Quantifying the Intrinsic Strength of C-H⋯O Intermolecular Interactions.

It has been recognized that the C-H⋯O structural motif can be present in destabilizing as well as highly stabilizing intermolecular environments. Thus, it should be of interest to describe the strength of the C-H⋯O hydrogen bond for constant structural factors so that this intrinsic strength can be quantified and compared to other types of interactions. This description is provided here for C2h-symmetric dimers of acrylic acid by means of the calculations that employ the coupled-cluster theory with singles, doubles, and perturbative triples [CCSD(T)] together with an extrapolation to the complete basis set (CBS) limit. Dimers featuring the C-H⋯O and O-H⋯O hydrogens bonds are carefully investigated in a wide range of intermolecular separations by the CCSD(T)/CBS approach, and also by the symmetry-adapted perturbation theory (SAPT) method, which is based on the density-functional theory (DFT) treatment of monomers. While the nature of these two types of hydrogen bonding is very similar according to the SAPT-DFT/CBS calculations and on the basis of a comparison of the intermolecular potential curves, the intrinsic strength of the C-H⋯O interaction is found to be about a quarter of its O-H⋯O counterpart that is less than one might anticipate.

A Cost Effective Scheme for the Highly Accurate Description of Intermolecular Binding in Large Complexes.

There has been a growing interest in quantitative predictions of the intermolecular binding energy of large complexes. One of the most important quantum chemical techniques capable of such predictions is the domain-based local pair natural orbital (DLPNO) scheme for the coupled cluster theory with singles, doubles, and iterative triples [CCSD(T)], whose results are extrapolated to the complete basis set (CBS) limit. Here, the DLPNO-based focal-point method is devised with the aim of obtaining CBS-extrapolated values that are very close to their canonical CCSD(T)/CBS counterparts, and thus may serve for routinely checking a performance of less expensive computational methods, for example, those based on the density-functional theory (DFT). The efficacy of this method is demonstrated for several sets of noncovalent complexes with varying amounts of the electrostatics, induction, and dispersion contributions to binding (as revealed by accurate DFT-based symmetry-adapted perturbation theory (SAPT) calculations). It is shown that when applied to dimeric models of poly(3-hydroxybutyrate) chains in its two polymorphic forms, the DLPNO-CCSD(T) and DFT-SAPT computational schemes agree to within about 2 kJ/mol of an absolute value of the interaction energy. These computational schemes thus should be useful for a reliable description of factors leading to the enthalpic stabilization of extended systems.

A computational inspection of the dissociation energy of mid-sized organic dimers.

The gas-phase value of the dissociation energy (D0) is a key parameter employed in both experimental and theoretical descriptions of noncovalent complexes. The D0 data were obtained for a set of mid-sized organic dimers in their global minima which was located using geometry optimizations that applied ample basis sets together with either the conventional second-order Møller-Plesset (MP2) method or several dispersion-corrected density-functional theory (DFT-D) schemes. The harmonic vibrational zero-point (VZP) and deformation energies from the MP2 calculations were combined with electronic energies from the coupled cluster theory with singles, doubles, and iterative triples [CCSD(T)] extrapolated to the complete basis set (CBS) limit to estimate D0 with the aim of inspecting values that were most recently measured, and an analogous comparison was performed using the DFT-D data. In at least one case (namely, for the aniline⋯methane cluster), the D0 estimate that employed the CCSD(T)/CBS energies differed from experiment in the way that could not be explained by a possible deficiency in the VZP contribution. Curiously, one of the DFT-D schemes (namely, the B3LYP-D3/def2-QZVPPD) was able to reproduce all measured D0 values to within 1.0 kJ/mol from experimental error bars. These findings show the need for further measurements and computations of some of the complexes. In order to facilitate such studies, the physical nature of intermolecular interactions in the investigated dimers was analyzed by means of the DFT-based symmetry-adapted perturbation theory.

Diversity of the TLR4 Immunity Receptor in Czech Native Cattle Breeds Revealed Using the Pacific Biosciences Sequencing Platform.

The allelic variants of immunity genes in historical breeds likely reflect local infection pressure and therefore represent a reservoir for breeding. Screening to determine the diversity of the Toll-like receptor gene TLR4 was conducted in two conserved cattle breeds: Czech Red and Czech Red Pied. High-throughput sequencing of pooled PCR amplicons using the PacBio platform revealed polymorphisms, which were subsequently confirmed via genotyping techniques. Eight SNPs found in coding and adjacent regions were grouped into 18 haplotypes, representing a significant portion of the known diversity in the global breed panel and presumably exceeding diversity in production populations. Notably, the ancient Czech Red breed appeared to possess greater haplotype diversity than the Czech Red Pied breed, a Simmental variant, although the haplotype frequencies might have been distorted by significant crossbreeding and bottlenecks in the history of Czech Red cattle. The differences in haplotype frequencies validated the phenotypic distinctness of the local breeds. Due to the availability of Czech Red Pied production herds, the effect of intensive breeding on TLR diversity can be evaluated in this model. The advantages of the Pacific Biosciences technology for the resequencing of long PCR fragments with subsequent direct phasing were independently validated.

Telomerase activity in pig granulosa cells proliferating and differentiating in vitro.

The aim of the work was to analyze the telomerase activity (TA) in two different populations of pig granulosa cells (GC) proliferating and differentiating in vitro: (a) in relatively undifferentiated granulosa cells isolated from small (1-2 mm) antral follicles and (b) in functionally advanced, differentiated cells obtained from large (5-7 mm) antral follicles. The proliferative potential in vitro of small follicle granulosa cells (SF-GC) was higher than that of large follicle granulosa cells (LF-GC). EGF stimulated significantly (p<0.01) proliferation in SF-GC as well as LF-GC. FSH did not have a stimulating effect on proliferation in both of the GC populations. Steroidogenesis was induced in both SF- and LF-GC in vitro. Significantly higher (p<0.01) levels of estradiol were measured in LF-GC cultures. In SF-GC, no significantly different effects of EGF and FSH on estradiol production were found. The production of progesterone in vitro was higher in LF-GC than in SF-GC and its production was specifically promoted by FSH in contrast to estradiol the synthesis of which in vitro was less dependent on culture conditions. Using the TRAP assay telomerase activity was detected in freshly isolated and in vitro cultured pig SF- and LF-GC. In EGF, but not FSH stimulated SF-GC, significantly enhanced (p<0.05) TA in comparison with the control was observed at an interval of 24 h of culture. After the 48 h in vitro, levels of TA in both EGF and FSH treated cells were comparable with control. In LF-GC, both EGF and FSH stimulated significantly (p<0.05) TA after the 24h of in vitro culture. At an interval of 48 h, no significant differences in the level of TA were observed between control, EGF and FSH stimulated LF-GC. Comparing the levels of TA in SF- and LF-GC, significantly higher levels of TA were found in control (p<0.05) and EGF (p<0.01) treated SF-GC after 24 h in vitro. On the other hand, absolutely, but not significantly, higher levels of TA were found in LF-GC versus SF-GC in all culture conditions after 48 h in vitro.

Cloning and expression of the haloalkane dehalogenase gene dhmA from Mycobacterium avium N85 and preliminary characterization of DhmA.

Haloalkane dehalogenases are microbial enzymes that catalyze cleavage of the carbon-halogen bond by a hydrolytic mechanism. Until recently, these enzymes have been isolated only from bacteria living in contaminated environments. In this report we describe cloning of the dehalogenase gene dhmA from Mycobacterium avium subsp. avium N85 isolated from swine mesenteric lymph nodes. The dhmA gene has a G+C content of 68.21% and codes for a polypeptide that is 301 amino acids long and has a calculated molecular mass of 34.7 kDa. The molecular masses of DhmA determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel permeation chromatography are 34.0 and 35.4 kDa, respectively. Many residues essential for the dehalogenation reaction are conserved in DhmA; the putative catalytic triad consists of Asp123, His279, and Asp250, and the putative oxyanion hole consists of Glu55 and Trp124. Trp124 should be involved in substrate binding and product (halide) stabilization, while the second halide-stabilizing residue cannot be identified from a comparison of the DhmA sequence with the sequences of three dehalogenases with known tertiary structures. The haloalkane dehalogenase DhmA shows broad substrate specificity and good activity with the priority pollutant 1,2-dichloroethane. DhmA is significantly less stable than other currently known haloalkane dehalogenases. This study confirms that a hydrolytic dehalogenase is present in the facultative pathogen M. avium. The presence of dehalogenase-like genes in the genomes of other mycobacteria, including the obligate pathogens Mycobacterium tuberculosis and Mycobacterium bovis, as well as in other bacterial species, including Mesorhizobium loti, Xylella fastidiosa, Photobacterium profundum, and Caulobacter crescentus, led us to speculate that haloalkane dehalogenases have some other function besides catalysis of hydrolytic dehalogenation of halogenated substances.