Phenotypic selection on nectar amino acid composition in the Lepidoptera pollinated orchid species Gymnadenia conopsea

Plant–pollinator interactions are potential drivers of evolution in floral traits. Because nectar chemical composition is known to mediate both plant–pollinator interactions and plant reproductive success, it can be expected that chemical composition of nectar is subjected to strong pollinator‐mediated selective forces. However, the extent of natural selection on different nectar components has not been studied so far. Using the Lepidoptera pollinated fragrant orchid Gymnadenia conopsea as a model species, we used high‐performance anion‐exchange chromatography (HPAEC) to characterize the sugar and amino acid composition of floral nectar in three calcareous grassland populations of G. conopsea. We then measured phenotypic selection on nectar composition and on other plant and floral traits through applying both linear regression and structural equation modelling. We demonstrate phenotypic selection on plant height, inflorescence height and on specific nectar amino acids, whereas spur length, total sugar and amino acid concentration were not direct targets of selection. Chemical nectar composition is thus indeed under selective pressure but nectar amino acids are much more important to fitness of G. conopsea, as compared to nectar sugars. Furthermore, as we found no evidence of selection on the total amino acid concentration, it is unlikely that amino acids increase pollinator attraction because they are a pollinator nitrogen source. To further unravel the evolutionary ecology of floral nectar, behavioural experiments with pollinators exposed to different nectar components and studies experimentally identifying the selective agents are recommended.     

Application of a computational model of natural deep eutectic solvents utilizing the COSMO-RS approach for screening of solvents with high solubility of rutin

The screening of natural deep eutectic solvents (NADES) to identify those with the ability to strongly solvate rutin was conducted using the COSMO-RS methodology. A NADES model was constructed that took into account the possible ionic and neutral forms of its constituents. The distributions of all forms were computed based on the equilibrium constants of neutralization reactions between amino and carboxylic acids. The proposed model was validated against the experimental solubilities of 15 NADES. A linear relationship between these data and the estimated activity coefficient values was found. The screening encompassed 126 different NADES. It was found that ten of them outperformed the best reference system. The most effective two-component solvent comprised proline combined with 2,3-diaminosuccinic acid, and the solubility of rutin in this solvent was found to be 130% greater than its solubility in the best reference system. The amino acids associated with the highest rutin solubilities were all cyclic, and the use of carboxylic acids with two carboxyl groups and a main chain consisting of two methylene groups with two amino substituents was observed to yield the best rutin solubilities. Because of the acidic properties of rutin, the presence of basic sites on the components of the NADES generally leads to enhanced solubility.     

Functional characterization of residues within the carnitine/acylcarnitine translocase RX2PANAAXF distinct motif

The mitochondrial carnitine/acylcarnitine carrier (CAC) is characterized by the presence of a distinct motif, RXXPANAAXF, within its sixth transmembrane α-helix. In this study, we analysed the role of the amino acids of this motif in the structure-function relationships of the human CAC by using two complementary approaches. First, we performed functional analysis in the model fungus Aspergillus nidulans of selected mutations with structural and functional relevance. Second, similar mutant human CACs were biochemically characterized after their reconstitution into liposomes. Both analyses have provided relevant information on the importance and role of the CAC motif residues in the activity and metabolic function of CAC. Only the two adjacent alanines, Ala281 and Ala282 in the human CAC, have been found not to be crucial for transport activity and in vivo function. Results obtained from amino acid substitutions of residues Arg275, Asn280 and Phe284 of human CAC together with structural analysis using molecular modelling of the carrier suggest that R275, N280 and F284 are involved in substrate binding during acylcarnitine/carnitine translocation. Furthermore, functional analysis of mutations of residues Pro278 and Ala279 in A. nidulans, together with kinetic data in reconstituted liposomes, suggest a predominant structural role for these amino acids.     

Nutritional Significance of Dietary Cystine for Maintaining the Hepatic Albumin mRNA Level in Rats Fed on a Soybean Diet

A new bacteriocin, gassericin A, was purified from the culture fluid of Lactobacillus gasseri LA39 mainly by reverse-phase (RP) chromatography. The purification of gassericin A from a modified MRS broth, in which Tween 80 had been replaced by oleic acid, resulted in a 4500-fold increase in specific activity with a 6% recovery. Gassericin A was eluted as a single peak on the chromatogram from RP-HPLC and migrated by SDS-PAGE as a single band with a molecular weight of ca. 3.8 kDa. Gassericin A, a highly hydrophobic bacteriocin, was slightly soluble in water, but its solubility was increased by adding alcohol and acetonitrile. An amino acid analysis revealed that it was composed of 45.7% hydrophobic amino acids in the total residues of 35 amino acids. Gassericin A produced in the MRS broth associated strongly with Tween 80, although several further trials of dissociation were unsuccessful.     

The Codon Usage in the Minimal Natural Cell

A statistical analysis of the variation in contents with the size of the current known smallest genomes, N. deltocephalinicola, C. ruddii, N. equitans, and M. genitalium, enabled the indication of a minimal set of codons capable of naturally building a modern-type free-living unicellular organism in an early stage of evolution. Using a linear regression model, the potential codon distribution in the minimal natural cell was predicted and compared to the composition of the smallest synthetic, JCVI-Syn3.0. The distribution of the molecular weight of potentially coded amino acids was also calculated. The main differences in the features of the minimal natural cell and H. Sapiens genome were analyzed. In this regard, the content percentage of respective amino acids and their polarization charge properties were reported and compared. The fractions of occurring nucleotides were calculated, too. Then, the estimated numbers of codons in a minimal natural cell were related to the expected numbers for random distribution. Shown increase, or decrease in the contents, relative to the calculated random filling was related to the evolutionary preferences, varying with the subsequent eras of the evolution of genetic code.

     

Water activity, pH and density of aqueous amino acids solutions

The water activity, pH and density of some aqueous amino acid solutions were determined at 25 degrees C in three different types of solvents. Previous published experimental data on water activity and solubility of amino acids in aqueous solutions were used together with data from this work to test the applicability of a group contribution model. The activity coefficients were estimated by the UNIFAC-Larsen model combined with the Debye-Hückel equation, taking also into account the partial dissociation phenomena of species in solution. Interaction energies between the charged species Na(+) and Cl(-) and the specific groups of amino acids (COOH and NH(2)) were adjusted using experimental solubility data.     

Topological modelling of analgesia

Analgesic activity (log IC) of a large series of 97 analgesics was modelled topologically using a series of distance-based topological indices. The results show that analgesic activity (log IC) exhibit inter familial correlation and these can only be modelled by splitting 97 analgesics into five different categories. The regression analyses of the data show that the Wiener (W)-, Branching (B)- and first-order connectivity (chi)- indices are the better topological indices for modelling the activity (log IC), and that W index gave the excellent results.     

Modern and prebiotic amino acids support distinct structural profiles in proteins

The earliest proteins had to rely on amino acids available on early Earth before the biosynthetic pathways for more complex amino acids evolved. In extant proteins, a significant fraction of the ‘late’ amino acids (such as Arg, Lys, His, Cys, Trp and Tyr) belong to essential catalytic and structure-stabilizing residues. How (or if) early proteins could sustain an early biosphere has been a major puzzle. Here, we analysed two combinatorial protein libraries representing proxies of the available sequence space at two different evolutionary stages. The first is composed of the entire alphabet of 20 amino acids while the second one consists of only 10 residues (ASDGLIPTEV) representing a consensus view of plausibly available amino acids through prebiotic chemistry. We show that compact conformations resistant to proteolysis are surprisingly similarly abundant in both libraries. In addition, the early alphabet proteins are inherently more soluble and refoldable, independent of the general Hsp70 chaperone activity. By contrast, chaperones significantly increase the otherwise poor solubility of the modern alphabet proteins suggesting their coevolution with the amino acid repertoire. Our work indicates that while both early and modern amino acids are predisposed to supporting protein structure, they do so with different biophysical properties and via different mechanisms.     

Structure-activity relationships of the N-methylcarbamate series in Salmonella typhimurium

Aromatic hydrocarbons of low molecular weight, hydroxy and N-methylcarbamate derivatives were tested for mutagenicity by the reversion of histidine-dependent Salmonella typhimurium TA98 and TA1535 in the presence of a rat-liver 9000 X g supernatant fraction. The presence of 2 or 3 aromatic rings resulted in a weak increase in revertants. Hydroxylation and carbamylation of aromatic rings increased the mutagenic activity of these aromatic compounds. In order to evaluate the structure-activity relationship, the specific molecular connectivity indices were calculated. A significant inverse relationship exists between mutagenicity and zero- and second-order specific molecular connectivity indices. Only compounds with second-order specific molecular connectivity indices lower than 0.300 increased mutagenic activity.     

Empirical assessment of the relation between ecological connectivity and land complexity based on information-theoretic metrics

Habitat fragmentation and connectivity loss pose significant threats to biodiversity at both local and landscape levels. Strategies to increase ecological connectivity and preserve strong connectivity are important for dealing with the potential threat of habitat degradation. Various metrics have been used to measure (i.e., quantify) landscape composition and configuration in landscape ecology. However, their relationship with ecological connectivity must be understood to interpret landscape patterns comprehensively. In the present study, correlations between ecological connectivity and land complexity are examined based on information-theory metrics. Two primary questions are explored: (1) to what extent are landscape mosaic measures of entropy correlated with ecological connectivity, with landscape gradient-based measures, and with each other? (2) are landscape gradient-based entropy measures correlated with ecological connectivity more than discrete entropy measures? Results show that all information theoretic metrics are statistically significant (p < 0.05) for modelling ecological connectivity. Among categorically-based indices, the relationship between ECI and joint entropy was the most significant, while a generalized additive model indicated that Boltzmann entropy could predict the ecological connectivity index, explaining ∼60% of the variance. Therefore, configurational entropy can be used for improving ecological connectivity models.     

Discovery of novel inhibitors of Mycobacterium tuberculosis MurG: homology modelling,structure based pharmacophore,molecular docking,and molecular dynamics simulations

MurG (Rv2153c) is a key player in the biosynthesis of the peptidoglycan layer in Mycobacterium tuberculosis (Mtb). This work is an attempt to highlight the structural and functional relationship of Mtb MurG, the three-dimensional (3D) structure of protein was constructed by homology modelling using Discovery Studio 3.5 software. The quality and consistency of generated model was assessed by PROCHECK, ProSA and ERRAT. Later, the model was optimized by molecular dynamics (MD) simulations and the optimized model complex with substrate Uridine-diphosphate-N-acetylglucosamine (UD1) facilitated us to employ structure-based virtual screening approach to obtain new hits from Asinex database using energy-optimized pharmacophore modelling (e-pharmacophore). The pharmacophore model was validated using enrichment calculations, and finally, validated model was employed for high-throughput virtual screening and molecular docking to identify novel Mtb MurG inhibitors. This study led to the identification of 10 potential compounds with good fitness, docking score, which make important interactions with the protein active site. The 25 ns MD simulations of three potential lead compounds with protein confirmed that the structure was stable and make several non-bonding interactions with amino acids, such as Leu290, Met310 and Asn167. Hence, we concluded that the identified compounds may act as new leads for the design of Mtb MurG inhibitors.     

Exploring the molecular basis for selective binding of Mycobacterium tuberculosis Asp kinase toward its natural substrates and feedback inhibitors: A docking and molecular dynamics study

Tuberculosis (TB) is still a major public health problem, compounded by the human immunodeficiency virus (HIV)-TB co-infection and recent emergence of multidrug-resistant (MDR) and extensively drug resistant (XDR)-TB. In this context, aspartokinase of mycobacterium tuberculosis has drawn attention for designing novel anti-TB drugs. Asp kinase is an enzyme responsible for the synthesis of 4-phospho-L-aspartate from L-aspartate and involved in the branched biosynthetic pathway leading to the synthesis of amino acids lysine, threonine, methionine and isoleucine. An intermediate of lysine biosynthetic branch, mesodiaminopimelate is also a component of the peptidoglycan which is a component of bacterial cell wall. To interfere with the production of all these amino acids and cell wall, it is possible to inhibit Asp kinase activity. This can be achieved using Asp kinase inhibitors. In order to design novel Asp kinase inhibitors as effective anti-TB drugs, it is necessary to have an understanding of the binding sites of Asp kinase. As no crystal structure of the enzyme has yet been published, we built a homology model of Asp kinase using the crystallized Asp kinase from M. Jannaschii, as template structures (2HMF and 3C1M). After the molecular dynamics refinement, the optimized homology model was assessed as a reliable structure by PROCHECK, ERRAT, WHAT-IF, PROSA2003 and VERIFY-3D. The results of molecular docking studies with natural substrates, products and feedback inhibitors are in agreement with the published data and showed that ACT domain plays an important role in binding to ligands. Based on the docking conformations, pharmacophore model can be developed by probing the common features of ligands. By analyzing the results, ACT domain architecture, certain key residues that are responsible for binding to feedback inhibitors and natural substrates were identified. This would be very helpful in understanding the blockade mechanism of Asp kinase and providing insights into rational design of novel Asp kinase inhibitors for M.tuberculosis.     

A topological model for the haemolysin translocator protein HlyD

Summary A topological model for HlyD is proposed that is based on results obtained with gene fusions of lacZ and phoA to hlyD. Active H1yD-LacZ fusion proteins were only generated when lacZ was fused to hlyD. within the first 180 by (60 amino acids). H1yD-PhoA proteins exhibiting alkaline phosphatase (AP) activity were obtained when phoA was inserted into hlyD. between nucleotides 262 (behind amino acid position 87) and 1405 (behind amino acid position 468, only 10 amino acids away from the C-terminus of HlyD Active insertions of phoA into the middle region of hlyD. were not observed on in vivo transposition but such fusions exhibiting AP activity could be constructed by in vitro techniques. A fusion protein that carried the PhoA part close to the C-terminal end of HlyD proved to be the most stable HlyD-PhoA fusion protein. In contrast to the other, rather unstable, HlyD-PhoA⁺ fusions, no proteolytic degradation product of this HlyD-PhoA protein was observed and nearly all the alkaline phosphatase activity was membrane bound. Protease accessibility and cell fractionation experiments indicated that the alkaline phosphatase moiety of this fusion protein was located in the periplasm as for all other HlyD-PhoA⁺ proteins. These data and computer-assisted predictions suggest a topological model for HlyD with the N-terminal 60 amino acids located in the cytoplasm, a single transmembrane segment from amino acids 60 to 80 and a large periplasmic region extending from amino acid 80 to the C-terminus. Neither the HlyD fusion proteins obtained nor a mutant HlyD protein that had lost the last 10 amino acids from the C-terminus of HlyD exhibited translocator activity for HlyA or other reporter proteins carrying the HlyA signal sequence. The C-terminal 10 amino acids of HlyD showed significant similarity with the corresponding sequences of other HlyD-related proteins involved in protein secretion.     

Probing the structural requirements of A-type Aurora kinase inhibitors using 3D-QSAR and molecular docking analysis

Aurora-A, the most widely studied isoform of Aurora kinase overexpressed aberrantly in a wide variety of tumors, has been implicated in early mitotic entry, degradation of natural tumor suppressor p53 and centrosome maturation and separation; hence, potent inhibitors of Aurora-A may be therapeutically useful drugs in the treatment of various forms of cancer. Here, we report an in silico study on a group of 220 reported Aurora-A inhibitors with six different substructures. Three-dimensional quantitative structure–activity relationship (3D-QSAR) studies were carried out using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) techniques on this series of molecules. The resultant optimum 3D-QSAR models exhibited an r _cv² value of 0.404-0.582 and their predictive ability was validated using an independent test set, ending in r _pred² 0.512-0.985. In addition, docking studies were employed to explore these protein–inhibitor interactions at the molecular level. The results of 3D-QSAR and docking analyses validated each other, and the key structural requirements affecting Aurora-A inhibitory activities, and the influential amino acids involved were identified. To the best of our knowledge, this is the first report on 3D-QSAR modeling of Aurora-A inhibitors, and the results can be used to accurately predict the binding affinity of related analogues and also facilitate the rational design of novel inhibitors with more potent biological activities.