Actinotignum schaalii is an emerging, opportunistic pathogen and its connection to non-infectious diseases and conditions, such as prostate or bladder cancer, or chronic inflammation has been proposed. Here, we analyzed 297 urine, ureteral and urinary catheter samples from 128 patients by Polymerase Chain Reaction followed by Denaturing Gradient Gel Electrophoresis and Sequencing (PCR-DGGE-S), and culture, and 29 of these samples also by 16S rRNA Illumina sequencing, to establish A. schaalii's prevalence in urinary tract-related samples, its relation to other bacteria, and its potential association with patients' conditions and samples' characteristics. A. schaalii-positive samples were significantly more diverse than A. schaalii negative and between-group diversity was higher than intra-group. Propionimicrobium lymphophilum, Fusobacterium nucleatum, Veillonella sp., Morganella sp., and Aerococcus sp. were significantly more often present in A. schaalii-positive samples; thus, we suggest these species are A. schaalii's concomitants, while Enterobacter and Staphylococcaceae were more often identified in A. schaalii-negative samples; therefore, we propose A. schaalii and these species are mutually exclusive. Additionally, a significantly higher A. schaalii prevalence in patients with ureter stricture associated hydronephrosis (p = 0.020) was noted. We suggest that A. schaalii could be an early polybacterial biofilm colonizer, together with concomitant species, known for pro-inflammatory features.

• Publikační typ
• časopisecké články MeSH

The dark web scene has been drawing the attention of law enforcement agencies and researchers alike. To date, most of the published works on the dark web are based on data gained by passive observation. To gain a more contextualized perspective, a study was conducted in which three vendors were selected on the "Dream Market" dark web marketplace, from whom subsequently several new psychoactive substances (NPS) were ordered. All transactions were documented from the initial drug deal solicitation to the final qualitative analysis of all received samples. From the selected vendors, a total of nine NPS samples was obtained, all of which were analyzed by NMR, HRMS, LC-UV, and two also by x-ray diffraction. According to our analyses, four of the five substances offered under already known NPS names contained a different NPS. The selected vendors therefore either did not know about their product, or deliberately deceived the buyers. Furthermore, two of three obtained samples of purportedly novel NPS were identified as already documented substances sold under a different name. However, the third characterized substance sold as "MPF-47700" was a novel, yet uncharacterized, NPS. Finally, we received a single undeclared substance, later identified as 5F-ADB. In addition to chemical analysis of the nine obtained NPS samples, the methodology used also yielded contextual information about the accessibility of NPS on the dark web, the associated purchase process, and the modus operandi of three NPS vendors. Direct participation in dark web marketplaces seems to provide additional layers of information useful for forensic studies.

• MeSH
• hmotnostní spektrometrie MeSH
• internet MeSH
• lidé MeSH
• magnetická rezonanční spektroskopie MeSH
• obchodování s drogami * MeSH
• odhalování abúzu drog MeSH
• psychotropní léky analýza   zásobování a distribuce   MeSH
• spektrofotometrie ultrafialová MeSH
• zakázané drogy analýza   zásobování a distribuce   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Fungal β-N-acetylhexosaminidases, though hydrolytic enzymes in vivo, are useful tools in the preparation of oligosaccharides of biological interest. The β-N-acetylhexosaminidase from Talaromyces flavus is remarkable in terms of its synthetic potential, broad substrate specificity, and tolerance to substrate modifications. It can be heterologously produced in Pichia pastoris in a high yield. The mutation of the Tyr470 residue to histidine greatly enhances its transglycosylation capability. The aim of this work was to identify the structural requirements of this model β-N-acetylhexosaminidase for its transglycosylation acceptors and formulate a structure-activity relationship study. Enzymatic reactions were performed using an activated glycosyl donor, 4-nitrophenyl N-acetyl-β-d-glucosaminide or 4-nitrophenyl N-acetyl-β-d-galactosaminide, and a panel of glycosyl acceptors of varying structural features (N-acetylglucosamine, glucose, N-acetylgalactosamine, galactose, N-acetylmuramic acid, and glucuronic acid). The transglycosylation products were isolated and structurally characterized. The C-2 N-acetamido group in the acceptor molecule was found to be essential for recognition by the enzyme. The presence of the C-2 hydroxyl moiety strongly hindered the normal course of transglycosylation, yielding unique non-reducing disaccharides in a low yield. Moreover, whereas the gluco-configuration at C-4 steered the glycosylation into the β(1-4) position, the galacto-acceptor afforded a β(1-6) glycosidic linkage. The Y470H mutant enzyme was tested with acceptors based on β-glycosides of uronic acid and N-acetylmuramic acid. With the latter acceptor, we were able to isolate and characterize one glycosylation product in a low yield. To our knowledge, this is the first example of enzymatic glycosylation of an N-acetylmuramic acid derivative. In order to explain these findings and predict enzyme behavior, a modeling study was accomplished that correlated with the acquired experimental data.

• MeSH
• beta-N-acetylhexosaminidasy chemie   metabolismus   MeSH
• glykosidy metabolismus   MeSH
• glykosylace MeSH
• kinetika MeSH
• konformace proteinů MeSH
• molekulární modely MeSH
• oligosacharidy metabolismus   MeSH
• substrátová specifita MeSH
• Talaromyces enzymologie   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH

This work aims to find the most suitable method that is practically applicable for the calculation of 31P NMR chemical shifts of Pt(II) complexes. The influence of various all-electron and ECP basis sets, DFT functionals, and solvent effects on the optimized geometry was tested. A variety of combinations of DFT functionals BP86, B3LYP, PBE0, TPSSh, CAM-B3LYP, and ωB97XD with all-electron basis sets 6-31G, 6-31G(d), 6-31G(d,p), 6-311G(d,p), and TZVP and ECP basis sets SDD, LanL2DZ, and CEP-31G were used. Chemical shielding constants were then calculated using BP86, PBE0, and B3LYP functionals in combination with the TZ2P basis. The magnitude of spin-orbit interactions was also evaluated.

• Publikační typ
• časopisecké články MeSH

β-N-Acetylhexosaminidases (EC 3.2.1.52) are typical of their dual activity encompassing both N-acetylglucosamine and N-acetylgalactosamine substrates. Here we present the isolation and characterization of a selective β-N-acetylhexosaminidase from the fungal strain of Aspergillus versicolor. The enzyme was recombinantly expressed in Pichia pastoris KM71H in a high yield and purified in a single step using anion-exchange chromatography. Homologous molecular modeling of this enzyme identified crucial differences in the enzyme active site that may be responsible for its high selectivity for N-acetylglucosamine substrates compared to fungal β-N-acetylhexosaminidases from other sources. The enzyme was used in a sequential reaction together with a mutant β-N-acetylhexosaminidase from Talaromyces flavus with an enhanced synthetic capability, affording a bioactive disaccharide bearing an azido functional group. The azido function enabled an elegant multivalent presentation of this disaccharide on an aromatic carrier. The resulting model glycoconjugate is applicable as a selective ligand of galectin-3 - a biomedically attractive human lectin. These results highlight the importance of a general availability of robust and well-defined carbohydrate-active enzymes with tailored catalytic properties for biotechnological and biomedical applications.

• MeSH
• Aspergillus enzymologie   MeSH
• beta-N-acetylhexosaminidasy chemie   genetika   izolace a purifikace   metabolismus   MeSH
• chromatografie iontoměničová MeSH
• disacharidy metabolismus   MeSH
• exprese genu MeSH
• katalytická doména MeSH
• konformace proteinů MeSH
• molekulární modely MeSH
• Pichia genetika   metabolismus   MeSH
• rekombinantní proteiny chemie   genetika   izolace a purifikace   metabolismus   MeSH
• Talaromyces enzymologie   MeSH
• Publikační typ
• časopisecké články MeSH

Alzheimer's disease (AD) is a multifactorial neurodegenerative condition of the central nervous system (CNS) that is currently treated by cholinesterase inhibitors and the N-methyl-d-aspartate receptor antagonist, memantine. Emerging evidence strongly supports the relevance of targeting butyrylcholinesterase (BuChE) in the more advanced stages of AD. Within this study, we have generated a pilot series of compounds (1-20) structurally inspired from belladine-type Amaryllidaceae alkaloids, namely carltonine A and B, and evaluated their acetylcholinesterase (AChE) and BuChE inhibition properties. Some of the compounds exhibited intriguing inhibition activity for human BuChE (hBuChE), with a preference for BuChE over AChE. Seven compounds were found to possess a hBuChE inhibition profile, with IC50 values below 1 μM. The most potent one, compound 6, showed nanomolar range activity with an IC50 value of 72 nM and an excellent selectivity pattern over AChE, reaching a selectivity index of almost 1400. Compound 6 was further studied by enzyme kinetics, along with in-silico techniques, to reveal the mode of inhibition. The prediction of CNS availability estimates that all the compounds in this survey can pass through the blood-brain barrier (BBB), as disclosed by the BBB score.

• MeSH
• acetylcholinesterasa chemie   MeSH
• alkaloidy amarylkovitých chemie   MeSH
• butyrylcholinesterasa chemie   MeSH
• cholinesterasové inhibitory chemie   farmakologie   MeSH
• lidé MeSH
• nádorové buňky kultivované MeSH
• neuroblastom farmakoterapie   patologie   MeSH
• počítačová simulace MeSH
• proliferace buněk MeSH
• simulace molekulového dockingu * MeSH
• tyramin analogy a deriváty   chemie   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH

BACKGROUND: Vertebrate meiotic recombination events are concentrated in regions (hotspots) that display open chromatin marks, such as trimethylation of lysines 4 and 36 of histone 3 (H3K4me3 and H3K36me3). Mouse and human PRDM9 proteins catalyze H3K4me3 and H3K36me3 and determine hotspot positions, whereas other vertebrates lacking PRDM9 recombine in regions with chromatin already opened for another function, such as gene promoters. While these other vertebrate species lacking PRDM9 remain fertile, inactivation of the mouse Prdm9 gene, which shifts the hotspots to the functional regions (including promoters), typically causes gross fertility reduction; and the reasons for these species differences are not clear. RESULTS: We introduced Prdm9 deletions into the Rattus norvegicus genome and generated the first rat genome-wide maps of recombination-initiating double-strand break hotspots. Rat strains carrying the same wild-type Prdm9 allele shared 88% hotspots but strains with different Prdm9 alleles only 3%. After Prdm9 deletion, rat hotspots relocated to functional regions, about 40% to positions corresponding to Prdm9-independent mouse hotspots, including promoters. Despite the hotspot relocation and decreased fertility, Prdm9-deficient rats of the SHR/OlaIpcv strain produced healthy offspring. The percentage of normal pachytene spermatocytes in SHR-Prdm9 mutants was almost double than in the PWD male mouse oligospermic sterile mutants. We previously found a correlation between the crossover rate and sperm presence in mouse Prdm9 mutants. The crossover rate of SHR is more similar to sperm-carrying mutant mice, but it did not fully explain the fertility of the SHR mutants. Besides mild meiotic arrests at rat tubular stages IV (mid-pachytene) and XIV (metaphase), we also detected postmeiotic apoptosis of round spermatids. We found delayed meiosis and age-dependent fertility in both sexes of the SHR mutants. CONCLUSIONS: We hypothesize that the relative increased fertility of rat versus mouse Prdm9 mutants could be ascribed to extended duration of meiotic prophase I. While rat PRDM9 shapes meiotic recombination landscapes, it is unnecessary for recombination. We suggest that PRDM9 has additional roles in spermatogenesis and speciation-spermatid development and reproductive age-that may help to explain male-specific hybrid sterility.

• MeSH
• chromatin MeSH
• dvouřetězcové zlomy DNA MeSH
• fertilita genetika   MeSH
• histonlysin-N-methyltransferasa genetika   MeSH
• krysa rodu rattus MeSH
• meióza * genetika   MeSH
• myši MeSH
• potkani inbrední SHR MeSH
• spermatogeneze genetika   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Decellularized tissue is an important source for biological tissue engineering. Evaluation of the quality of decellularized tissue is performed using scanned images of hematoxylin-eosin stained (H&E) tissue sections and is usually dependent on the observer. The first step in creating a tool for the assessment of the quality of the liver scaffold without observer bias is the automatic segmentation of the whole slide image into three classes: the background, intralobular area, and extralobular area. Such segmentation enables to perform the texture analysis in the intralobular area of the liver scaffold, which is crucial part in the recellularization procedure. Existing semi-automatic methods for general segmentation (i.e., thresholding, watershed, etc.) do not meet the quality requirements. Moreover, there are no methods available to solve this task automatically. Given the low amount of training data, we proposed a two-stage method. The first stage is based on classification of simple hand-crafted descriptors of the pixels and their neighborhoods. This method is trained on partially annotated data. Its outputs are used for training of the second-stage approach, which is based on a convolutional neural network (CNN). Our architecture inspired by U-Net reaches very promising results, despite a very low amount of the training data. We provide qualitative and quantitative data for both stages. With the best training setup, we reach 90.70% recognition accuracy.

• MeSH
• játra * diagnostické zobrazování   MeSH
• neuronové sítě MeSH
• počítačové zpracování obrazu * MeSH
• sémantika * MeSH
• Publikační typ
• dopisy MeSH

In this work, we studied the biochemical properties and evolutionary histories of catalase (CAT) and ascorbate peroxidase (APX), two central enzymes of reactive oxygen species detoxification, across the highly diverse clade Eugenozoa. This clade encompasses free-living phototrophic and heterotrophic flagellates, as well as obligate parasites of insects, vertebrates, and plants. We present evidence of several independent acquisitions of CAT by horizontal gene transfers and evolutionary novelties associated with the APX presence. We posit that Euglenozoa recruit these detoxifying enzymes for specific molecular tasks, such as photosynthesis in euglenids and membrane-bound peroxidase activity in kinetoplastids and some diplonemids.

• Publikační typ
• časopisecké články MeSH

Every cell is protected by a semipermeable membrane. Peptides with the right properties, for example Antimicrobial peptides (AMPs), can disrupt this protective barrier by formation of leaky pores. Unfortunately, matching peptide properties with their ability to selectively form pores in bacterial membranes remains elusive. In particular, the proline/glycine kink in helical peptides was reported to both increase and decrease antimicrobial activity. We used computer simulations and fluorescence experiments to show that a kink in helices affects the formation of membrane pores by stabilizing toroidal pores but disrupting barrel-stave pores. The position of the proline/glycine kink in the sequence further controls the specific structure of toroidal pore. Moreover, we demonstrate that two helical peptides can form a kink-like connection with similar behavior as one long helical peptide with a kink. The provided molecular-level insight can be utilized for design and modification of pore-forming antibacterial peptides or toxins.

• MeSH
• biologické modely MeSH
• buněčná membrána chemie   metabolismus   MeSH
• hydrofobní a hydrofilní interakce MeSH
• kationické antimikrobiální peptidy chemie   metabolismus   MeSH
• konformace proteinů * MeSH
• metoda Monte Carlo MeSH
• molekulární modely MeSH
• poriny chemie   metabolismus   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH