Assessment of Tractable Cysteines for Covalent Targeting by Screening Covalent Fragments

Targeted covalent inhibition and the use of irreversible chemical probes are important strategies in chemical biology and drug discovery. To date, the availability and reactivity of cysteine residues amenable for covalent targeting have been evaluated by proteomic and computational tools. Herein, we present a toolbox of fragments containing a 3,5-bis(trifluoromethyl)phenyl core that was equipped with chemically diverse electrophilic warheads showing a range of reactivities. We characterized the library members for their reactivity, aqueous stability and specificity for nucleophilic amino acids. By screening this library against a set of enzymes amenable for covalent inhibition, we showed that this approach experimentally characterized the accessibility and reactivity of targeted cysteines. Interesting covalent fragment hits were obtained for all investigated cysteine-containing enzymes.     

Attachment and proliferation of human osteoblast-like cells (MG-63) on laser-ablated titanium implant material

Demand is increasing for shortening the long (3–6 months) osseointegration period to rehabilitate patients' damaged chewing apparatus in as short a time as possible. For dental implants, as for biomaterials in general, the bio- and osseointegration processes can be controlled at molecular and cellular levels by modification of the implant surface. One of the most promising of such surface modifications is laser ablation, as demonstrated by our previous results [46]. Commercially pure (CP4) sand-blasted, acid-etched titanium disks (Denti® System Ltd., Hungary) were irradiated with a KrF excimer laser (248 nm, fluence 0.4 J/cm², FWHM 18 ns, 2000 pulses), or with a Nd:YAG laser (532 nm, 1.3 J/cm², 10 ns, 200 pulses) then examined by SEM, AFM, and XPS. In vitro attachment (24 h) and proliferation (72 h) of MG-63 osteoblast cells were investigated via dimethylthiazol-diphenyl tetrazolium bromide (MTT), alamarBlue (AB) assays alkaline phosphatase quantification (ALP) and SEM. SEM and AFM revealed significant changes in morphology and roughness. XPS confirmed the presence of TiO₂ on each sample; after Nd:YAG treatment a reduced state of Ti (Ti^3 +) was also observed. MTT, AB and ALP measurements detected an increase in the number of cells between the 24- and 72 hour observations; however, laser treatment did not affect cell attachment and proliferation significantly.     

Molecular characterization and antibiotic resistance profiling of Campylobacter isolated from cattle in Polish slaughterhouses

A total of 812 samples from bovine hides and the corresponding carcasses collected at the slaughterhouse level in the eastern part of Poland were examined for the presence of Campylobacter jejuni and Campylobacter coli. Recovered isolates were confirmed using species-specific PCR, characterized by the presence of 11 putative virulence genes and antimicrobial susceptibility was determined using a microbroth dilution method. Furthermore, the genotypic relatedness of the isolates was determined by PFGE profiling and virulence pattern cluster analysis. The prevalence of Campylobacter was 25.6% and 2.7% in bovine hide and carcass samples, respectively. The presence of virulence markers varied between C. jejuni and C. coli species however, the majority of strains possessed the cadF, flhA, flaA genes, irrespective of the bacterial species and origin. The lower number of the strains was positive for the invasive associated markers – virB11 and wlaN. Antibiotic profiling showed that campylobacters were most frequently resistant to quinolones and fluoroquinolones (nalidixic acid and ciprofloxacin, 38.3% of each, respectively) followed by streptomycin (24.3%) and tetracycline (20.9%). Resistance to erythromycin and gentamicin was demonstrated in 4.3% and 2.6% of strains, respectively. Comparisons of the PFGE and virulence marker profiles of the isolates reflected the high genetic diversity of Campylobacter tested. Moreover, a poor correlation between the PFGE type, pathogenic gene marker and antimicrobial resistance patterns was observed.     

MicroRNAs and Extracellular Vesicles as Distinctive Biomarkers of Precocious and Advanced Stages of Breast Cancer Brain Metastases Development

Triple negative breast cancer presents higher mortality and poorer survival rates than other breast cancer (BC) types, due to the proneness to brain metastases formation, which are usually diagnosed at advanced stages. Therefore, the discovery of BC brain metastases (BCBM) biomarkers appears pivotal for a timely intervention. With this work, we aimed to disclose microRNAs (miRNAs) and extracellular vesicles (EVs) in the circulation as biomarkers of BCBM formation. Using a BCBM animal model, we analyzed EVs in plasma by nanoparticle tracking analysis and ascertained their blood-brain barrier (BBB) origin by flow cytometry. We further evaluated circulating miRNAs by RT-qPCR and their brain expression by in situ hybridization. In parallel, a cellular model of BCBM formation, combining triple negative BC cells and BBB endothelial cells, was used to differentiate the origin of biomarkers. Established metastases were associated with an increased content of circulating EVs, particularly of BBB origin. Interestingly, deregulated miRNAs in the circulation were observed prior to BCBM detection, and their brain origin was suggested by matching alterations in brain parenchyma. In vitro studies indicated that miR-194-5p and miR-205-5p are expressed and released by BC cells, endothelial cells and during their interaction. These results highlight miRNAs and EVs as biomarkers of BCBM in early and advanced stages, respectively.     

A Theoretical Study of the Half-Sandwich Silicon(II) Cation [(Me<Subscript>5</Subscript>C<Subscript>5</Subscript>)Si]<Superscript>+</Superscript> and of the Cationic Complex [(Me<Subscript>5</Subscript>C<Subscript>5</Subscript>)Si (DME)]<Superscript>+</Superscript>

Ab-initio calculations have been performed for the half-sandwich cation [(Me₅C₅)Si]⁺ and its DME complex [(Me₅C₅)Si(DME)]⁺. For these cations, the ground state energies, the complexation energy, the frontier orbitals, the vertical singlet-triplet excitation energies, and the natural atomic charges have been calculated. In both cations, the “lone-pair” at silicon does not represent the HOMO. The nature of the weak dative O → Si bond can best be described in terms of electrostatic and attractive dispersion interactions. The DME coordination destabilizes the cluster orbitals and slightly enhances the positive charge at silicon.     

Biomass productivity and phytoremediation potential of Salix alba and Salix viminalis

The aim of this work was to determine selected Salix clones’ capacities for biomass production and accumulation of heavy metal ions. Determination of the relationship between sorption of metals and biomass productivity was a further purpose of this study. Eight Salix viminalis cultivars and one Salix alba cultivar were analyzed. The taxa characterized by greatest biomass production were S. alba var. Chermesina and S. viminalis ‘1056’ (respectively 6.8 and 4.3 kg of fresh mass per shrub per year).The results have revealed significant differences among clones. The clones most effective in accumulating all five metals were S. viminalis ‘1154’ and ‘1054’. The studied Salix population was significantly diverse as regards accumulation efficiency. The differences between the highest and lowest heavy metal content in extreme clones were for: Cd 84%, Cu 90%, Hg 167%, Pb 190% and Zn 36%. At the same time, significant differences were observed in Salix structure. The greatest cellulose content was observed in S. viminalis ‘Sprint’ (49.69%) and the lowest in S. viminalis ‘1059’ (42.09%).     

Indirect,non-competitive amperometric immunoassay for accurate quantification of calpastatin,a meat tenderness marker,in bovine muscle

A method is described for quantification of the beef tenderness marker, calpastatin, in meat samples by amperometric detection. Using a novel bovine recombinant partial calpastatin protein as standard antigen a low detection limit of 0.2 ng/mL was achieved. The influence of the complex matrix was minimised by heat pretreatment and dilution of the samples prior to detection of calpastatin. The relative error between the direct sample measurement and standard addition methods was 5.89%, confirming the accuracy of the developed amperometric immunoassay.