Synthesis of imidazoline and imidazo[2,1-c][1,2,4]triazole aryl derivatives containing the methylthio group as possible antibacterial agents

1-Arylimidazolidine-2-thiones (1a-g) were synthesized by the condensation reaction of N-arylethylenediamines with carbon disulfide in xylene medium. Their further alkylation with methyl iodide led to the formation of some biologically active 1-aryl-2-methylthio-imidazolines (2a-g). The 7-(4-methylphenyl)-3-methylthio-5H-6,7-dihydroimidazo[2,1-c][1,2,4]triazole (4b) was obtained by the alkylation of the respective 7-(4-methylphenyl)-2,5,6,7-tetrahydroimidazo[2,1-c][1,2,4]triazol-3(H)-thione (3b) with methyl iodide. Antimicrobial activities of 1-aryl-2-methylthio-imidazolines (2a-g) and the 7-(4-methylphenyl)-3- methylthio-5H-6,7-dihydroimidazo[2,1-c][1,2,4]triazole (4b) are presented. All tested compounds showed MIC in the range of 11.0-89.2 microM. Compounds 2a,e were found to be equipotent to chloramphenicol in vitro, whereas 2a,c,e-g and 4b showed superior activity (MIC) to ampicillin.     

Metabolic changes in fruits of the tomato dx mutant

The tomato DWARF cytochrome P450 protein catalyzes the C-6 oxidation of 6-deoxo-castasterone to castasterone. The d(x) mutant does not produce a functional DWARF enzyme, and d(x) shoots display severe symptoms of brassinosteroid-deficiency. However, fruits express the CYP85A3 protein which compensates for the deficiency of the DWARF protein and produce bioactive brassinosteroids. Here, we report on the metabolic characterization of d(x) fruits. Fruit size, fresh weight, and pigment content were not altered. However, d(x) fruits showed reduced dry mass content. Levels of starch and various sugars were reduced, amino acid levels were elevated. BR application to d(x) leaves partially normalized dry mass content, sugar and amino acid levels in d(x) fruits. The data demonstrate that brassinosteroid in shoots is required for fruit development in tomato.     

A high molecular arabinogalactan from Ribes nigrum L.: influence on cell physiology of human skin fibroblasts and keratinocytes and internalization into cells via endosomal transport

An arabinogalactan protein (F2) was isolated in 1.5% yield from the seeds of Ribes nigrum L. (Grossulariaceae) by aqueous extraction and a one-step anion exchange chromatography on DEAE-Sephacel with 24% galactose, 43% arabinose, and 20% xylose as main carbohydrate residues. Methylation analysis revealed the presence of a 1,3-/1,3,6-galactose backbone, side chains from arabinose in different linkages, and terminal xylose residues. The polysaccharide which turned out to be an arabinogalactan protein had a molecular weight of >10⁶ Da and deaggregated under chaotropic conditions. The cellular dehydrogenase activities (MTT and WST-1 tests) of human skin cells (fibroblasts, keratinocytes) as well as the proliferation rate of keratinocytes (BrdU incorporation ELISA) were significantly stimulated by the polymer at 10 and 100 μg/mL. F2 had no influence on differentiation status of keratinocytes and did not exhibit any cytotoxic potential (LDH test). The biological activity of F2 was not dependent on the high molecular weight. Influence of the polysaccharide on the gene expression of specific growth factors, growth factor receptors, signal proteins and marker proteins for skin cell proliferation, and differentiation by RT-PCR could not be shown. Gene array investigations indicated an increased expression of various genes encoding for catabolic enzymes, DNA repair, extracellular matrix proteins, and signal transduction factors. Removal of terminal arabinose residues by α-l-arabinofuranosidase did not influence the activity toward skin cells, while the treatment with β-d-galactosidase yielded an inactive polysaccharide. The FITC-labeled polysaccharide was incorporated in a time-dependent manner into human fibroblasts (laser scanning microscopy) via endosomal transport. This internalization of the polysaccharide was inhibited by Cytochalasin B.     

Exploring the molecular linkage of protein stability traits for enzyme optimization by iterative truncation and evolution

The stability of proteins is paramount for their therapeutic and industrial use and, thus, is a major task for protein engineering. Several types of chemical and physical stabilities are desired, and discussion revolves around whether each stability trait needs to be addressed separately and how specific and compatible stabilizing mutations act. We demonstrate a stepwise perturbation-compensation strategy, which identifies mutations rescuing the activity of a truncated TEM β-lactamase. Analyses relating structural stress with the external stresses of heat, denaturants, and proteases reveal our second-site suppressors as general stability centers that also improve the full-length enzyme. A library of lactamase variants truncated by 15 N-terminal and three C-terminal residues (Bla-NΔ15CΔ3) was subjected to activity selection and DNA shuffling. The resulting clone with the best in vivo performance harbored eight mutations, surpassed the full-length wild-type protein by 5.3 °C in T(m), displayed significantly higher catalytic activity at elevated temperatures, and showed delayed guanidine-induced denaturation. The crystal structure of this mutant was determined and provided insights into its stability determinants. Stepwise reconstitution of the N- and C-termini increased its thermal, denaturant, and proteolytic resistance successively, leading to a full-length enzyme with a T(m) increased by 15.3 °C and a half-denaturation concentration shifted from 0.53 to 1.75 M guanidinium relative to that of the wild type. These improvements demonstrate that iterative truncation-optimization cycles can exploit stability-trait linkages in proteins and are exceptionally suited for the creation of progressively stabilized variants and/or downsized proteins without the need for detailed structural or mechanistic information.     

Phylogenetic position of Sipuncula derived from multi‐gene and phylogenomic data and its implication for the evolution of segmentation

The phylogenetic position of Sipuncula, a group of unsegmented marine worms, has been controversial for several decades: Especially based on morphological data, closer relationships to Mollusca or Annelida were among the most favoured hypotheses. Increasing amounts of molecular data in recent years have consistently placed Sipuncula either in close affinity to or even within Annelida, the segmented worms, and rejected a close relationship to Mollusca. Yet, it remained uncertain whether Sipuncula is the sister group of Annelida or an annelid subtaxon. Therefore, herein we gathered data for five nuclear genes, which have been rarely used regarding Annelida and Sipuncula, and combined these with data for six previously used genes to further elucidate the phylogenetic position of Sipuncula. We also compiled a data set for 78 ribosomal proteins from publicly available genomic data sets. These are the two largest data sets for annelids with more than 10 taxa to date. All analyses placed Sipuncula within Annelida. For the first time, topology tests significantly rejected the possibility that Sipuncula is sister to Annelida. Thus, our analyses revealed that Sipuncula had secondarily lost segmentation. Given that unsegmented Echiura is also an annelid subtaxon, segmentation, a key character of Annelida, is much more variable than previously thought. Yet, this conclusion does not support the hypothesis that the last common ancestor of Annelida, Arthropoda and Chordata was segmented, assuming several losses along the branches leading to them. As yet no traces of segmentation could be shown in taxa exhibiting serially organized organ systems such as certain Mollusca, while in Sipuncula and Echiura such traces could be demonstrated. An independent origin of segmentation in Annelida, Arthropoda and Chordata thus appears to be more plausible and parsimonious.     

Pannexin-1 channel “fuels” by releasing ATP from bone marrow cells a state of sterile inflammation required for optimal mobilization and homing of hematopoietic stem cells

An efficient harvest of hematopoietic stem/progenitor cells (HSPCs) after pharmacological mobilization from the bone marrow (BM) into peripheral blood (PB) and subsequent proper homing and engraftment of these cells are crucial for clinical outcomes from hematopoietic transplants. Since extracellular adenosine triphosphate (eATP) plays an important role in both processes as an activator of sterile inflammation in the bone marrow microenvironment, we focused on the role of Pannexin-1 channel in the secretion of ATP to trigger both egress of HSPCs out of BM into PB as well as in reverse process that is their homing to BM niches after transplantation into myeloablated recipient. We employed a specific blocking peptide against Pannexin-1 channel and noticed decreased mobilization efficiency of HSPCs as well as other types of BM-residing stem cells including mesenchymal stroma cells (MSCs), endothelial progenitors (EPCs), and very small embryonic-like stem cells (VSELs). To explain better a role of Pannexin-1, we report that eATP activated Nlrp3 inflammasome in Gr-1⁺ and CD11b⁺ cells enriched for granulocytes and monocytes. This led to release of danger-associated molecular pattern molecules (DAMPs) and mitochondrial DNA (miDNA) that activate complement cascade (ComC) required for optimal egress of HSPCs from BM. On the other hand, Pannexin-1 channel blockage in transplant recipient mice leads to a defect in homing and engraftment of HSPCs. Based on this, Pannexin-1 channel as a source of eATP plays an important role in HSPCs trafficking.

     

Comparison of morphological,DNA barcoding,and metabarcoding characterizations of freshwater nematode communities

Biomonitoring approaches and investigations of many ecological questions require assessments of the biodiversity of a given habitat. Small organisms, ranging from protozoans to metazoans, are of great ecological importance and comprise a major share of the planet's biodiversity but they are extremely difficult to identify, due to their minute body sizes and indistinct structures. Thus, most biodiversity studies that include small organisms draw on several methods for species delimitation, ranging from traditional microscopy to molecular techniques. In this study, we compared the efficiency of these methods by analyzing a community of nematodes. Specifically, we evaluated the performances of traditional morphological identification, single‐specimen barcoding (Sanger sequencing), and metabarcoding in the identification of 1500 nematodes from sediment samples. The molecular approaches were based on the analysis of the 28S ribosomal large and 18S small subunits (LSU and SSU). The morphological analysis resulted in the determination of 22 nematode species. Barcoding identified a comparable number of operational taxonomic units (OTUs) based on 28S rDNA (n = 20) and fewer OTUs based on 18S rDNA (n = 12). Metabarcoding identified a higher OTU number but fewer amplicon sequence variants (AVSs) (n = 48 OTUs, n = 17 ASVs for 28S rDNA, and n = 31 OTUs, n = 6 ASVs for 18S rDNA). Between the three approaches (morphology, barcoding, and metabarcoding), only three species (13.6%) were shared. This lack of taxonomic resolution hinders reliable community identifications to the species level. Further database curation will ensure the effective use of molecular species identification.     

Temperature Dependence of the Krokinobacter rhodopsin 2 Kinetics

We investigated the temperature-dependent kinetics of the light-driven Na⁺ pump Krokinobacter rhodopsin 2 (KR2) at Na⁺-pumping conditions. The recorded microsecond flash photolysis data were subjected to detailed global target analysis, employing Eyring constraints and spectral decomposition. The analysis resulted in the kinetic rates, the composition of the different photocycle equilibria, and the spectra of the involved photointermediates. Our results show that with the temperature increase (from 10 to 40°C), the overall photocycle duration is accelerated by a factor of 6, with the L-to-M transition exhibiting an impressive 40-fold increase. It follows from the analysis that in KR2 the chromophore and the protein scaffold are more kinetically decoupled than in other microbial rhodopsins. We link this effect to the rigidity of the retinal protein environment. This kinetic decoupling should be considered in future studies and could potentially be exploited for fine-tuning biotechnological applications.