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The aim of our study was to develop a novel method for nanocarriers’ preparation as a fluorine magnetic resonance imaging (19F MRI)-detectable drug delivery system. The novelty of the proposed approach is based on the application of fluorinated polyelectrolyte Nafion as a contrast agent since typical MRI contrast agents are based on paramagnetic gadolinium or ferro/superparamagnetic iron oxide compounds. An advantage of using an 19F-based tracer comes from the fact that the 19F image is detected at a different resonance frequency than the 1H image. In addition, the close to zero natural concentration of 19F nuclei in the human body makes fluorine atoms a promising MRI marker without any natural background signal. That creates the opportunity to localize and identify only exogenous fluorinated compounds with 100% specificity. The nanocarriers were formed by the deposition of polyelectrolytes on nanoemulsion droplets via the layer-by-layer technique with the saturation approach. The polyelectrolyte multilayer shell was composed of Nafion, the fluorinated ionic polymer used for labeling by 19F nuclei, and poly-l-lysine (PLL). The surface of such prepared nanocarriers was further pegylated by adsorption of pegylated polyanion, poly-l-glutamic acid (PGA). The 19F MRI-detectable hydrophobic nanocarriers with an average size of 170 nm and a sufficient signal-to-noise ratio have been developed and optimized to be used for passive tumor targeting and drug delivery.

To maintain its functional abilities, the mature brain obtains energy from glucose produced in carbohydrate metabolism. When carbohydrates are eliminated from the diet, the energy comes from the oxidation of fatty acids. In this metabolic state called ketosis, ketone bodies are formed: β-hydroxybutyric acid (bHb), acetone, and acetoacetate as alternative source of energy passing through the blood–brain barrier easily. The ketosis state can be achieved through various strategies like caloric restriction, supplementation with medium-chain triglycerides, intense physical training, or ketogenic diet (KD). Using KD, drug-resistant epilepsy has been successfully treated in children and adults. It can also exert neuroprotective influences in cases of brain damage, glioblastoma multiforme, and Alzheimer's or Parkinson's diseases. Although many possible mechanisms of KD activity have been proposed, newer hypotheses appear with the research progress, mostly characterizing the brain under pathological but not normal conditions. Since different pathological conditions may affect the mechanism of KD action differently, additional research on the normal brain appears reasonable. For this purpose, young adult rats were treated with 4-month-lasting KD. Then, MRI structural measurements, spectroscopy, and tractography were performed. The procedures revealed significant increases in the concentration of glutamine, glutamate, glutathione and NAA, accompanied by changes in the pattern of neuronal connections of the striatum and hippocampal formation. This implies a possible involvement of these structures in the functional changes occurring in the brain after KD application. Thus, the investigations on the normal brain add important details concerning mechanisms underlying KD effects without their possible modification by a pathological status.

Cancer is one of the most important health problems of our population, and one of the common anticancer treatments is chemotherapy. The disadvantages of chemotherapy are related to the drug’s toxic effects, which act on cancer cells and the healthy part of the body. The solution of the problem is drug encapsulation and drug targeting. The present study aimed to develop a novel method of preparing multifunctional 5-Fluorouracil (5-FU) nanocarriers and their in vitro characterization. 5-FU polyaminoacid-based core@shell nanocarriers were formed by encapsulation drug-loaded nanocores with polyaminoacids multilayer shell via layer-by-layer method. The size of prepared nanocarriers ranged between 80–200 nm. Biocompatibility of our nanocarriers as well as activity of the encapsulated drug were confirmed by MTT tests. Moreover, the ability to the real-time observation of developed nanocarriers and drug accumulation inside the target was confirmed by fluorine magnetic resonance imaging (19F-MRI).

There are huge demands on multifunctional nanocarriers to be used in nanomedicine. Herein, we present a simple and efficient method for the preparation of multifunctional magnetically responsive polymeric-based nanocarriers optimized for biomedical applications. The hybrid delivery system is composed of drug-loaded polymer nanoparticles (poly(caprolactone), PCL) coated with a multilayer shell of polyglutamic acid (PGA) and superparamagnetic iron oxide nanoparticles (SPIONs), which are known as bio-acceptable components. The PCL nanocarriers with a model anticancer drug (Paclitaxel, PTX) were formed by the spontaneous emulsification solvent evaporation (SESE) method, while the magnetically responsive multilayer shell was formed via the layer-by-layer (LbL) method. As a result, we obtained magnetically responsive polycaprolactone nanocarriers (MN-PCL NCs) with an average size of about 120 nm. Using the 9.4 T preclinical magnetic resonance imaging (MRI) scanner we confirmed, that obtained MN-PCL NCs can be successfully used as a MRI-detectable drug delivery system. The magnetic hyperthermia effect of the MN-PCL NCs was demonstrated by applying a 25 mT radio-frequency (f = 429 kHz) alternating magnetic field. We found a Specific Absorption Rate (SAR) of 55 W g−1. The conducted research fulfills the first step of investigation for biomedical application, which is mandatory for the planning of any in vitro and in vivo studies.

The purpose of the study was to assess the variability between 16 wild thyme populations introduced into ex situ conditions, in terms of morphological traits and chemical profiles. The observations were carried out on 2-year-old plants. Total content of essential oil and phenolic compounds (flavonoids, phenolic acids and tannins) was determined according to Polish Pharmacopoeia 6th. The composition of essential oil was carried out by GC/MS and GC/FID. Populations differed in respect of examined traits, i.e.: plant’s height (from 50 to 90 mm), plant diameter (from 21.3 to 53.0 cm), length of stems (from 8.1 to 20.5 cm). Fresh mass of herb per plant ranged from 36.0 to 307.0 g, while dry mass from 15.63 to 73.11 g (CV 55.0 and 47.5%, respectively). Total content of essential oil varied from 0.23 to 0.97%. Three chemotypes were distinguished within investigated populations: geranyl acetate + β-terpineol + β-myrcene, geranyl acetate + β-terpineol + borneol and pure linalool type. The total content of phenolic compounds ranged, as following: flavonoids (0.17–0.26%), tannins (0.57–1.32%), phenolic acids (0.28–0.61%). To sum up, investigated populations differed significantly in respect of developmental features as well as content and composition of identified compounds. Obtained results may be used in future works on selection and breeding of wild thyme, providing a high-quality raw material characterized by specific chemical composition and pharmacological activity.

In biology and medicine, cell counting is one of the most important elements of cytometry, with applications to research and clinical practice. For instance, the complete cell count could help to determine conditions for which cancer cells could grow or not. However, cell counting is a laborious and time-consuming process, and its automatization is highly demanded. Here, we propose use of a Convolutional Neural Network-based regressor, a regression model trained end-to-end, to provide the cell count. First, unlike most of the related work, we formulate the problem of cell counting as the regression task rather than the classification task. This allows not only to reduce the required annotation information (i.e., the number of cells instead of pixel-level annotations) but also to reduce the burden of segmenting potential cells and then classifying them. Second, we propose use of xResNet, a successful convolutional architecture with residual connection, together with transfer learning (using a pretrained model) to achieve human-level performance. We demonstrate the performance of our approach to real-life data of two cell lines, human osteosarcoma and human leukemia, collected at the University of Amsterdam (133 training images, and 32 test images). We show that the proposed method (deep learning and transfer learning) outperforms currently used machine learning methods. It achieves the test mean absolute error equal 12 (±15) against 32 (±33) obtained by the deep learning without transfer learning, and 41 (±37) of the best-performing machine learning pipeline (Random Forest Regression with the Histogram of Gradients features).

Hydrogel wound dressings are highly effective in the therapy of wounds. Yet, most of them do not contain any active ingredient that could accelerate healing. The aim of this study was to prepare hydrophilic active dressings loaded with an anti-inflammatory compound - trans-resveratrol (RSV) of hydrophobic properties. A special attention was paid to select such a technological strategy that could both reduce the risk of irritation at the application site and ensure the homogeneity of the final hydrogel. RSV dissolved in Labrasol was combined with an aqueous sol of poly(vinyl) alcohol (PVA), containing propylene glycol (PG) as a plasticizer. This sol was transformed into a gel under six consecutive cycles of freezing (-80 °C) and thawing (RT). White, uniform and elastic membranes were successfully produced. Their critical features, namely microstructure, mechanical properties, water uptake and RSV release were studied using SEM, DSC, MRI, texture analyser and Franz-diffusion cells. The cryogels made of 8 % of PVA showed optimal tensile strength (0.22 MPa) and elasticity (0.082 MPa). The application of MRI enabled to elucidate mass transport related phenomena in this complex system at the molecular (detection of PG, confinement effects related to pore size) as well as at the macro level (swelling). The controlled release of RSV from membranes was observed for 48 h with mean dissolution time of 18 h and dissolution efficiency of 35 %. All in all, these cryogels could be considered as a promising new active wound dressings. Supplementary Information The online version contains supplementary material available at 10.1208/s12249-021-01976-1.

An efficient treatment against a COVID-19 disease, caused by the novel coronavirus SARS-CoV-2 (CoV2), remains a challenge. The papain-like protease (PLpro) from the human coronavirus is a protease that plays a critical role in virus replication. Moreover, CoV2 uses this enzyme to modulate the host’s immune system to its own benefit. Therefore, it represents a highly promising target for the development of antiviral drugs. We used Approximate Bayesian Computation tools, molecular modelling and enzyme activity studies to identify highly active inhibitors of the PLpro. We discovered organoselenium compounds, ebselen and its structural analogues, as a novel approach for inhibiting the activity of PLproCoV2. Furthermore, we identified, for the first time, inhibitors of PLproCoV2 showing potency in the nanomolar range. Moreover, we found a difference between PLpro from SARS and CoV2 that can be correlated with the diverse dynamics of their replication, and, putatively to disease progression.

Methods of spatiotemporal characterization of nonequilibrated polymer based matrices are still immature and imperfect. The purpose of the study was to develop the methodology for the spatiotemporal characterization of water transport and properties in alginate tablets under hydration. The regions of low water content were spatially and temporally sampled using Karl Fisher and Differential Scanning Callorimetry (spatial distribution of freezing/nonfreezing water) with spatial resolution of 1 mm. In the regions of high water content, where sampling was infeasible due to gel/sol consistency, magnetic resonance imaging (MRI) enabled characterization with an order of magnitude higher spatial resolution. The minimally hydrated layer (MHL), infiltration layer (IL) and fully hydrated layer (FHL) were identified in the unilaterally hydrated matrices. The MHL gained water from the first hour of incubation (5–10% w/w) and at 4 h total water content was 29–39% with nonfreezing pool of 28–29%. The water content in the IL was 45–47% and at 4 h it reached ~50% with the nonfreezing pool of 28% and T2 relaxation time &lt, 10 ms. The FHL consisted of gel and sol layer with water content of 85–86% with a nonfreezing pool of 11% at 4 h and T2 in the range 20–200 ms. Hybrid destructive/nondestructive analysis of alginate matrices under hydration was proposed. It allowed assessing the temporal changes of water distribution, its mobility and interaction with matrices in identified layers.

The purpose was to show, using destructive/nondestructive methods, that the interplay between water, tablet structure, and composition determine the unique spatiotemporal hydration pattern of polymer-based matrices. The tablets containing a 1:1 w/w mixture of sodium alginate with salicylic acid (ALG/SA) or sodium salicylate (ALG/SNA) were studied using Karl Fischer titration, differential scanning calorimetry, X-ray microtomography, and magnetic resonance imaging. As the principal results, matrix specific features were detected, e.g., “locking” of the internal part of the matrix (ALG/SA), existence of lamellar region associated with detection of free/freezing water (ALG/SA), existence of water penetrating the matrix forming specific region preceding infiltration layer (ALG/SNA), switch in the onset temperature of endothermic water peak associated with an increase in the fraction of non-freezing water weight per dry matrix weight in the infiltration layer (ALG/SNA). The existence of complicated spatiotemporal hydration patterns influenced by matrix composition and molecular properties of constituents has been demonstrated.

Izabela Czyzynska-Cichon,1,* Małgorzata Janik-Hazuka,2,* Joanna Szafraniec-Szczęsny,2,3 Krzysztof Jasinski,4 Władysław P Węglarz,4 Szczepan Zapotoczny,2 Stefan Chlopicki1,5 1Jagiellonian University, Jagiellonian Centre for Experimental Therapeutics (JCET), Krakow, 30-348, Poland; 2Jagiellonian University, Faculty of Chemistry, Krakow, 30-387, Poland; 3Jagiellonian University Medical College, Faculty of Pharmacy, Department of Pharmaceutical Technology and Biopharmaceutics, Krakow, 30-688, Poland; 4Institute of Nuclear Physics Polish Academy of Sciences, Department of Magnetic Resonance Imaging, Krakow, 31-342, Poland; 5Jagiellonian University Medical College, Faculty of Medicine, Department of Pharmacology, Krakow, 31-531, Poland*These authors contributed equally to this workCorrespondence: Stefan Chlopicki; Szczepan Zapotoczny Email stefan.chlopicki@jcet.eu; zapotocz@chemia.uj.edu.plBackground: Vascular drug delivery becomes a promising direction in the development of novel therapeutic strategies in the treatment of cardiovascular pathologies, such as hypertension. However, targeted delivery of hydrophobic substances, with poor bioavailability, remains a challenge. Here, we described the hypotensive effects of a low dose of curcumin delivered to the vascular wall using hyaluronic acid-based nanocapsules.Methods: The group of hypertensive TGR(m-Ren2)27 rats, was administrated respectively with the vehicle, curcumin solution or curcumin delivered using hyaluronic acid-based nanocapsules (HyC12-Cur), for 7 days each, maintaining the wash-out period between treatments. Arterial blood pressure (systolic - SBP, diastolic – DBP) and heart rate (HR) were monitored continuously using a telemetry system (Data Science International), and Mean Arterial Pressure (MAP) was calculated from SBP and DBP.Results: In hypertensive rats, a low dose of curcumin (4.5 mg/kg) administrated in HyC12-Cur for 7 days resulted in a gradual inhibition of SBP, DBP and MAP increase without an effect on HR. At the end of HyC12-Cur – based treatment changes in SBP, DBP and MAP amounted to − 2.0± 0.8 mmHg, − 3.9± 0.7 mmHg and − 3.3± 0.7 mmHg, respectively. In contrast, the administration of a curcumin solution (4.5 mg/kg) did not result in a significant hypotensive effect and the animals constantly developed hypertension. Vascular delivery of capsules with curcumin was confirmed using newly developed fluorine-rich nanocapsules (HyFC10-PFOB) with a shell based on a HA derivative and similar size as HyC12-Cur. HyFC10-PFOB gave fluorine signals in rat aortas analyzed ex vivo with a 19F NMR technique after a single intragastric administration.Conclusion: These results suggest that nanocapsules based on hyaluronic acid, the ubiquitous glycosaminoglycan of the extracellular matrix and an integral part of endothelial glycocalyx, may represent a suitable approach to deliver hydrophobic, poorly bioavailable compounds, to the vascular wall.Keywords: nanocapsules, hyaluronic acid, 19F NMR, curcumin, hypertension, targeted vascular delivery

The purpose of the study was to determine the differences between two subspecies: O. vulgare L. subsp. hirtum (Link) Ietswaart (Greek oregano) and O. vulgare L. subsp. vulgare (common oregano) growing in cultivation conditions within temperate climate of Central Europe. The characteristic of the subspecies was undertaken in terms of selected morphological parameters and the quality of the raw material. The herb of both subspecies was evaluated on the content and composition of essential oil by hydrodistillation followed by GC-MS and GC-FID (gas chromatography coupled with mass spectrometry and flame ionization detector), the total content of phenolic acids (according to PP 6th ed.) and the content of rosmarinic acid (by HPLC). The sensory evaluation (QDA) was performed, as well. Greek oregano was distinguished by visibly higher number of glandular trichomes on the leaves (up to 4.85 per 1 mm2) followed by higher content of essential oil in the herb (up to 3.36 g &times, 100 g&minus, 1 DW) in comparison to common oregano. Based on the essential oil composition, Greek oregano was classified as mixed carvacrol/&gamma, terpinene chemotype, while common oregano as mixed sabinyl/cymyl type rich in sesquiterpenes. Greek oregano was also characterized by higher total content of phenolic acids (up to 6.16 g &times, 1 DW) and rosmarinic acid (up to 6787.2 mg &times, 1 DW) than common oregano. Essential oil content reached the maximum at the beginning of blooming (common oregano) and at the full blooming stage (Greek oregano). In turn, the amount of phenolic acids followed by rosmarinic acid was the highest at the beginning of seed-setting stage, in the case of both subspecies. The differences between subspecies concerning chemical composition (especially essential oil) were reflected in the sensory attributes, where both odor and taste notes were found at higher level for Greek oregano. Results of our work indicate that Greek oregano is well adapted to grow in the temperate zone conditions. Such adaptation was reflected mainly in the satisfied yield and maintaining characters typical for the Mediterranean plant, e.g., a high essential oil content followed by high carvacrol share, traits the most important from practice viewpoint.

The application of the Three-Dimensional Ultra-Short Echo Time (3D UTE) pulse sequence at a high magnetic field for visualization of the distribution of 19F loaded theranostic core-shell nanocapsules with Nafion&reg, (1,1,2,2-tetrafluoroethene, 1,1,2,2-tetrafluoro-2- [1,1,1,2,3,3-hexafluoro-3-(1,2,2-trifluoroethenoxy)propan-2-yl] oxyethanesulfonic acid) incorporated into the shell is presented. The nanocarriers were formed via the layer-by-layer technique with biodegradable polyelectrolytes: PLL (Poly-L-lysine), and with Nafion&reg, polymer with high 19F content. Before imaging, an MR (magnetic resonance) spectroscopy and T1 and T2 measurements were performed, resulting in values of T2 between 1.3 ms and 3.0 ms, depending on the spectral line. To overcome limitations due to such short T2, the 3D UTE pulse sequence was applied for 19F MR imaging. First Nafion&reg, solutions of various concentrations were measured to check the detection limit of our system for the investigated molecule. Next, the imaging of a phantom containing core-shell nanocapsules was performed to assess the possibility of visualizing their distribution in the samples. Images of Nafion&reg, containing samples with SNR &ge, 5 with acquisition time below 30 minutes for 19F concentration as low as 1.53&middot, 10&minus, 2 mmol 19F/g of sample, were obtained. This is comparable with the results obtained for molecules, which exhibit more preferable MR characteristics.

The phytochemical diversity of Melittis melissophyllum was investigated in terms of seasonal changes and age of plants including plant organs diversity. The content of phenolics, namely: coumarin, 3,4-dihydroxycoumarin, o-coumaric acid 2-O-glucoside, verbascoside, apiin, luteolin-7-O-glucoside, and o-coumaric, p-coumaric, chlorogenic, caffeic, ferulic, cichoric acids, was determined using HPLC-DAD. Among these, luteolin-7-O-glucoside, verbascoside, chlorogenic acid, and coumarin were the dominants. The highest content of flavonoids and phenolic acids was observed in 2-year-old plants, while coumarin in 4-year-old plants (272.06 mg 100 g&ndash, 1 DW). When considering seasonal changes, the highest content of luteolin-7-O-glucoside was observed at the full flowering, whereas verbascoside and chlorogenic acid were observed at the seed-setting stage. Among plant organs, the content of coumarin and phenolic acids was the highest in leaves, whereas verbascoside and luteolin-7-O-glucoside were observed in flowers. The composition of essential oil was determined using GC-MS/GC-FID. In the essential oil from leaves, the dominant was 1-octen-3-ol, whilst from flowers, the dominant was &alpha, pinene.

A collection of twelve organoselenium compounds, structural analogues of antioxidant drug ebselen were screened for inhibition of the papain-like protease (PLpro) from the acute respiratory syndrome coronavirus 2 (SARS-CoV-2, CoV2). This cysteine protease, being responsible for the hydrolysis of peptide bonds between specific amino acids, plays a critical role in CoV2 replication and in assembly of new viral particles within human cells. The activity of the PLpro CoV2 is essential for the progression of coronavirus disease 2019 (COVID-19) and it constitutes a key target for the development of anti-COVID-19 drugs. Here, we identified four strong inhibitors that bind favorably to the PLpro CoV2 with the IC50 in the nanomolar range.

Since December 2019 a novel a coronavirus identified as SARS-CoV-2 or COV2 has been spreading around the world. On the 16th of May around 4.5 million people got infected and over 300,000 died due to the infection of COV2. The effective treatment remains a challenge. Targeted therapeutics are still under investigation. The papain-like protease (PLPro) from the human SARS-CoV-2 coronavirus is a cysteine protease that plays a critical role in virus replication. Its activity is required to process the viral polyprotein into functional, mature subunits. Moreover, COV2 uses this enzyme to modulate the host’s immune system to its own benefit. Therefore, it represents a highly promising target for the development of antiviral drugs.In this work, we discovered that ebselen, a synthetic organoselenium drug molecule with anti-inflammatory, anti-oxidant and cytoprotective activity in mammalian cells and cytotoxicity in lower organisms, is a highly active inhibitor of PLProCoV2. We proved that ebselen is a covalent, fast-binding inhibitor of PLProCoV2 exhibiting a low micromolar potency. Furthermore, we identified a difference between PLPro from SARS-CoV-1 (the corona virus which caused the 2002–2004 outbreak, SARS) and SARS-CoV-2 that allows to explain the difference in dynamics of the replication, and, thus, the disease progression. Namely, we present that they show differences in the binding affinity of substrates that we observed through kinetics and molecular docking studies. Using a novel Approximate Bayesian Computation method we were able to find kinetic constants for both enzymes. Molecular modeling study on the structure of the active site and binding mode of the ebselen with SARS and COV2 showed also significant differences that could explain our observation that ebselen is less active and slower bounding with SARS than COV2.In conclusion, we show that ebselen inhibits the activity of the essential viral enzyme papain-like protease (PLpro) from SARS-COV-2 in low micromolar range.

Spinal cord injury (SCI) causes disturbances of motor skills. Free radicals have been shown to be essential for the development of spinal cord trauma. Despite some progress, until now no effective pharmacological therapies against SCI have been verified. The purpose of our experiment was to investigate the neuroprotective effects of ebselen on experimental SCI. Twenty-two rats subjected to SCI were randomly subjected to SCI with no further treatment (n = 10) or intragastric administration of ebselen (10 mg/kg) immediately and 24 hours after SCI. Behavioral changes were assessed using the Basso, Beattie, and Bresnahan locomotor scale and footprint test during 12 weeks after SCI. Histopathological and immunohistochemical analyses of spinal cords and brains were performed at 12 weeks after SCI. Magnetic resonance imaging analysis of spinal cords was also performed at 12 weeks after SCI. Rats treated with ebselen presented only limited neurobehavioral progress as well as reduced spinal cord injuries compared with the control group, namely length of lesions (cysts/scars) visualized histopathologically in the spinal cord sections was less but cavity area was very similar. The same pattern was found in T2-weighted magnetic resonance images (cavities) and diffusion-weighted images (scars). The number of FluoroGold retrogradely labeled neurons in brain stem and motor cortex was several-fold higher in ebselen-treated rats than in the control group. The findings suggest that ebselen has only limited neuroprotective effects on injured spinal cord. All exprimental procedures were approved by the Local Animal Ethics Committee for Experiments on Animals in Katowice (Katowice, Poland) (approval No. 19/2009).

Purpose Although anorexia nervosa is classified as a psychiatric disorder associated with socio-environmental and psychological factors, a deeper insight into the dominant neurobiological basis is needed to develop a more effective approach of treatment. Given the high contribution of genetic predisposition and the underlying pathophysiology of neurohormonal circuits, it seems that pharmacological targeting of these mechanisms may provide us with better therapeutic outcomes. Methods 1 H-NMR spectroscopy was used to measure concentrations of the hypothalamus and brain stem metabolites in an activity-based rodent model (ABA) after subcutaneous administration of kisspeptin-10. Because anorexia mainly affects young women and often leads to hypogonadotropic-hypogonadism, we investigated the influence of this neuropeptide, which is involved in reproductive function by regulating the hypothalamic-pituitary-gonadal axis, on the ABA model development. Results Kisspeptin reinforced food consumption in an activity-based rodent model of anorexia changing a pattern of weight loss. 1 H-NMR spectroscopy of the hypothalamus and brain stem of ABA rats revealed a statistically significant change in the concentration of creatine (Cr; decreased, P = 0.030), phosphocreatine (PCr; increased, P = 0.030), γ-aminobutyric acid (GABA; decreased, P = 0.011), glutathione (GSH; increased, P = 0.011) and inositol (INS; increased, P = 0.047) compared to the control group. Subcutaneous administration of kisspeptin reversed the decrease in GABA (P = 0.018) and Cr (P = 0.030) levels in the hypothalamus as well as restored glutamate (GLU; P = 0.040) level in the brain stem. Conclusions We suspect that kisspeptin through modulation of hypothalamic GABAergic signaling increases food intake, and thus positively alters brain metabolism.

The potent transcription inhibitor Actinomycin D is used with several cancers. Here, we report the discovery that this naturally occurring antibiotic inhibits two human neutral aminopeptidases, the cell-surface alanine aminopeptidase and intracellular methionine aminopeptidase type 2. These metallo-containing exopeptidases participate in tumor cell expansion and motility and are targets for anticancer therapies. We show that the peptide portions of Actinomycin D and Actinomycin X2 are not required for effective inhibition, but the loss of these regions changes the mechanism of interaction. Two structurally less complex Actinomycin D analogs containing the phenoxazone chromophores, Questiomycin A and Actinocin, appear to be competitive inhibitors of both aminopeptidases, with potencies similar to the non-competitive macrocyclic parent compound (Ki in the micromolar range). The mode of action for all four compounds and both enzymes was demonstrated by molecular modeling and docking in the corresponding active sites. This knowledge gives new perspectives to Actinomycin D’s action on tumors and suggests new avenues and molecules for medical applications.

The term “theranostic” has been designated for a material that combines the modalities of therapy and diagnostics. For example, theranostic nanoparticles deliver therapeutic drugs and diagnostic imaging agents at the same time within one dose. The aim of our work was to develop the method of formation of polyelectrolyte nanocapsules containing iron oxide nanoparticles as MRI visible drug delivery system. The nanocapsules were prepared by encapsulation of nanoemulsion droplets in hybrid shell formed by the layer-by-layer deposition of biocompatible polyelectrolytes, poly- l -glutamic acid (PGA), poly- l -lysine (PLL) and Fe2O3 nanoparticles. The outer layer of nanocapsules was tailored for biomedical applications by the adsorption of pegylated polyelectrolyte (PGA-g-PEG). The average size of synthesized nanocapsules was around 150 nm and they were stable in cell culture medium containing serum. Using T2 and T1 NMR relaxation measurements as well as relaxation time weighted images with 9.4T preclinical MRI scanner we demonstrated that hybrid nanocapsules can be detected due to a locally altered contrast in the MR image. Thus, they may become a promising platform for future theranostic applications.

In the presented work 2 at% thulium doped yttrium oxide precursor powders were synthesized by the co-precipitation method using ammonium bicarbonate as a precipitation agent. The main aim of this work was to investigate the influence of co-precipitation parameters such as the process temperature (25°C–70 °C) and molar concentration of reagents on the morphology and luminescence properties of Tm:Y 2 O 3 . The lowest average particle size of about 20 nm and the highest value of the specific surface of 55.2 m 2 /g were obtained for powder co-precipitated at a temperature of 70 °C at a molar concentration of 0.1 M for thulium and yttrium nitrate. The emission spectra of the outcome powders were measured and compared and fluorescence dynamics behavior was presented and discussed.

Pressure ulcers belong to the most chalenging clinical problems. As hydration level of such wounds is important for optimal healing, preparation of new wound dressing (WD) materials for pressure ulcers requires thorough in vitro evaluation as prerequisite to final in vivo testing. The aims of the study were to: (a) develop a simple method of preparation of asymmetric polymeric membrane, (b) to propose a set of in vitro methods for membrane characterization during hydration. A polyvinyl alcohol asymmetric membrane with homogeneous skin layer and porous spongy layer was developed with nonadhesive properties and ability to absorb and retain the water. Complementary methods, including magnetic resonance imaging, allowed quantitative assessment of spatiotemporal aspects of membrane hydration, that is, global water uptake; swelling; local hydration in terms of proton density mapping; spatial distribution of T2 relaxation time; Young's modulus; piercing resistance. The proposed method of initial wound dressing evaluation seems to be promising to compare various WD formulations, to assess the time required to prepare WD membrane to be applied to the wound and to assess how long WD retains desired working properties. The developed asymmetric membrane seems to be a good candidate for further evaluation. It was found that: Young's modulus of hydrated membrane was comparable to those of human skin; asymmetrical structure was retained during the entire hydration period; each layer had its own distinct, hydration related, properties and their spatiotemporal evolution; relatively slow changes of membrane properties during the potential WD application time-span of several hours was observed. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 843-853, 2018.

The Michaelis-Menten equation is one of the most extensively used models in biochemistry for studying enzyme kinetics. However, this model requires at least a couple (e.g., eight or more) of measurements at different substrate concentrations to determine kinetic parameters. Here, we report the discovery of a novel tool for calculating kinetic constants in the Michaelis-Menten equation from only a single enzymatic assay. As a consequence, our method leads to reduced costs and time, primarily by lowering the amount of enzymes, since their isolation, storage and usage can be challenging when conducting research.

Colorectal cancer ranks 3rd in terms of cancer incidence. Growth and development of colon cancer cells may be affected by juice and extracts from Saposhnikovia divaricata root. The objective of the research was to analyze the effect of S. divaricata juice and extracts on the viability, membrane integrity and types of cell death of Caco-2 cells. Juice and extracts were analyzed using Ultra-High Performance Liquid Chromatography-Mass Spectrometry (UHPLC-MS) and in respect of the presence of antioxidants, total carbohydrates, protein, fat and polyphenols. The contents of cimifugin &beta, D-glucopyranoside, cimifugin, 4&prime, O-glucopyranosyl-5-O-methylvisamminol, imperatorin and protein were the highest in juice. 50% Hydroethanolic extract had the greatest antioxidant potential, concentration of polyphenols and fat. Water extract was characterized by the highest content of glutathione. Juice and 75% hydroethanolic extract contained the most carbohydrates. After the application of juice, 50% extract and the juice fraction containing the molecules with molecular weights &gt, 50 kDa, a decrease of the cell viability was noted. Juice and this extract exhibited the protective properties in relation to the cell membranes and they induced apoptosis. The knowledge of further mechanisms of anticancer activity of the examined products will allow to consider their use as part of combination therapy.

Here, we designed a novel Gadolinium (Gd) labeled drug-loaded polyelectrolyte nanocarriers for theranostics. The nanocarriers were formed via layer-by-layer technique with biodegradable polyelectrolytes: PLL (Poly-L-lysine), PLL-Gd (Gadolinium-labeled Poly-L-lysine) and PGA (Poly-L-glutamic acid). Anticancer drug (Paclitaxel) was encapsulated in the formed nanocarriers. The average size of synthesized nanocarriers was around 150 nm. The empty gadolinium labeled nanocarriers did not show any deleterious effects on tested cells (CT26-CEA, B16F10, 4T1 and PBMC), whereas encapsulated paclitaxel retained its cytotoxic/cytostatic activity. Using T2 and T1 NMR relaxation measurements with 9.4 T preclinical MRI scanner, we demonstrated that gadolinium labeled nanocarriers can be detected due to a locally altered contrast in the MR image. Thus, they may become a promising platform for future theranostic applications.

Peptide foldamers containing both cis-β-aminocyclopentanecarboxylic acid and α-amino acid residues combined in various sequence patterns (ααβ, αααβ, αβααβ, and ααβαααβ) were screened using CD and NMR spectroscopy for the tendency to form helices. ααβ-Peptides were found to fold into an unprecedented and well-defined 16/17/15/18/14/17-helix. By extending the length of the sequence or shifting a fragment of the sequence from one terminus to another in ααβ-peptides, the balance between left-handed and right-handed helix populations present in the solution can be controlled. Engineering of the peptide sequence could lead to compounds with either a strong propensity for the selected helix sense or a mixture of helical conformations of opposite senses.

A collection of twenty-six organoselenium compounds, ebselen and its structural analogues, provided a novel approach for inhibiting the activity of human methionine aminopeptidase 2 (MetAP2). This metalloprotease, being responsible for the removal of the amino-terminal methionine from newly synthesized proteins, plays a key role in angiogenesis, which is essential for the progression of diseases, including solid tumor cancers. In this work, we discovered that ebselen, a synthetic organoselenium drug molecule with anti-inflammatory, anti-oxidant and cytoprotective activity, inhibits one of the main enzymes in the tumor progression pathway. Using three-step synthesis, we obtained twenty-five ebselen derivatives/analogues, ten of which are new, and tested their inhibitory activity toward three neutral aminopeptidases (MetAP2, alanine and leucine aminopeptidases). All of the tested compounds proved to be selective, slow-binding inhibitors of MetAP2. Similarly to ebselen, most of its analogues exhibited a moderate potency (IC50=1-12μM). Moreover, we identified three strong inhibitors that bind favorably to the enzyme with the half maximal inhibitory concentration in the submicromolar range.

Zero echo time (ZTE) imaging at 9.4T was used to assess local water saturation level in the tight sandstone rocks. The results were compared with the industry standard porosity estimation basing on T2 relaxation analysis at 0.05T. A linear dependence between the two was achieved. This suggests the possibility to use 3D ZTE method for assessment of local amount of water in rocks. The method can be applicable in investigation of water saturation processes in tight rocks, where imaging methods based on spin echo like RARE failed due to short T2, while single point imaging (SPI) is impractical due to long acquisition time.

Greek oregano and common oregano were compared in respect of the antioxidant and antibacterial activity of the corresponding essential oils and hydroethanolic extracts in relation with their chemical profile. The chemical composition of essential oils was determined by GC-MS and GC-FID, while extracts (phenolic acids and flavonoids fractions) were analyzed by HPLC-DAD. Based on given volatiles, the investigated subspecies represented two chemotypes: a carvacrol/γ-terpinene/p-cymene type in the case of Greek oregano and a sabinyl/cymyl type rich in terpinen-4-ol in common oregano. Within non-volatile phenolic compounds, rosmarinic acid appeared to dominate in both subspecies. Lithospermic acid B, chlorogenic acid and isovitexin were present only in Greek oregano extracts. However, the total content of flavonoids was higher in common oregano extracts. The essential oil and extract of Greek oregano revealed visibly stronger antibacterial activity (expressed as MIC and MBC) than common oregano, whereas the antioxidant potential (determined by DPPH, ABTS and FRAP) of these extracts was almost equal for both subspecies. In the case of Origanum plants, the potential application of essential oils and extracts as antiseptic and antioxidant agents in the food industry should be preceded by subspecies identification followed by recognition of their chemotype concerning both terpene and phenolics composition.

Common thyme is regarded as one of the most important culinary plants. The purpose of the work was to determine the intraspecific variability of common thyme with respect to morphological and chemical characters including the content and composition of essential oil and phenolic compounds in the herb. The objects of the study were 12 clones, vegetatively multiplied from randomly selected individual plants of cultivar &lsquo, Standard Winter&rsquo, The morphological observations and harvest of raw materials were carried out in the first year plants&rsquo, vegetation. The highest differences between clones were on fresh and dry weight of herb (CV = 0.38 and 0.36, respectively), width of leaves (CV = 0.21), and density of glandular trichomes on the abaxial surface of leaves (CV = 0.29). Examined clones were also differentiated as to the chemical features. Essential oil content (performed by hydrodestillation) and composition (by GC-MS and GC-FID) were determined and they ranged from 2.10 to 4.38 g &times, 100 g&minus, 1 DW. Here, thymol, &gamma, terpinen, and p-cymen were the dominant compounds. Clone no 4 was distinctive as to the highest content of essential oil followed by the highest share of thymol (54.59%). The total content of phenolic acids and flavonoids (determined according to PPh 6th) also differed among clones (CV = 0.38 and 0.36, respectively). Using a validated HPLC-DAD method, the following compounds were identified: caffeic, rosmarinic, p-coumaric acids, luteolin 7-O-glucoside, naryngenin, and (-)-epicatechin. Here, rosmarinic acid followed by luteolin 7-O-glucoside were present in the highest amounts (611.47&ndash, 2675.59 and 46.77&ndash, 325.11 mg &times, 1 DW, respectively). The highest differences between clones were the contents of p-coumaric acid (CV = 0.59), luteolin 7-O-glucoside (CV = 0.50) and rosmarinic acid (CV = 0.40). Such a high range of variability can provide problems with raw material standardization. Nevertheless, it opens possibilities for breeders, whereas individual plants/clones may become valuable components for breeding.

The aim of the study was to assess the effects of Melittis melissophyllum shading on its development and accumulation of phenolics. Their content (verbascoside, apiin, luteolin-7-O-glucoside, coumarin, 3,4-dihydroxycoumarin, o-coumaric acid 2-O-glucoside as well as o-coumaric, p-coumaric, chlorogenic, caffeic, ferulic and cichoric acid) was determined in the herb using HPLC-DAD. The results showed that the content of abovementioned flavonoids and phenolic acids was highest in plants grown under full sunlight. On the other hand, a higher content of coumarin was observed in shaded plants, especially after the seed-setting stage. A similar tendency was noted for the amount of chlorophyll a and b. The content of hydrogen peroxide and malondialdehyde, the activity of polyphenol oxidase and catalase and the antioxidant capacity of plant extracts (measured using DPPH, ABTS and FRAP assays) were found to be the highest in the plants grown in full sunlight. However, the plants grown in moderate (30%) shade were found to thrive best.

Purpose The purpose of the study was initial evaluation of applicability of metal organic framework (MOF) Fe-MIL-101-NH2 as a theranostic carrier of antituberculous drug in terms of its functionality, i.e. drug loading, drug dissolution, magnetic resonance imaging (MRI) contrast and cytotoxic safety. Methods Fe-MIL-101-NH2 was characterized using X-ray powder diffraction, FTIR spectrometry and scanning electron microscopy. The particle size analysis was determined using laser diffraction. Magnetic resonance relaxometry and MRI were carried out on phantoms of the MOF system suspended in polymer solution. Drug dissolution studies were conducted using Franz cells. For MOF cytotoxicity, commercially available fibroblasts L929 were cultured in Eagle’s Minimum Essential Medium supplemented with 10% fetal bovine serum. Results MOF particles were loaded with 12% of isoniazid. The particle size (3.37–6.45 μm) depended on the micronization method used. The proposed drug delivery system can also serve as the MRI contrast agent. The drug dissolution showed extended release of isoniazid. MOF particles accumulated in the L929 fibroblast cytoplasmic area, suggesting MOF release the drug inside the cells. The cytotoxicity confirmed safety of MOF system. Conclusions The application of MOF for extended release inhalable system proposes the novel strategy for delivery of standard antimycobacterial agents combined with monitoring of their distribution within the lung tissue.

De novo designed helix-loop-helix peptide foldamers containing cis-2-aminocyclopentanecarboxylic acid residues were evaluated for their conformational stability and possible use in enzyme mimetic development. The correlation between hydrogen bond network size and conformational stability was demonstrated through CD and NMR spectroscopies. Molecules incorporating a Cys/His/Glu triad exhibited enzyme-like hydrolytic activity.

The aim of the study was to investigate the chemical variability of small-leaved lime ( Tilia cordata Mill.) growing wild in the eastern area of Poland, in terms of content and composition of phenolic compounds (phenolic acids and flavonoids), composition of essential oil and content of mucilage in flowers. Analyzed populations differed significantly when regards the accumulation of biologically active compounds. The total content of phenolic acids ranged from 0.17 to 1.87 g × 100 g −1 , flavonoids from 0.09 to 0.52 g × 100 g −1 , and mucilage from 0.6 to 3.0 g × 100 g −1 . Using validated HPLC-DAD method, the chemical composition of phenolic acids and flavonoids were determined. Among phenolic acids, four compounds were identified with vanillic acid as the dominant one (391.5–1718.0 mg × 100 g −1 ). Among flavonoids, nine compounds were found. Rutoside and (−)-epicatechin (tannin precursor) were present in the highest amounts (1264.1–4654.6 mg × 100 g −1 and 1000.3–4745.6 mg × 100 g −1 , respectively). GC–MS and GC-FID were used to perform the qualitative and quantitative analyze of the essential oil. The major compounds were alkanes: tricosane (5.7–20.7%), pentacosane (3.0–6.1%) and heneicosane (1.1–6.1%), as well as acetophenon (3.5–6.9%), nonanal (1.3–7.8%) and nonanoic acid (3.9–18.2%).

This paper presents the results of the application of freeze granulation to the production of transparent neodymium-doped yttrium–aluminum garnet ceramics. Aqueous suspensions of aluminium oxide, yttrium oxide and neodymium oxide powders were prepared based on nanometric or submicronic powders which were either commercially available or prepared by precipitation. The relations between the composition of suspension, the properties of granulate and the final properties of ceramics (microstructure, optical transmission and emission spectra) were studied.

The aim of this work has been to obtain transparent Tm, Ho:YAG ceramics (thulium doping range: 2–6 at.%, holmium doping range: 0.1–1.0 at.%) by reaction sintering using commercial powders. It has been proved that the particle size, purity and degree of agglomeration of the powders used are crucial from the point of view of the optical quality of ceramics. The spectroscopic measurements of Tm, Ho:YAG ceramics with different concentration of active ions (including transmission and emission spectra measurements) have been presented and discussed. As has been found, both concentration of holmium and thulium separately as well as the balance between them are of great importance. Energy transfer between Tm and Ho ions has been demonstrated.

The aim of this work was to investigate the influence of precipitation parameters on the morphology of obtained thulium oxide powders. Tm2O3 precursor powders were synthesized by precipitation method using 0.1–0.25M water solutions of thulium nitrate and 1.5M ammonium hydrogen carbonate water solution as a precipitation agent. The processes were conducted at different temperatures (25–50°C). The result showed that the morphology of the obtained thulium oxide (Tm2O3) powders depends both on the molar concentration of thulium nitrate and the temperature of precipitation. Small, round, loosely agglomerated Tm2O3 nanoparticles were obtained after air calcination of precursor precipitated at room temperature with the use of 0.1M thulium nitrate solution.

The objective of this study was to investigate the composition of polyphenolic compounds of water and ethanol extracts from leather bergenia leaves (LBL) and to determine the antimicrobial properties of pullulan coating enriched with these extracts. The result showed that both extracts consisted mainly of flavonols and phenolic acids. The antimicrobial activity of pullulan coating solution was determined at different concentrations: 0.4, 1, 2, 5, 10, 20% (w/v) of extracts. The incorporation of LBL extracts showed antimicrobial activities of the films against both Gram-positive and Gram-negative bacteria and molds. Pullulan films incorporated with 20% of LBL extracts showed the strongest antimicrobial activity. In the next part of the study pullulan coating solution with a 20% concentration of water LBL extract was chosen and used as coating on pepper and apple surfaces. Non-coated samples of food were also used as controls. Samples coated with the antimicrobial solutions showed advantages in reducing microbial growth by 1 log CFU/g as compared to the control samples. Physico-chemical and organoleptic observations were also evaluated after storage. The application of the pullulan film enabled an extension of the storage time of the peppers and apples as a result of reduced weight losses and helped to maintain their hardness.

α,β-Diaminoethylphosphonic acids are efficient transition-state analogue inhibitors of alanyl aminopeptidase and endoplasmic reticulum aminopeptidase 2, biomedically important members of the M01 family of proteases. In this work, the individual enantiomers of selected inhibitors were separated and their inhibitory potency verified. The synthesis involved the use of a chiral α-methylbenzylamine to obtain diastereomeric aziridinephosphonates as key intermediates of the multistep process. The diastereomeric aziridines were resolved chromatographically, and the heterocyclic ring opened with the appropriate amines. The inhibition study revealed an interesting correlation between the absolute configuration and inhibitory activity. Certain enantiomeric phosphonic amino acid analogues exhibited inhibition constants in the nanomolar range.