Double Substitution Research Articles

Nonadditive functional interactions between ligand-binding sites of the multidrug efflux pump AdeB from Acinetobacter baumannii.

Multidrug efflux is one of the major mechanisms of antibiotic resistance identified in clinical isolates of the human pathogen Acinetobacter baumannii. The multiple antibiotic resistance in this species is often enabled by the overproduction of the tripartite efflux pump AdeABC. In this pump, AdeB is the inner membrane transporter from the resistance-nodulation-division (RND) superfamily of proteins, which is responsible for the recognition and efflux of multiple structurally unrelated compounds. Like other RND transporters, AdeB is a trimeric protein with ligand-binding sites located in the large periplasmic domains. Previous structural studies, however, highlighted the uniqueness of AdeB interactions with ligands. Up to three ligand molecules were bound to one protomer of AdeB, mapping its substrate translocation path. In this study, we introduced single and double substitutions in the identified ligand-binding sites of AdeB. Our results show that the mechanism of substrate translocation by AdeB is different from that of other characterized RND transporters and that the functional interactions between the sites are nonadditive. We identified AdeB mutants with both the loss and the gain of antibiotic susceptibility phenotypes, as well as AdeB mutations making A. baumannii cells overproducing such pump variants even more susceptible to multiple antibiotics than efflux-deficient cells. IMPORTANCE Multidrug efflux pumps of the resistance-nodulation-division superfamily of proteins are important contributors to various aspects of bacterial physiology and antibiotic resistance. Studies of the best-characterized model transporter AcrB from Escherichia coli suggested that these transporters operate by a functional rotation mechanism in which various substrates bind to at least two different binding sites. This study suggests that the mechanism of AdeB is distinct and that the binding sites in this transporter are functionally linked.

Laves phase double substitution alloy design and device filling modification for Ti-based metal hydride hydrogen compressors

The ambition of carbon neutrality has become a common pursuit for researchers worldwide, and hydrogen as a green and clean energy carrier is expected to contribute to this achievement. Since hydrogen fuel cell vehicles require a high input hydrogen pressure, compressors in hydrogen refueling stations are thus indispensable for the large-scale application of hydrogen energy. Compared to conventional mechanical hydrogen compressors, metal hydride based hydrogen compressors have the advantages of high security and low cost, providing a viable solution for hydrogen refueling station development. Herein, two series of Ti0.95Zr0.07Cr1.3-xMnxFe0.4 (x = 0.2, 0.3, 0.4, 0.6) and Ti0.95Zr0.07Cr1.0Mn0.6Fe0.4-yCuy (y = 0.1, 0.15, 0.2, 0.3) alloys were designed to achieve 8–22 MPa hydrogen compression in water bath at 283–353 K. All the as-prepared alloys exhibit the single hexagonal C14 Laves phase structure. In particular, the double substitution of Cr and Fe by Cu and Mn elements in Ti–Zr–Cr–Mn–Fe alloy can enlarge its hydrogen storage capacity without varying the equilibrium pressure. For instance, Ti0.95Zr0.07Cr1.0Mn0.6Fe0.25Cu0.15 alloy has a hydrogenation plateau pressure of 6.16 MPa at 283 K and a dehydrogenation plateau pressure of 24.23 MPa at 353 K, with the max hydrogen capacity of 1.72 wt%. Furthermore, the relative high density molding method of metal hydride bed with 20 g of alloy powder was developed. The binder epoxy resin and the thermal conductor flake graphite were selected and added into the alloys to form metal hydride compacts (MHCs). The structural stability of modified MHCs can be enhanced due to the formation of a three-dimensional grid structure, with a higher resistance to pulverization and degradation. The improved thermal conductivity involving a filling compact strategy can significantly shorten hydrogenation reaction time by 50% and lower the abrupt temperature of metal hydride bed by 20 K with excellent stability over 100 cycles, and provide new insights of the development of safe and efficient hydride hydrogen compressor.

Exploration of the novel fluoroquinolones with high inhibitory effect against quinolone-resistant DNA gyrase of Salmonella Typhimurium.

Quinolone-resistant nontyphoidal Salmonella, one of the prominent pathogens causing acute gastroenteritis, has become a public health concern globally. The World Health Organization has ranked fluoroquinolone-resistant Salmonella as a high-priority pathogen for researching and developing new antibiotics. WQ-3034 and WQ-3154 are relatively new synthetic fluoroquinolones with distinctive structures. WQ-3034 has 6-amino-3,5-difluoropyridine-2-yl at R1, 3-hydroxyazetidinyl at R7, and the addition of chlorine atom at R8. WQ-3154 has a similar basic pharmacophore to WQ-3034 except for the modification at R8 with a methyl group. In this study, the inhibitory effect and DNA cleavage effect against wild-type (WT) and mutant Salmonella Typhimurium DNA gyrases of WQ-3034 and WQ-3154 were examined along with WQ-3810 and ciprofloxacin by measuring the drug concentration that inhibits half of the enzyme activity (IC50) and the drug concentration that induces 25% of maximum DNA cleavage (CC25). The minimum inhibitory concentration (MIC) of the compounds was assessed against Salmonella Typhimurium and Salmonella Enteritidis. Among four compounds, WQ-3034 demonstrated the highest inhibitory effect against both WT and mutant Salmonella Typhimurium DNA gyrases with amino acid substitution at codon 83 and/or 87, while ciprofloxacin showed the lowest inhibitory effect. Remarkably, WQ-3034 and WQ-3154 exhibited a significantly higher inhibitory effect than ciprofloxacin against Salmonella Typhimurium DNA gyrase with double amino acid substitution, Ser83Phe-Asp87Asn. Similarly, CC25 of WQ-3034 against mutant Salmonella Typhimurium DNA gyrase was lower than ciprofloxacin. Notably, MICs of WQ-3034 and WQ-3154 were higher than ciprofloxacin. In conclusion, this study revealed that WQ-3034 and WQ-3154 could potentially be effective therapeutic agents against quinolone-resistant nontyphoidal Salmonella. IMPORTANCE Quinolone-resistant nontyphoidal Salmonella is a pressing public health concern, demanding the exploration of novel treatments. In this study, we focused on two innovative synthetic fluoroquinolones, WQ-3034 and WQ-3154. Our findings revealed that these new compounds demonstrate potent inhibitory effects, even against mutant strains that cause resistance to existing quinolones. Hence, WQ-3034 and WQ-3154 could potentially be effective therapeutic agents against quinolone-resistant Salmonella Typhimurium. Furthermore, the data obtained in this study will be baseline information for antimicrobial drug development.

OR0304 Novel Dual Antagonists Of The Growth Hormone Receptor And The Prolactin Receptor

Abstract Disclosure: R. Basu: None. J.J. Kopchick: None. An excess of the human growth hormone (hGH) is associated with multiple pathophysiology including acromegaly, diabetes, cancer, renal dysfunction and age-associated cognitive impairment. On the other hand, a congenital lack of GH action in humans and mice render a protective effect from the above co-morbidities. Consequently, there exists significant pharmaceutical interest in developing antagonists of GH action - rendered via binding and activating the GHR, as well as the prolactin receptor (PRLR), which is particularly relevant for PRLR-rich cancers of the breast and prostate. Our laboratory has pioneered the discovery of the first and only FDA approved GHR antagonist - Somavert (pegvisomant for injection, Pfizer Inc., USA) - currently prescribed for the treatment of acromegaly. Here we present two new and unique designs of antagonists which block the complete spectrum of GH action at both GHR and PRLR binding. The first GHR antagonist design is a full-length G120K-hGH peptide at the core, incorporating targeted cysteine (single or double) substitutions at carefully selected solvent-accessible residues distal to the binding sites. To these substituted cysteines, maleimide activated discrete PEGs (MAL-dPEG; neutral or anionic, branched, different sizes) were specifically conjugated to build a library of multiple putative GHR antagonists. Binding studies and biochemical activities reveal multiple promising GHRA candidates with high binding affinities to both GHR and PRLR, while the biological assays show robust inhibition of GH driven intracellular signaling and oncogenic processes in cultured human cancer cell lines. Intraperitoneal administration of a selected candidate (compound-G) in mice significantly reduced serum IGF1 and IGFBP3 as well, confirming in vivo efficacy. The second candidate antagonist termed ‘S1H’ is a 16-amino-acid peptide designed as a direct sequence mimetic of a unique fragment of the site-1 binding helix of the hGH molecule. To this end we have characterized S1H with mass spectrometry, circular dichroism spectropolarimetry, and in vitro biological assays using human cell lines expressing GHR. S1H inhibited GH as well as PRL induced STAT5 phosphorylation in a dose-dependent manner in multiple cell lines. Moreover, using alanine scanning mutagenesis studies, we identified a strong correlation between the helical propensity and biological activity of S1H - allowing extensive structure activity relationship studies. Thus, we present two distinct designs, preparation and characterization of dual antagonists of the GH action at the level of GHR and PRLR, with prospective applications in pathologies associated with a GH excess and also as valuable compounds to study the nature of human GH-GHR interactions. We currently continue to develop both compounds for pre-clinical validations. Presentation: Thursday, June 15, 2023

The Impact of Substitutions at Positions 1 and 8 of Fluoroquinolones on the Activity Against Mutant DNA Gyrases of Salmonella Typhimurium.

Although many drug-resistant nontyphoidal Salmonella (NTS) infections are reported globally, their treatment is challenging owing to the ineffectiveness of the currently available antimicrobial drugs against resistant bacteria. It is therefore essential to discover novel antimicrobial drugs for the management of these infections. In this study, we report high inhibitory activities of the novel fluoroquinolones (FQs; WQ-3810 and WQ-3334) with substitutions at positions R-1 by 6-amino-3,5-difluoropyridine-2-yl and R-8 by methyl group or bromine, respectively, against wild-type and mutant DNA gyrases of Salmonella Typhimurium. The inhibitory activities of these FQs were assessed against seven amino acid substitutions in DNA gyrases conferring FQ resistance to S. Typhimurium, including high-level resistant mutants, Ser83Ile and Ser83Phe-Asp87Asn by in vitro DNA supercoiling assay. Drug concentrations of WQ compounds with 6-amino-3,5-difluoropyridine-2-yl that suppressed DNA supercoiling by 50% (IC50) were found to be ∼150-fold lower than ciprofloxacin against DNA gyrase with double amino acid substitutions. Our findings highlight the importance of the chemical structure of an FQ drug on its antimicrobial activity. Particularly, the presence of 6-amino-3,5-difluoropyridine-2-yl at R-1 and either methyl group or bromine at R-8 of WQ-3810 and WQ-3334, respectively, was associated with improved antimicrobial activity. Therefore, WQ-3810 and WQ-3334 are promising candidates for use in the treatment of patients infected by FQ-resistant Salmonella spp.

Extragenic suppression of an HSV-1 UL34 nuclear egress mutant reveals role for pUS9 as an inhibitor of epithelial cell-to-cell spread.

Herpesviruses are able to disseminate in infected hosts despite development of a strong immune response. Their ability to do this relies on a specialized process called cell-to-cell spread in which newly assembled virus particles are trafficked to plasma membrane surfaces that abut adjacent uninfected cells. The mechanism of cell-to-cell spread is obscure, and little is known about whether or how it is regulated in different cells. We show here that a viral protein with a well-characterized role in promoting spread from neurons has an opposite, inhibitory role in other cells.

A protective multiple gene-deleted African swine fever virus genotype II, Georgia 2007/1, expressing a modified non-haemadsorbing CD2v protein

ABSTRACT African swine fever virus is a complex DNA virus that causes high fatality in pigs and wild boar and has a great socio-economic impact. An attenuated genotype II strain was constructed by replacing the gene for wildtype CD2v protein with versions in which single or double amino acid substitutions were introduced to reduce or abrogate the binding to red blood cells and reduce virus persistence in blood. The mutant CD2v proteins were expressed at similar levels to the wildtype protein on the surface of infected cells. Three recombinant viruses also had K145R, EP153R, and in one virus DP148R genes deleted. Following immunization of pigs, the virus with a single amino acid substitution in CD2v, Q96R, induced moderate levels of replication, and 100% protection against virulent ASFV. Two additional recombinant viruses had two amino acid substitutions in CD2v, Q96R, and K108D, and induced no binding to red blood cells in vitro. In immunized pigs, reduced levels of virus in blood and strong early ASFV-specific antibody and cellular responses were detected. After challenge low to moderate replication of challenge virus was observed. Reduced clinical signs post-challenge were observed in pigs immunized with the virus from which DP148R gene was deleted. Protection levels of 83–100% were maintained across a range of doses. Further experiments with virus GeorgiaΔDP148RΔK145RΔEP153R-CD2v_mutantQ96R/K108D showed low levels of virus dissemination in tissue and transient clinical signs at high doses. The results support further evaluation of GeorgiaΔDP148RΔK145RΔEP153R-CD2v_mutantQ96R/K108D as a vaccine candidate.

Brick-concrete recycled aggregate/powder synergistic preparation of pervious concrete

In order to improve the resource utilization rate of brick-concrete construction waste (BC-CW) and meet the demand of 'sponge city' construction for pervious concrete, a kind of brick-concrete recycled pervious concrete (BC-RPC) was prepared by brick-concrete recycled aggregate (BC-RA) / powder (BC-RP). The design was carried out from the two levels of cementitious material pastes and aggregate. Firstly, BC-RP was used as the main silicon-aluminum raw material to prepare a brick-concrete recycled powder based geopolymer (BC-RPG) for pervious concrete. It was determined that the content of BC-RP in BC-RPG was 50%, and the alkali content and modulus of the best alkali activator were 12% and 1.2, respectively. Then, the effect of BC-RA gradation and substitution rate on the mechanical properties of BC-RPC was studied by using BC-RPG as cementitious material and BC-RA as aggregate. The method of 'double grain composite substitution' was proposed to control the strength loss rate of BC-RPC. At the same time, the volume structure parameters (design porosity and pastes-aggregate ratio) of BC-RPC were optimized, and the optimum design porosity and pastes-aggregate ratio for BC-RPC preparation were determined to be 27% and 0.39, respectively. Finally, BC-RPC with coordinated mechanical and water permeability properties was prepared.

Structure of bound water layer on montmorillonite surface: The role of trans- and cis- vacant sites

Bound water on the surface of clay minerals determines the nature of a wide class of phenomena, from the diffusion of interlayer cations to synthesizing prebiotic macromolecules during the early Earth. However, the nature of forming the bound water layer's spatial structure and properties has yet to be fully investigated. Many factors, including peculiarities of structural charge distribution, surface adsorption centers, and OH-groups of the octahedral sheet of the clay minerals, can affect it in natural clays. In this work the role of hydroxyl ligands and isomorphic substitutions of the octahedral sheet in trans- and cis- vacant sites in the formation of the bound water layer structure on the basal surface of montmorillonite was studied by the Density Functional Theory. It was found out that in the case of single isomorphic substitution in the octahedral sheet OH-groups in the cis-vacant sites provide higher adsorption energy of water molecules on the basal surface as compared to OH-groups in the trans-vacant sites. The double isomorphic substitution in the octahedral sheet creates a mutual enhancement effect due to the difference in the structural arrangement of the OH-groups. This leads to an increase in the interaction with adsorbed water molecules. The maximum increase in adsorption energy is observed for the case with isomorphic substitutions of Mg-cis and Fe-trans in the octahedral sheet. The results of the theoretical study contribute to explaining the experimental data available in the literature.

Variations on the Bergman Cyclization Theme: Electrocyclizations of Ionic Penta-, Hepta-, and Octadiynes.

The Bergman cyclization of (Z)-hexa-3-ene-1,5-diyne to form the aromatic diradical p-benzyne has garnered attention as a potential antitumor agent due to its relatively low cyclization barrier and the stability of the resulting diradical. Here, we present a theoretical investigation of several ionic extensions of the fundamental Bergman cyclization: electrocyclizations of the penta-1,4-diyne anion, hepta-1,6-diyne cation, and octa-1,7-diyne dication, leveraging the spin-flip formulation of the equation-of-motion coupled cluster theory with single and double substitutions (EOM-SF-CCSD). Though the penta-1,4-diyne anion exhibits a large cyclization barrier of +66 kcal mol-1, cyclization of both the hepta-1,6-diyne cation and octa-1,7-diyne dication along a previously unreported triplet pathway requires relatively low energy. We also identified the presence of significant aromaticity in the triplet diradical products of these two cationic cyclizations.

Human γS-Crystallin Mutation F10_Y11delinsLN in the First Greek Key Pair Destabilizes and Impairs Tight Packing Causing Cortical Lamellar Cataract.

Aromatic residues forming tyrosine corners within Greek key motifs are critical for the folding, stability, and order of βγ-crystallins and thus lens transparency. To delineate how a double amino acid substitution in an N-terminal-domain tyrosine corner of the CRYGS mutant p.F10_Y11delinsLN causes juvenile autosomal dominant cortical lamellar cataracts, human γS-crystallin c-DNA was cloned into pET-20b (+) and a p.F10_Y11delinsLN mutant was generated via site-directed mutagenesis, overexpressed, and purified using ion-exchange and size-exclusion chromatography. Structure, stability, and aggregation properties in solution under thermal and chemical stress were determined using spectrofluorimetry and circular dichroism. In benign conditions, the p.F10_Y11delinsLN mutation does not affect the protein backbone but alters its tryptophan microenvironment slightly. The mutant is less stable to thermal and GuHCl-induced stress, undergoing a two-state transition with a midpoint of 60.4 °C (wild type 73.1 °C) under thermal stress and exhibiting a three-state transition with midpoints of 1.25 and 2.59 M GuHCl (wild type: two-state transition with Cm = 2.72 M GuHCl). The mutant self-aggregates upon heating at 60 °C, which is inhibited by α-crystallin and reducing agents. Thus, the F10_Y11delinsLN mutation in human γS-crystallin impairs the protein's tryptophan microenvironment, weakening its stability under thermal and chemical stress, resulting in self-aggregation, lens opacification, and cataract.

Functional involvement of a conserved motif in the middle region of the human ribosomal protein eL42 in translation

Ribosomal protein eL42 (formerly known as L36A), a small protein of the large (60S) subunit of the eukaryotic ribosome, is a component of its exit (E) site. The residue K53 of this protein resides within the motif QSGYGGQTK mainly conserved in eukaryotes, and it is located in the immediate vicinity of the CCA-terminus of the ribosome-bound tRNA in the hybrid P/E state. To examine the role of this eL42 motif in translation, we obtained HEK293T cells producing the wild-type FLAG-tagged protein or its mutant forms with either single substitutions of conserved amino acid residues in the above motif, or simultaneous replacements in positions 45 and 51 or 45 and 53. Examination of the level of exogenous eL42 in fractions of polysome profiles from the target protein-producing cells by the Western blotting revealed that neither single substitution affects the assembly of 60S ribosomal subunits and 80S ribosomes or critically decreases the level of polysomes, but the latter was observed with the double replacements. Analysis of tRNAs bound to 80S ribosomes containing eL42 with double substitutions and examination their peptidyl transferase activity enabled estimation the stage of the elongation cycle, in which amino acid residues of the conserved eL42 motif are involved. We clearly show that cooperative interactions implicating the eL42 residues Q45, Q51, and K53 play a critical role in the ability of the human ribosome to perform properly elongation cycle at the step of deacylated tRNA dissociation from the E site in the human cell.

The Effect of Aliovalent Substitution on Magnetic Properties of PolyCrystalline Ca/K-1144

Among IBSC superconductors, the 1144 family ( <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">AAE</i> Fe4As4 with <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">A</i> =alkali metal, <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">AE</i> =alkaline-earth metal), has attracted considerable interest in recent years because of their propensity to form lattice defects that positively influence the flux pinning properties. Extensive research is underway to optimize this class of materials for the low-temperature high-field regime, both in the form of single crystal and polycrystalline powder. The latter is of particular interest because the 1144 material has been shown to be suitable for wire production through the easy and well-assessed Powder In Tube (PIT) process. <p xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">In our recent experiments, it has been shown that the 1144 structure can be tailored to obtain a doped compound with a different chemical formula (<i>A</i>_x<i>AE</i>_1-x)(<i>AE</i>_y<i>A</i>_1-y)Fe₄As₄, that is characterized by unaltered critical temperature values despite the high level of substitution up to 15 at.%. In order to unveil the influence of the double substitution on the grain boundary and pinning properties of doped samples, we recently started an extensive characterization campaign whose preliminary results are presented here. Both structural and morphological properties of the compound are not affected by the simultaneous doping with 10 at.% Na and 5 at.% Ba, as revealed by XRD and SEM analyses. On the contrary, the study of magnetic properties shows that although there is no improvement in transport properties, the aliovalent substitution induces a clear change in the in-field behavior which deserves further investigations.

Single opsin driven white noise ERGs in mice.

Electroretinograms elicited by photopigment isolating white noise stimuli (wnERGs) in mice were measured. The dependency of rod- and cone-opsin-driven wnERGs on mean luminance was studied. Temporal white noise stimuli (containing all frequencies up to 20 Hz, equal amplitudes, random phases) that modulated either rhodopsin, S-opsin or L*-opsin, using the double silent substitution technique, were used to record wnERGs in mice expressing a human L*-opsin instead of the native murine M-opsin. Responses were recorded at 4 mean luminances (MLs).Impulse response functions (IRFs) were obtained by cross-correlating the wnERG recordings with the corresponding modulation of the photopigment excitation elicited by the stimulus. So-called modulation transfer functions (MTFs) were obtained by performing a Fourier transform on the IRFs.Potentials of two repeated wnERG recordings at corresponding time points were plotted against each other. The correlation coefficient (r2repr) of the linear regression through these data was used to quantify reproducibility. Another correlation coefficient (r2ML) was used to quantify the correlations of the wnERGs obtained at different MLs with those at the highest (for cone isolating stimuli) or lowest (for rod isolating stimuli) ML. IRFs showed an initial negative (a-wave like) trough N1 and a subsequent positive (b-wave like) peak P1. No oscillatory potential-like components were observed. At 0.4 and 1.0 log cd/m2 ML robust L*- and S-opsin-driven IRFs were obtained that displayed similar latencies and dependencies on ML. L*-opsin-driven IRFs were 2.5-3 times larger than S-opsin-driven IRFs. Rhodopsin-driven IRFs were observed at -0.8 and - 0.2 log cd/m2 and decreased in amplitude with increasing ML. They displayed an additional pronounced late negativity (N2), which may be a correlate of retinal ganglion cell activity.R2repr and r2ML values increased for cones with increasing ML whereas they decreased for rods. For rhodopsin-driven MTFs at low MLs and L*-opsin-driven MTFs at high MLs amplitudes decreased with increasing frequency, with much faster decreasing amplitudes for rhodopsin. A delay was calculated from MTF phases showing larger delays for rhodopsin- vs. low delays for L*-opsin-driven responses. Opsin-isolating wnERGs in mice show characteristics of different retinal cell types and their connected pathways.

Design of Protegrin-1 Analogs with Improved Antibacterial Selectivity.

Protegrin-1 (PG-1) is a cationic β-hairpin pore-forming antimicrobial peptide having a membranolytic mechanism of action. It possesses in vitro a potent antimicrobial activity against a panel of clinically relevant MDR ESKAPE pathogens. However, its extremely high hemolytic activity and cytotoxicity toward mammalian cells prevent the further development of the protegrin-based antibiotic for systemic administration. In this study, we rationally modulated the PG-1 charge and hydrophobicity by substituting selected residues in the central β-sheet region of PG-1 to design its analogs, which retain a high antimicrobial activity but have a reduced toxicity toward mammalian cells. In this work, eight PG-1 analogs with single amino acid substitutions and five analogs with double substitutions were obtained. These analogs were produced as thioredoxin fusions in Escherichia coli. It was shown that a significant reduction in hemolytic activity without any loss of antimicrobial activity could be achieved by a single amino acid substitution, V16R in the C-terminal β-strand, which is responsible for the PG-1 oligomerization. As the result, a selective analog with a ≥30-fold improved therapeutic index was obtained. FTIR spectroscopy analysis of analog, [V16R], revealed that the peptide is unable to form oligomeric structures in a membrane-mimicking environment, in contrast to wild-type PG-1. Analog [V16R] showed a reasonable efficacy in septicemia infection mice model as a systemic antibiotic and could be considered as a promising lead for further drug design.

Ab Initio Investigation of the Auger Spectra of Methane, Ethane, Ethylene, and Acetylene.

We present an ab initio computational study of the Auger spectra of methane, ethane, ethylene, and acetylene. Auger spectroscopy is an established technique to probe the electronic structure of molecules and exploits the Auger-Meitner effect that core-ionized states undergo. We compute partial decay widths using coupled-cluster theory with single and double substitutions (CCSD) and equation-of-motion CCSD theory combined with complex-scaled basis functions and Feshbach-Fano projection. We generate Auger spectra from these partial widths and draw conclusions about the strength of particular decay channels and trends among the four molecules. A connection to experimental results about fragmentation pathways of the electronic states produced by Auger decay is also made.

N-N(+) Bond-Forming Intramolecular Cyclization of O-Tosyloxy β-Aminopropioamidoximes and Ion Exchange Reaction for the Synthesis of 2-Aminospiropyrazolilammonium Chlorides and Hexafluorophosphates.

Our research area is related to the spiropyrazolinium-containingcompounds, which are insufficiently studied compared with pyrazoline-containing compounds. Nitrogen-containing azoniaspiromolecules have also been well studied. In drug design and other areas, they are a priori important structures, since rigid spirocyclic scaffolds with the reduced conformational entropy are able to organize a closely spaced area. Azoniaspirostructures are currently of wide practical interest as ionic liquids, current sources (membranes), structure-directing agents in organocatalysis, and in the synthesis of ordered ceramics. Our goal was the synthesis of 2-aminospiropyrazolilammonium chlorides and hexafluorophosphates. Our methodology is based on the tosylation of β-aminopropioamidoximes with six-membered N-heterocycles (piperidine, morpholine, thiomorpholine, and phenylpiperazine) at the β-position. 2-Aminospiropyrazolilammonium chlorides and hexafluorophosphates were obtained by the reaction of double ion substitution in the reaction of toluenesulfonates of 2-aminospiropyrazolinium compounds with an ethereal solution of HCl in ethanol and with ammonium hexafluorophosphate in ethanol in quantitative yields of 55-97%. The physicochemical characteristics of the synthesized compounds and their IR and NMR spectra are presented. The obtained salts were additionally characterized by the single-crystal XRD analysis. The presence of both axial and equatorial conformations of spirocations in solids was confirmed. 2-Aminospiropyrazolilammonium chlorides and hexafluorophosphates have weak in vitro antimicrobial activity on Gram-positive and Gram-negative bacterial lines.

N-terminal lid swapping contributes to the substrate specificity and activity of thermophilic lipase TrLipE.

TrLipE is a thermophilic lipase that has potential commercial applications because of its catalytic ability under extreme conditions. Consistent with most lipases, the lid of TrLipE is located over the catalytic pocket, controls the substrate channel to the active center, and regulates the substrate specificity, activity, and stability of the enzyme through conformational changes. TrLipE from Thermomicrobium roseum has potential industrial applications, which is hindered by its weak enzymatic activity. Here, 18 chimeras (TrL1-TrL18) were reconstructed by N-terminal lid swapping between TrLipE and structurally similar enzymes. The results showed that the chimeras had a similar pH range and optimum pH as wild TrLipE but a narrower temperature range of 40-80°C, and TrL17 and the other chimeras showed lower optimum temperatures of 70°C and 60°C, respectively. In addition, the half-lives of the chimeras were lower than those of TrLipE under optimum temperature conditions. Molecular dynamics simulations indicated that chimeras had high RMSD, RMSF, and B-factor values. When p-nitrophenol esters with different chains were used as substrates, compared with TrLipE, most of the chimeras had a low Km and high kcat value. The chimeras TrL2, TrL3, TrL17, and TrL18 could specifically catalyze the substrate 4-nitrophenyl benzoate, with TrL17 showing the highest kcat/Km value of 363.88 ± 15.83 L⋅min-1⋅mmol-1. Mutants were then designed by investigating the binding free energies of TrL17 and 4-nitrophenyl benzoate. The results indicated that single, double, and triple substitution variants (M89W and I206N; E33W/I206M and M89W/I206M; and M89W/I206M/L21I and M89W/I206N/L21I, respectively) presented approximately 2- to 3-fold faster catalysis of 4-nitrophenyl benzoate than the wild TrL17. Our observations will facilitate the development of the properties and industrial applications of TrLipE.

Repartitioned Brillouin-Wigner perturbation theory with a size-consistent second-order correlation energy.

Second-order Møller-Plesset perturbation theory (MP2) often breaks down catastrophically in small-gap systems, leaving much to be desired in its performance for myriad chemical applications such as noncovalent interactions, thermochemistry, and dative bonding in transition metal complexes. This divergence problem has reignited interest in Brillouin-Wigner perturbation theory (BWPT), which is regular at all orders but lacks size consistency and extensivity, severely limiting its application to chemistry. In this work, we propose an alternative partitioning of the Hamiltonian that leads to a regular BWPT perturbation series that, through the second order, is size-extensive, size-consistent (provided its Hartree-Fock reference is also), and orbital invariant. Our second-order size-consistent Brillouin-Wigner (BW-s2) approach can describe the exact dissociation limit of H2 in a minimal basis set, regardless of the spin polarization of the reference orbitals. More broadly, we find that BW-s2 offers improvements relative to MP2 for covalent bond breaking, noncovalent interaction energies, and metal/organic reaction energies, although rivaling coupled-cluster with single and double substitutions for thermochemical properties.

SOME OBSERVATIONS ON THE LEXICAL VARIATION WITHIN THE PHRASEOLOGICAL UNIT ИМАМ КОЗ В РЪКАВА IN BULGARIAN PHRASEOLOGY

The present text discusses the lexical variation of the phraseological unit имам коз в ръкава in the Bulgarian phraseological system. That idiom is attested in many languages and in Bulgarian phraseology functions as a calque. It belongs to the group of idioms representing the idea for life as a gambling game. Three main types of lexical transformation within the idiom are observed: variation by substituting the verb имам, by substitution of lexical element коз and lexical variation by insertion. The variation is commonly based on substitution of the near-synonymous lexical elements and elements associatively connected. But there are some restrictions described in the analysis. The verb and the noun may also co-vary and that double lexical substitution is attested very often. The variation by lexical insertion within the idiom имам коз в ръкава leаds to variants where the adjective modifier (силен, скрит) does not change the idiom semantics, but intensifies the meaning. In the actual utterances lexical variation is not separable from the other type of phraseological modification - morphological, syntactic, they co-occur in the extracted examples. The lexical variants share semantic equivalence and common stylistic and functional features. The text finds that substitution is more widespread than the lexical insertion. The present research describes and some other occasional utterances.