Raman micro-spectroscopy technique offers a combination of relatively high spatial resolution with identification of components or mixtures of components in different sample areas, e.g. on the surface or the cross-section of a sample. This study is focused on the analysis of the tablets from pharmaceutical development with different technological parameters: (1) the manufacturing technology, (2) the particle size of the input API (active pharmaceutical ingredient) and (3) the quantitative composition of the individual excipients. These three mentioned parameters represent the most frequently solved problems in the field of reverse engineering in pharmacy. The investigation aims to distinguish tablets with the above-described technological parameters with limited subjective steps by Raman microscopy. Furthermore, non-subjective methods of Raman data analysis using advanced statistical analysis have been proposed, namely Principal Component Analysis, Soft Independent Modelling of Class Analogy and Linear Discriminant Analysis. The methods successfully distinguished and identified even very small differences in the analysed tablets within our study and provided objective statistic evaluation of Raman maps. The information on component and particle size distribution including their small differences, which is the critical parameter in the development of the original and generic products, was obtained due to combination of these methods. Even though each of these chemometric methods evaluates the data set from a different perspective, their mutual application on the problem of Raman maps evaluation confirmed and specified results on level that would be unattainable with the use of only one them.

• Klíčová slova
• Linear Discriminant Analysis, Multivariate analysis, Principal Component Analysis, Raman mapping, Soft Independent Modelling of Class Analogy, Tablets,
• MeSH
• chemometrika MeSH
• farmacie * MeSH
• léčivé přípravky * MeSH
• pomocné látky MeSH
• Ramanova spektroskopie MeSH
• tablety MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• léčivé přípravky * MeSH
• pomocné látky MeSH
• tablety MeSH

The wide variety of protein structures and functions results from the diverse properties of the 20 canonical amino acids. The generally accepted hypothesis is that early protein evolution was associated with enrichment of a primordial alphabet, thereby enabling increased protein catalytic efficiencies and functional diversification. Aromatic amino acids were likely among the last additions to genetic code. The main objective of this study was to test whether enzyme catalysis can occur without the aromatic residues (aromatics) by studying the structure and function of dephospho-CoA kinase (DPCK) following aromatic residue depletion. We designed two variants of a putative DPCK from Aquifex aeolicus by substituting (a) Tyr, Phe and Trp or (b) all aromatics (including His). Their structural characterization indicates that substituting the aromatics does not markedly alter their secondary structures but does significantly loosen their side chain packing and increase their sizes. Both variants still possess ATPase activity, although with 150-300 times lower efficiency in comparison with the wild-type phosphotransferase activity. The transfer of the phosphate group to the dephospho-CoA substrate becomes heavily uncoupled and only the His-containing variant is still able to perform the phosphotransferase reaction. These data support the hypothesis that proteins in the early stages of life could support catalytic activities, albeit with low efficiencies. An observed significant contraction upon ligand binding is likely important for appropriate organization of the active site. Formation of firm hydrophobic cores, which enable the assembly of stably structured active sites, is suggested to provide a selective advantage for adding the aromatic residues.

• Klíčová slova
• aromatic amino acids, catalysis evolution, genetic code evolution, protein disorder, protein structure evolution,
• MeSH
• Aquifex enzymologie   genetika   MeSH
• bakteriální proteiny chemie   genetika   MeSH
• fosfotransferasy s alkoholovou skupinou jako akceptorem chemie   genetika   MeSH
• katalytická doména MeSH
• katalýza MeSH
• mutageneze cílená MeSH
• sekundární struktura proteinů MeSH
• substituce aminokyselin MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• dephospho-CoA kinase MeSH Prohlížeč
• fosfotransferasy s alkoholovou skupinou jako akceptorem MeSH

Reverse engineering is the process of creating a digital version of an existing part without any knowledge in advance about the design intent. Due to 3D printing, the reconstructed part can be rapidly fabricated for prototyping or even for practical usage. To showcase this combination, this study presents a workflow on how to restore a motorcycle braking pedal from material SS316L with the Powder Bed Fusion (PBF) technology. Firstly, the CAD model of the original braking pedal was created. Before the actual PBF printing, the braking pedal printing process was simulated to identify the possible imperfections. The printed braking pedal was then subjected to quality control in terms of the shape distortion from its CAD counterpart and strength assessments, conducted both numerically and physically. As a result, the exterior shape of the braking pedal was restored. Additionally, by means of material assessments and physical tests, it was able to prove that the restored pedal was fully functional. Finally, an approach was proposed to optimize the braking pedal with a lattice structure to utilize the advantages the PBF technology offers.

• Klíčová slova
• 3D scanning, SS316L, electronic speckle pattern interferometry, lattice structure, powder bed fusion, printing simulation, reverse engineer,
• Publikační typ
• časopisecké články MeSH

A novel fractographic approach based on a combination of (i) mechanical behavior of cured rubber in uniaxial tensile loading and (ii) spectroscopy of fracture on a ruptured surface was experimentally validated. This approach related the migration of paraffin oil from a matrix to the ruptured rubber surface, to the tearing energy related to the deformation speed responsible for total rubber sample rupture, and the approach itself was configured experimentally. It was evaluated on cured natural rubber (NR) for two different paraffin oil concentrations. Single edge notched tensile (SENT) samples were subjected to uniaxial tensile loadings at two different deformation speeds. First, the tearing energy as a function of deformation speed was determined for each defined oil concentration. Secondly, at specific locations on the ruptured surfaces, infrared (IR) spectroscopy was performed to quantify a characteristic absorbance peak height of migrated paraffin oil during the rupture process. The results of the IR analyses were related to the deformation speed to understand the relation between the amount of migrated paraffin oil during the fracture process and the deformation speed which brought about such a fracture. This novel approach enhanced the reverse engineering process of rubber fracture related to the cause of tearing energies during critical failure.

• Klíčová slova
• deformation speed, fractography, fracture, oil, rubber, spectroscopy, tearing energy, uniaxial tensile,
• Publikační typ
• časopisecké články MeSH

Electrochemical synthesis of nickel-nitrilotriacetic acid (Ni-NTA) chelators, for subsequent immobilization of (His)(6)-tagged proteins (Photosystem II (PSII) as model molecule), on Au or Au-graphite electrodes is compared to chemical synthesis. Results show: (i) higher Ni-NTA surface density, (ii) shorter treatment time (1-12 min vs. 16 h normally needed for self-assembled monolayer (SAM)), (iii) possibility of addressing the chelator to only one Au electrode, in a sensor micro-array.

• MeSH
• adsorpce MeSH
• biokompatibilní potahované materiály chemie   MeSH
• biosenzitivní techniky metody   MeSH
• chelátory chemie   MeSH
• elektrochemie metody   MeSH
• elektrody MeSH
• fotosystém II (proteinový komplex) analýza   chemie   genetika   účinky záření   MeSH
• kyselina nitrilotrioctová analogy a deriváty   chemie   MeSH
• organokovové sloučeniny chemie   MeSH
• proteinové inženýrství metody   MeSH
• rekombinantní proteiny chemie   účinky záření   MeSH
• světlo MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• biokompatibilní potahované materiály MeSH
• chelátory MeSH
• fotosystém II (proteinový komplex) MeSH
• kyselina nitrilotrioctová MeSH
• nickel nitrilotriacetic acid MeSH Prohlížeč
• organokovové sloučeniny MeSH
• rekombinantní proteiny MeSH

Reversed-phase ultrahigh-performance liquid chromatography-mass spectrometry (RP-UHPLC/MS) method is optimized for the quantitation of a large number of lipid species in biological samples, primarily in human plasma and serum. The method uses a C18 bridged ethylene hybrid (BEH) column (150 × 2.1 mm; 1.7 μm) for the separation of lipids from 23 subclasses with a total run time of 25 min. Lipid species separation allows the resolution of isobaric and isomeric lipid forms. A triple quadrupole mass spectrometer is used for targeted lipidomic analysis using multiple reaction monitoring (MRM) in the positive ion mode. Data are evaluated by Skyline software, and the concentrations of analytes are determined using internal standards per each individual lipid class.

• Klíčová slova
• High-throughput lipidomics, Mass spectrometry, Plasma, Quantitation, Reversed-phase, Serum, Ultrahigh-performance liquid chromatography,
• MeSH
• chromatografie s reverzní fází * metody   MeSH
• hmotnostní spektrometrie metody   MeSH
• kapalinová chromatografie-hmotnostní spektrometrie MeSH
• lidé MeSH
• lipidomika * metody   MeSH
• lipidy * analýza   MeSH
• rychlé screeningové testy metody   MeSH
• software MeSH
• tandemová hmotnostní spektrometrie metody   MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• lipidy * MeSH

Fibrin plays an important role during wound healing and skin regeneration. It is often applied in clinical practice for treatment of skin injuries or as a component of skin substitutes. We prepared electrospun nanofibrous membranes made from poly(l-lactide) modified with a thin fibrin nanocoating. Fibrin surrounded the individual fibers in the membrane and also formed a thin fibrous mesh on several places on the membrane surface. The cell-free fibrin nanocoating remained stable in the cell culture medium for 14 days and did not change its morphology. On membranes populated with human dermal fibroblasts, the rate of fibrin degradation correlated with the degree of cell proliferation. The cell spreading, mitochondrial activity, and cell population density were significantly higher on membranes coated with fibrin than on nonmodified membranes, and this cell performance was further improved by the addition of ascorbic acid in the cell culture medium. Similarly, fibrin stimulated the expression and synthesis of collagen I in human dermal fibroblasts, and this effect was further enhanced by ascorbic acid. The expression of beta1-integrins was also improved by fibrin, and on pure polylactide membranes, it was slightly enhanced by ascorbic acid. In addition, ascorbic acid promoted deposition of collagen I in the form of a fibrous extracellular matrix. Thus, the combination of nanofibrous membranes with a fibrin nanocoating and ascorbic acid seems to be particularly advantageous for skin tissue engineering.

• Klíčová slova
• ascorbic acid, beta1-integrins, collagen I synthesis, electrospun nanofibers, fibrin, fibroblasts, nanocoating, nanomedicine, nanotechnology, skin tissue engineering,
• MeSH
• buněčná diferenciace MeSH
• elektrochemie metody   MeSH
• extracelulární matrix metabolismus   MeSH
• fibrin chemie   metabolismus   MeSH
• fibroblasty cytologie   metabolismus   MeSH
• fluorescenční protilátková technika MeSH
• imunoenzymatické techniky MeSH
• kolagen genetika   metabolismus   MeSH
• kultivované buňky MeSH
• kůže cytologie   metabolismus   MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• lidé MeSH
• messenger RNA genetika   MeSH
• nanovlákna chemie   MeSH
• polyestery chemie   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• proliferace buněk MeSH
• regenerace fyziologie   MeSH
• tkáňové inženýrství metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fibrin MeSH
• kolagen MeSH
• messenger RNA MeSH
• poly(lactide) MeSH Prohlížeč
• polyestery MeSH

Due to the technical advances of mass spectrometers, particularly increased scanning speed and higher MS/MS resolution, the use of data-independent acquisition mass spectrometry (DIA-MS) became more popular, which enables high reproducibility in both proteomic identification and quantification. The current DIA-MS methods normally cover a wide mass range, with the aim to target and identify as many peptides and proteins as possible and therefore frequently generate MS/MS spectra of high complexity. In this report, we assessed the performance and benefits of using small windows with, e.g., 5-m/z width across the peptide elution time. We further devised a new DIA method named RTwinDIA that schedules the small isolation windows in different retention time blocks, taking advantage of the fact that larger peptides are normally eluting later in reversed phase chromatography. We assessed the direct proteomic identification by using shotgun database searching tools such as MaxQuant and pFind, and also Spectronaut with an external comprehensive spectral library of human proteins. We conclude that algorithms like pFind have potential in directly analyzing DIA data acquired with small windows, and that the instrumental time and DIA cycle time, if prioritized to be spent on small windows rather than on covering a broad mass range by large windows, will improve the direct proteome coverage for new biological samples and increase the quantitative precision. These results further provide perspectives for the future convergence between DDA and DIA on faster MS analyzers.

• Klíčová slova
• Data-independent acquisition, Isolation windows, Maxquant, Spectronaut, pFind,
• MeSH
• chromatografie s reverzní fází MeSH
• hmotnostní spektrometrie metody   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• peptidy analýza   MeSH
• proteiny analýza   MeSH
• proteomika metody   MeSH
• software MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• peptidy MeSH
• proteiny MeSH

Nine new dihydrochloride salts of 3-(4-arylpiperazin-1-yl)-2-hydroxypropyl 4-alkoxyethoxybenzoates were designed and synthesized. The physicochemical properties such as lipophilicity index (log kw) and dissociation constant (pKa) were experimentally determined and compared to the software calculated data. The lipophilicity index was determined by means of reversed-phase high performance liquid chromatography (RP-HPLC). The pKa values were determined by means of capillary zone electrophoresis. The "drug-likeness" properties according to the Lipinski Rule of Five and prediction of possible blood-brain barrier penetration were computed and discussed.

• Klíčová slova
• Lipinski rules, arylcarbonyloxyaminopropanols, blood–brain barrier, lipophilicity index, pKa determination, phenylpiperazines,
• MeSH
• benzoáty chemická syntéza   chemie   MeSH
• chemické jevy MeSH
• chromatografie s reverzní fází metody   MeSH
• hydrofobní a hydrofilní interakce MeSH
• koncentrace vodíkových iontů MeSH
• lipidy chemie   MeSH
• software * MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• benzoáty MeSH
• lipidy MeSH

The fast motion of the snap-traps of the terrestrial Venus flytrap (Dionaea muscipula) have been intensively studied, in contrast to the tenfold faster underwater snap-traps of its phylogenetic sister, the waterwheel plant (Aldrovanda vesiculosa). Based on biomechanical and functional-morphological analyses and on a reverse biomimetic approach via mechanical modelling and computer simulations, we identify a combination of hydraulic turgor change and the release of prestress stored in the trap as essential for actuation. Our study is the first to identify and analyse in detail the motion principle of Aldrovanda, which not only leads to a deepened understanding of fast plant movements in general, but also contributes to the question of how snap-traps may have evolved and also allows for the development of novel biomimetic compliant mechanisms.

• Klíčová slova
• Aldrovanda, Dionaea, finite-element model, mechanical modelling, plant movement, reverse biomimetics,
• MeSH
• biologické modely MeSH
• biomechanika MeSH
• biomimetika MeSH
• Droseraceae fyziologie   MeSH
• listy rostlin fyziologie   MeSH
• masožravci MeSH
• počítačová simulace MeSH
• pohyb těles MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH