototype macrolide erythromycin with additional hydrophobic side chain. In our recent study, we have discovered that the side chain of dirithromycin forms lone pair-π stacking interaction with the aromatic imidazole ring of the His69 residue in ribosomal protein uL4 of the Thermus thermophilus 70S ribosome. In the current work, we found that neither the presence of the side chain, nor the additional contact with the ribosome, improve the binding affinity of dirithromycin to the ribosome. Nevertheless, we found that dirithromycin is a more potent inhibitor of in vitro protein synthesis in comparison with its parent compound, erythromycin. Using high-resolution cryo-electron microscopy, we determined the structure of the dirithromycin bound to the translating Escherichia coli 70S ribosome, which suggests that the better inhibitory properties of the drug could be rationalized by the side chain of dirithromycin pointing into the lumen of the nascent peptide exit tunnel, where it can interfere with the normal passage of the growing polypeptide chain.

Influence of the spacer region between the Shine-Dalgarno box and the start codon for fine-tuning of the translation efficiency in Escherichia coli.

Translation efficiency contributes several orders of magnitude difference in the overall yield of exogenous gene expression in bacteria. In diverse bacteria, the translation initiation site, whose sequence is the primary determinant of the translation performance, is comprised of the start codon and the Shine-Dalgarno box located upstream. Here, we have examined how the sequence of a spacer between these main components of the translation initiation site contributes to the yield of synthesized protein. We have created a library of reporter constructs with the randomized spacer region, performed fluorescently activated cell sorting and applied next-generation sequencing analysis (the FlowSeq protocol). As a result, we have identified sequence motifs for the spacer region between the Shine-Dalgarno box and AUG start codon that may modulate the translation efficiency in a 100-fold range.

Oligoglutamylation of E. coli ribosomal protein S6 is under growth phase control.

Ribosomal protein S6 in Escherichia coli is modified by ATP-dependent glutamate ligase RimK. Up to four glutamate residues are added to the C-terminus of S6 protein. In this work we demonstrated that unlike the majority of ribosome modifications in E. coli, oligoglutamylation of S6 protein is regulated and happens only in the stationary phase of bacterial culture. Only S6 protein incorporated into assembled small ribosomal subunits, but not newly made free S6 protein is a substrate for RimK protein. Overexpression of the rimK gene leads to the modification of S6 protein even in the exponential phase of bacterial culture. Thus, it is unlikely that any stationary phase specific factor is needed for the modification. We propose a model that S6 modification is regulated solely via the rate of ribosome biosynthesis at limiting concentration of RimK enzyme.