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Free radical formation in two diuretics: indapamide and torasemide was examined during UV irradiation and storage at higher temperatures using X-band (9.3 GHz) electron paramagnetic resonance spectroscopy (EPR). The aim of this study was to investigate the possibility of storing indapamide and torasemide under UV irradiation and at higher temperatures, which may occur during exposure to light. The diuretic samples were exposed to UVA irradiation for 15, 30 and 45 minutes, and stored at temperatures of 40 ° C and 50 ° C by 30 minutes. The EPR spectra were analyzed to determine the amplitudes (A), linewidths (ΔB pp ), and integral intensities (I) and g factors. The concentrations of free radical (N) in the diuretic samples were also determined. The influence of microwave power on amplitudes, linewidths and the asymmetry parameter were evaluated. The result showed that the tested indapamide and torasemide samples exhibited high free radical concentrations in the range of 10 18 -10 19 spin/g after UV irradiation and heat treatment. Therefore, due to the significant free radical formation indapamide and torasemide should not be stored under UV light and at temperatures of 40 ° C and 50 ° C. The complex character of free radical systems in the diuretic samples was proved as evidenced by the changes of the asymmetry parameters of the EPR lines with increasing microwave power. Fast spin-lattice relaxation processes were observed in all tested diuretic samples, regardless of the storage conditions. Electron paramagnetic resonance spectroscopy is proposed as a useful method in pharmacy to determine the appropriate storage conditions for diuretics., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Prostate cancer is a common cancer in men in older age groups. The WHO forecasts an increase in the incidence of prostate cancer in the coming years. Patients may not respond to treatment, and may not tolerate the side effects of chemotherapy. Compounds of natural origin have long been used in the prevention and treatment of cancer. Flavonoids obtained from natural products, e.g., propolis, are compounds with proven antibacterial and antiviral efficacy which modulate the immune response and may be useful as adjuvants in chemotherapy. The main aim of the present study was to evaluate the cytotoxic and pro-apoptotic properties of selected flavonoids on prostate cancer cells of the LNCaP line. The compounds used in this study were CAPE, curcumin (CUR), and quercetin (QUE). Mitochondrial and lysosome metabolism was assessed by the XTT-NR-SRB triple assay as well as by the fluorescent staining techniques. Staining for reactive oxygen species was performed as well. The experiment showed that each of the tested compounds has a cytotoxic effect on the LNCaP cell line. Different types of cell death were induced by the tested compounds. Apoptosis was induced by quercetin, while autophagy-specific changes were observed after using CAPE. Compounds obtained from other bee products have antiproliferative and cytotoxic activity against LNCaP prostate cancer cells.

The current review focuses on the effect of phenothiazine derivatives, tested in vitro, on necrosis and necroptosis, the latter constitutes one of the kinds of programmed cell death. Necroptosis is a necrotic and inflammatory type of programmed cell death. Phenothiazines are D1 and D2-like family receptor antagonists, which are used in the treatment of schizophrenia. Necroptosis begins from TNF-α, whose synthesis is stimulated by dopamine receptors, thus it can be concluded that phenothiazine derivatives may modulate necroptosis. We identified 19 papers reporting in vitro assays of necroptosis and necrosis in which phenothiazine derivatives, and both normal and cancer cell lines were used. Chlorpromazine, fluphenazine, levomepromazine, perphenazine, promethazine, thioridazine, trifluoperazine, and novel derivatives can modulate necroptosis and necrosis. The type of a drug, concentration and a cell line have an impact on the ultimate effect. Unfortunately, the authors confirmed both processes on the basis of TNF-α and ATP levels as well as the final steps of necrosis/necroptosis related to membrane permeability (PI staining, LDH release, and HMGB1 amount), which makes it impossible to understand the complete mechanism of phenothiazines impact on necroptosis and necrosis. Studies analyzing the effect of phenothiazines on RIPK1, RIPK3, or MLKL has not been performed yet. Only the analysis of the expression of those proteins as well as necrosis and necroptosis inhibitors can help us to comprehend how phenothiazine derivatives act, and how to improve their therapeutic potential., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Michal Otreba reports financial support was provided by Medical University of Silesia. Anna Rzepecka-Stojko reports financial support was provided by Medical University of Silesia. Conflict of interest The authors declare that they have no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

In this review, we summarized the current literature on the impact of phenothiazine derivatives on autophagy in vitro. Phenothiazines are antipsychotic drugs used in the treatment of schizophrenia, which is related to altered neurotransmission and dysregulation of neuronal autophagy. Thus, phenothiazine derivatives can impact autophagy. We identified 35 papers, where the use of the phenothiazines in the in vitro autophagy assays on normal and cancer cell lines, Caenorhabditis elegans, and zebrafish were discussed. Chlorpromazine, fluphenazine, mepazine, methotrimeprazine, perphenazine, prochlorperazine, promethazine, thioridazine, trifluoperazine, and novel derivatives can modulate autophagy. Stimulation of autophagy by phenothiazines may be either mammalian target of rapamycin (mTOR)-dependent or mTOR-independent. The final effect depends on the used concentration as well as the cell line. A further investigation of the mechanisms of autophagy regulation by phenothiazine derivatives is required to understand the biological actions and to increase the therapeutic potential of this class of drugs., (© 2022 John Wiley & Sons Ltd.)

Glioblastoma multiforme is the most frequent type of malignant brain tumor, and is one of the most lethal and untreatable human tumors with a very poor survival rate. Therefore, novel and effective strategies of treatment are required. Integrins play a crucial role in the regulation of cellular adhesion and invasion. Integrins and α-tubulin are very important in cell migration, whereas E-cadherin plays a main role in tumor metastasis. Notably, drugs serve a crucial role in glioblastoma treatment; however, they have to penetrate the blood-brain barrier (BBB) to be effective. ABC transporters, including ATP binding cassette subfamily B member 1 (ABCB1) and ATP binding cassette subfamily G member 2 (ABCG2), are localized in the brain endothelial capillaries of the BBB, have a crucial role in the development of multidrug resistance and are modulated by phenothiazine derivatives. The impact of perphenazine and prochlorperazine on the motility of human Uppsala 87 malignant glioma (U87-MG) cells was evaluated using a wound-healing assay, cellular migration and invasion were assessed by Transwell assay, and the protein expression levels of ABCB1, ABCG2, E-cadherin, α-tubulin and integrins were determined by western blotting. The present study explored the effects of perphenazine and prochlorperazine on the levels of ABCB1, ABCG2, E-cadherin, α-tubulin and integrins (α3, α5, and β1), as well as on the migratory and invasive ability of U87-MG cells. The results suggested that perphenazine and prochlorperazine may modulate the expression levels of multidrug resistance proteins (they decreased ABCB1 and increased ABCG2 expression), E-cadherin, α-tubulin and integrins, and could impair the migration and invasion of U-87 MG cells. In conclusion, the decrease in migratory and invasive ability following treatment with phenothiazine derivatives due to the increase in ABCG2 and E-cadherin expression, and decrease in α-tubulin and integrins expression, may suggest that research on perphenazine and prochlorperazine in the treatment of glioblastoma is worth continuing., Competing Interests: The authors declare that they have no competing interests., (Copyright: © Otręba et al.)

Propolis composition depends on several factors. The classification of propolis is based on its geographical location, color and agricultural characteristics. It is also classified according to the flora where the bees collect the resins, which represent the raw material for propolis production. Propolis possesses high antioxidant activity determined by its phenolic compounds. Due to diverse composition and possible impact on human health, eight samples of propolis were evaluated for their phenolic composition and antioxidant activity. Samples of Polish, Romanian, Turkish and Uruguayan origin propolis were used for phenolic spectrum determination using high performance liquid chromatography and photodiode array detection and in vitro DPPH and ABTS methods were used to determine the antioxidant activity of the extracts. PCA and HCA models were applied to evaluate the correlation between isolated polyphenols and antioxidant activity. The results confirmed variability in propolis composition depending on the geographical region of collection and the plant sources, and correlation between chemical composition and antioxidant activity. Results of PCA and HCA analyses confirm that Polish propolis is similar to that from different provinces of Romania, while Turkish and Uruguay are completely different. Polish and Romanian propolis belong to the poplar type. The assessed phenolic compounds of propolis samples used in the study are responsible for its antioxidant effect. The observed antioxidant activity of the analyzed samples may suggest directing subsequent research on prophylactic and therapeutic properties concerning cardiovascular, metabolic, neurodegenerative, and cancerous diseases, which are worth continuing.

Melatonin exerts its effects through a series of target proteins/receptors and enzymes. Its antioxidant capacity might be due to its capacity to inhibit a quinone reductase (NQO2) at high concentration (50 μM). Demonstrating the existence of a complex between a compound and a protein is often not easy. It requires either that the compound is an inhibitor-and the complex translates by an inhibition of the catalytic activity-or the compound is radiolabeled-and the complex translates in standard binding approaches, such as in receptology. Outside these two cases, the detection of the protein:small molecule complexes by mass spectrometry has recently been made possible, thanks to the development of so-called native mass spectrometry. Using this approach, one can measure masses corresponding to an intact noncovalent complex between a compound and its target, usually after titration or competition experiments. In the present chapter, we detail the characterization of NQO2:melatonin interaction using native mass spectrometry., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Posttranslational modifications of proteins are catalyzed by a large family of enzymes catalyzing many chemical modifications. One can hijack the natural use of those enzymes to modify targeted proteins with synthetic chemical moieties. The lipoic acid ligase LplA mutants can be used to introduce onto the lysine sidechain lipoic acid moiety synthetic analogues. Substrate protein candidates of the ligase must obey a few a priori rules., Methods and Results: In the present report, we technically detailed the use of a cell line stably expressing both the ligase and a model protein (thioredoxin). Although the goal can be reach, and the protein visualized in situ, many experimental difficulties must be fixed. The sequence of events comprises (i) in cellulo labeling of the target protein with a N 3 -lipoic acid derivative catalyzed by the mutant ligase, (ii) the further introduction by click chemistry onto this lysine sidechain of a fluorophore and (iii) the following of the labeled protein in living cells. One of the main difficulties was to assess the click chemistry step onto the living cells, because images from both control and experimental cells were similar. Alternatively, we describe at that stage, the preferred use of another technique: the Halo-Tag one that led to the obtention of clear images of the targeted protein in its cellular context. Although the ligase-mediated labeling of protein in situ is a rich domain for which many cellular tools must be developed, many difficulties must be considered before entering a systematic use of this approach., Conclusions: In the present contribution, we added several steps of analytical characterization, both in vitro and in cellulo that were previously lacking. Furthermore, we show that the use of the click chemistry should be manipulated with care, as the claimed specificity might be not complete whenever living cells are used. Finally, we added another approach-the Halo Tag-to complete the previously suggested approaches for labelling proteins in cells, as we found difficult to strictly apply the previously reported methodology., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Oral diseases affect a very large number of people, and the applied pharmacological methods of treatment and/or prevention have serious side effects. Therefore, it is necessary to search for new, safer methods of treatment. Natural bee products, such as honey, royal jelly, and bee venom, can be a promising alternative in the treatment of oral cavity bacterial infections. Thus, we performed an extensive literature search to find and summarize all articles about the antibacterial activity of honey, royal jelly, and bee venom. Our analysis showed that these bee products have strong activity against the bacterial strains causing caries, periodontitis, gingivitis, pharyngitis, recurrent aphthous ulcers, supragingival, and subgingival plaque. An analysis of average MIC values showed that honey and royal jelly have the highest antimicrobial activity against Porphyromonas gingivalis and Fusobacterium nucleatum . In turn, bee venom has an antibacterial effect against Streptococcus mutans . Streptococcus sobrinus and Streptoccus pyogenes were the most resistant species to different types of honey, and royal jelly, respectively. Moreover, these products are safer in comparison to the chemical compounds used in the treatment of oral cavity bacterial infections. Since the antimicrobial activity of bee products depends on their chemical composition, more research is needed to standardize the composition of these compounds before they could be used in the treatment of oral cavity bacterial infections.

VHH stands for the variable regions of heavy chain only of camelid IgGs. The VHH family forms a set of interesting proteins derived from antibodies that maintain their capacity to recognize the antigen, despite their relatively small molecular weight (in the 12,000 Da range). Continuing our exploration of the possibilities of those molecules, we chose to design alternative molecules with maintained antigen recognition, but enhanced capacity, by fusing four VHH with one Fc, the fragment crystallizable region of antibodies. In doing so, we aimed at having a molecule with superior quantitative antigen recognition (×4) while maintaining its size below the 110 kDa. In the present paper, we described the building of those molecules that we coined VHH 2 -Fc-VHH 2 . The structure of VHH 2 -Fc-VHH 2 in complex with HER2 antigen was determined using electronic microscopy and modeling. The molecule is shown to bind four HER2 proteins at the end of its flexible arms. VHH 2 -Fc-VHH 2 also shows an internalization capacity via HER2 receptor superior to the reference anti-HER2 monoclonal antibody, Herceptin®, and to a simple fusion of two VHH with one Fc (VHH 2 -Fc). This new type of molecules, VHH 2 -Fc-VHH 2 , could be an interesting addition to the therapeutic arsenal with multiple applications, from diagnostic to therapy., (© 2021 The Protein Society.)

Polyphenols have recently gained popularity among the general public as products and diets classified as healthy and containing naturally occurring phenols. Many polyphenolic extracts are available on the market as dietary supplements, functional foods, or cosmetics, taking advantage of clients' desire to live a healthier and longer life. However, due to the difficulty of discovering the in vivo functions of polyphenols, most of the research focuses on in vitro studies. In this review, we focused on the cardioprotective activity of different polyphenols as possible candidates for use in cardiovascular disease therapy and for improving the quality of life of patients. Thus, the studies, which were mainly based on endothelial cells, aortic cells, and some in vivo studies, were analyzed. Based on the reviewed articles, polyphenols have a few points of action, including inhibition of acetylcholinesterase, decrease in reactive oxygen species production and endothelial tube formation, stimulation of acetylcholine-induced endothelium-derived mediator release, and others, which lead to their cardio- and/or vasoprotective effects on endothelial cells. The obtained results suggest positive effects of polyphenols, but more long-term in vivo studies demonstrating effects on mechanism of action, sensitivity, and specificity or efficacy are needed before legal health claims can be made.

The selection of dressing is crucial for the wound healing process. Traditional dressings protect against contamination and mechanical damage of an injured tissue. Alternatives for standard dressings are regenerating systems containing a polymer with an incorporated active compound. The aim of this research was to obtain a biodegradable wound dressing releasing propolis in a controlled manner throughout the healing process. Dressings were obtained by electrospinning a poly(lactide- co -glycolide) copolymer (PLGA) and propolis solution. The experiment consisted of in vitro drug release studies and in vivo macroscopic treatment evaluation. In in vitro studies released active compounds, the morphology of nonwovens, chemical composition changes of polymeric material during degradation process, weight loss and water absorption were determined. For in vivo research, four domestic pigs, were used. The 21-day experiment consisted of observation of healing third-degree burn wounds supplied with PLGA 85/15 nonwovens without active compound, with 5 wt % and 10 wt % of propolis, and wounds rinsed with NaCl. The in vitro experiment showed that controlling the molar ratio of lactidyl to glycolidyl units in the PLGA copolymer gives the opportunity to change the release profile of propolis from the nonwoven. The in vivo research showed that PLGA nonwovens with propolis may be a promising dressing material in the treatment of severe burn wounds.

Ovarian cancer has the worst prognosis among all gynecological cancers. Therefore, it seems reasonable to seek new drugs that may improve the effectiveness of treatment or mitigate the adverse effects of chemotherapy. Caffeic acid phenethyl ester (CAPE) has many beneficial biological properties. The aim of the study was to assess the anticancer properties of CAPE against serum ovarian carcinoma cells. The morphology of the cells was evaluated in H-E staining and in transmission electron microscopy. The cytotoxic and proapoptotic activity of CAPE was investigated by using the XTT-NR-SRB assay, qRT-PCR analysis of BAX/BCL2 expression, and by cytometric evaluation. CAPE causes constriction in OV7 cells, numerous granulomas were observed in the cytoplasm, the cell nuclei were pyknotic. Autophagosomal vacuoles could suggest the occurrence of aponecrosis. CAPE significantly decreased the lysosomal activity and the total synthesis of cellular proteins. CAPE exhibited, dose and time dependent, cytotoxic activity against OV7 serum ovarian cancer cells. In OV7 cells CAPE induced apoptosis via dysregulation of BAX/BCL2 balance, while activated proapoptotic BAX gene expression level was 10 times higher than BCL2.

The evolution of the paramagnetic center system in blood during the healing of skin burn wounds dressed with a biodegradable apitherapeutic nanofiber dressing was examined. The aim of this study was to determine the changes in paramagnetic centers in blood during the influence of apitherapeutic nanofiber dressings on the healing process. The blood samples were tested before burn infliction (day 0) and, respectively, on the 10 th and 21 st days of the experiment. Paramagnetic centers in the blood of the pig used as the model animal were examined with an X-band (9.3 GHz) electron paramagnetic resonance spectroscopy. The EPR spectra were measured with Bruker spectrometer at 230 K with a modulation frequency of 100 kHz. The EPR lines of the high spin Fe 3+ in methemoglobin, high spin Fe 3+ in transferrin, Cu 2+ in ceruloplasmin, and free radicals were observed in the multicomponent spectra of blood. For the application of the apitherapeutic nanofiber dressing, the amplitudes of the EPR signals of Fe 3+ in methemoglobin were similar up to 10 days. For the experiment with the apitherapeutic formulation, the heights of EPR signals of Fe 3+ in transferrin were lower after 10 days and 21 days of therapy, compared to day 0. For the application of the apitherapeutic formulation the signals of Cu 2+ in ceruloplasmin and free radicals, strongly decreased after 10 days of therapy, and after 21 days it increased to the initial values characteristic for day 0. The apitherapeutic formulation caused that after 21 days the EPR spectrum of Cu 2+ in ceruloplasmin and free radicals was considerably high. The apitherapeutic formulation interaction after 10 days and after 21 days of therapy resulted in the low EPR lines of Fe 3+ in methemoglobin. EPR spectra of blood may be useful for presentation of the changes in its paramagnetic centers during the healing process of the burn wounds., Competing Interests: The authors declare no conflict of interest., (Copyright © 2020 Pawel Olczyk et al.)

The present study assesses the in vitro and in vivo bioavailability of genistein derivatives, hydroxyalkyl- and glycosyl alkyl ethers (glycoconjugates). Studies were carried out using compounds that exhibit higher in vitro antiproliferative activity in comparison with the parent isoflavone. Based on in vitro experiments using the Parallel Artificial Membrane Permeability Assay (PAMPA) and the Caco-2 cell monolayer permeability model, we found that modification of the isoflavone structure by O-alkylation improved bioavailability in comparison to genistein. Additionally, the structure of the substituent and its position on genistein influenced the type of mechanism involved in the transport of compounds through biological membranes. The PAMPA assay showed that the structure of glycoconjugates had a significant influence on the passive transport of the genistein synthetic derivatives through a biological membrane. Preferentially the glycoconjugates containing O-glycosidic bond were transported and the transport rate decreased as the carbon linker increased. For glycoconjugates, determination of their transport and metabolism through the Caco-2 membrane was not possible due to interaction with the membrane surface, probably by the change of compound structure caused by contact with the cells or degradation in medium. The intestinal absorption and metabolism of genistein and three derivatives, Ram-3, Ram'-3 and Ram-C-4α (Fig. 1), were tested in vivo in rats. We found that in comparison to genistein, glycoconjugates were metabolized more slowly and to a lesser extent. As part of the in vivo research, we performed analysis of compound levels in plasma samples after enzymatic hydrolysis, but in the collected samples, analytes were not observed. We hypothesize that glycoconjugates compounds bind plasma proteins and were removed from the sample. In conclusion, we show that O-functionalization of the natural, biologically active isoflavone genistein can affect biological activity, bioavailability, and the rate of compound metabolism. The position of the substituent, the length of the linker and the structure of sugar moieties provides a tool for the optimization of the derivative's biological properties., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Honey, propolis, bee pollen, bee bread, royal jelly, beeswax and bee venom are natural products which have been used in medicine since ancient times. Nowadays, studies indicate that natural bee products can be used for skin treatment and care. Biological properties of these products are related to flavonoids they contain like: chrysin, apigenin, kaempferol, quercetin, galangin, pinocembrin or naringenin. Several pharmacological activities of phenolic acids and flavonoids, and also 10-hydroxy- trans -2-decenoic acid, which is present in royal jelly, have been reported. Royal jelly has multitude of pharmacological activities: antibiotic, antiinflammatory, antiallergenic, tonic and antiaging. Honey, propolis and pollen are used to heal burn wounds, and they possess numerous functional properties such as: antibacterial, anti-inflammatory, antioxidant, disinfectant, antifungal and antiviral. Beeswax is used for production of cosmetics and ointments in pharmacy. Due to a large number of biological activities, bee products could be considered as important ingredients in medicines and cosmetics applied to skin., Competing Interests: The authors declare no conflict of interest.

Among the biological approaches to therapeutics, are the cells, such as CAR-T cells engineered or not, the antibodies armed or not, and the smaller protein scaffolds that can be modified to render them specific of other proteins, à la façon of antibodies. For several years, we explored ways to substitute antibodies by nanobodies (also known as VHHs), the smallest recognizing part of camelids' heavy-chain antibodies: production of those small proteins in host microorganisms, minute analyses, characterization, and qualification of their affinity towards designed targets. Here, we present three standard VHHs described in the literature: anti-albumin, anti-EGF receptor and anti-HER2, a typical cancer cell surface -associated protein. Because they differ slightly in global structure, they are good models to assess our body of analytical methodologies. The VHHs were expressed in several bacteria strains in order to identify and overcome the bottlenecks to obtain homogeneous preparations of this protein. A large panel of biophysical tools, ranging from spectroscopy to mass spectrometry, was here combined to assess VHH structural features and the impact of the disulfide bond. The routes are now ready to move to more complex VHHs raised against specific targets in numerous areas including oncology., (Copyright © 2019. Published by Elsevier Inc.)

Mesenchymal stem cells (MSCs) are objects of interest in regenerative medicine. They are used for various therapies such as for the regeneration of bone, chondrocytes and other tissues. Adipose derived stem cells (ADSCs) inter alia are particularly easy to access, they are relatively abundant in fat tissue. ADSCs could be differentiated into many types of cells. To date, it has been proven that ADSCs only differentiate into mesodermal cell lineages. In this study, we present the differentiation of ADSCs into the corneal epithelium. Human ADSCs were placed in a co-culture with porcine limbal epithelial stem cells (LESCs). After 14 days of cultivation, total RNA was extracted for the analysis of the molecular markers (expression of genes of interest). The gene expression was assessed by real-time RT-qPCR. The expression of the surface molecular markers of ADSCs is modulated after co-culturing. We have observed the decrease in CD73, CD90 and CD105 mRNA expression, while the expression of mRNA coding for CK3 and CK12 mRNA was increased in ADSCs co-cultured with porcine limbal epithelial stem cells as compared to the control. We conclude that the co-culture of LESCs and ADSCs changed ADSCs' molecular markers gene expression indicating initiation of differentiation towards limbal cells., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

In the continuous exploration of the VHH chemistry, biochemistry and therapeutic future use, we investigated two different production strategies of this small antibody-like protein, using an anti-HER2 VHH as a model. The total chemical synthesis of the 125 amino-acid peptide was performed with reasonable yield, even if optimization will be necessary to upgrade this kind of production. In parallel, we expressed the same sequence in two different hosts: Escherichia coli and Pichia pastoris. Both productions were successful and led to a fair amount of VHHs. The integrity and conformation of the VHH were characterized by complementary mass spectrometry approaches, while surface plasmon resonance experiments were used to assess the VHH recognition capacity and affinity toward its "antigen." Using this combination of orthogonal techniques, it was possible to show that the three VHHs-whether synthetic or recombinant ones-were properly and similarly folded and recognized the "antigen" HER2 with similar affinities, in the nanomolar range. This opens a route toward further exploration of modified VHH with unnatural amino acids and subsequently, VHH-drug conjugates., (© 2019 The Protein Society.)

The significant increase in the number of new psychoactive substances on the drug market has recently been a serious problem. The manuscript presents a fatal case of suicide poisoning with 3-MMC (3-methylmethcathinone). The biological material collected during the autopsy of a 19-year-old woman, transferred to the toxicological Laboratory in Katowice ToxLab, was subjected to a chemical and toxicological analysis. The toxicological analysis of blood, vitreous humor and gastric contents revealed 3-methylmetcatinone at a concentration of 800 ng/ml, 153 ng/ml and 5,5 mg, respectively. The presence of 3-MMC has also been confirmed in physical evidence secured on site. 3-methylmethcathinone is a dangerous psychoactive substance that caused the death of the 19-year-old., (Copyright © 2019. Published by Elsevier B.V.)

The multicomponent electron paramagnetic resonance spectra of the blood during healing of skin burned wounds treated with a new generation biodegradable dressings containing poly(lactide-co-glycolide) were analysed. The evolution of different types of paramagnetic centers in the blood with time of healing was determined. The EPR spectra of the blood samples at 230 K temperature were measured at 1, 10, and 21 days after burning of the pig skin. The EPR lines of the following paramagnetic centers: the high-spin Fe 3+ in methemoglobin (line I), high-spin Fe 3+ in transferrin (line II), and Cu 2+ in ceruloplasmin and free radicals (line III) were observed in the X-band (9.3 GHz) spectra of the blood. The multicomponent structure of the EPR spectra of the tested blood samples depended on the time of the healing of the burned wounds. The amount of the high-spin Fe 3+ in methemoglobin (line I) in the blood decreased after 21 days of the healing of the burned wounds. The amount of the high-spin Fe 3+ in transferrin (line II) slightly increased after 21 days of therapy with the basis. The amount of Cu 2+ in ceruloplasmin and free radicals (line III) in the blood was very high after 10 days of therapy. At the first day of the healing of the burned wounds, the highest amount of the high-spin Fe 3+ in methemoglobin (line I), the relatively lower amounts of the high-spin Fe 3+ in transferrin (line II), and Cu 2+ in ceruloplasmin and free radicals (line III) existed in the blood. In the medium phase (after 10 days) of the healing of the burned wounds, the extremely higher amounts of Cu 2+ in ceruloplasmin and free radicals (line III) appeared in the blood. In the last phase (after 21 days), only the low differences between the amounts of the high-spin Fe 3+ in methemoglobin (line I), the high-spin Fe 3+ in transferrin (line II), and Cu 2+ in ceruloplasmin and free radicals (line III) were observed. The present study may serve as a starting point for the development of a new technique for monitoring molecular complexes containing iron Fe 3+ (methemoglobin, transferrin) or copper Cu 2+ ions (ceruloplasmin) and free radicals in the blood during wound healing.

Quinone reductase 2 (QR2, E.C. 1.10.5.1) is an enzyme with a feature that has attracted attention for several decades: in standard conditions, instead of recognizing NAD(P)H as an electron donor, it recognizes putative metabolites of NADH, such as N -methyl- and N -ribosyl-dihydronicotinamide. QR2 has been particularly associated with reactive oxygen species and memory, strongly suggesting a link among QR2 (as a possible key element in pro-oxidation), autophagy, and neurodegeneration. In molecular and cellular pharmacology, understanding physiopathological associations can be difficult because of a lack of specific and powerful tools. Here, we present a thorough description of the potent, nanomolar inhibitor [2-(2-methoxy-5 H -1,4b,9-triaza(indeno[2,1-a]inden-10-yl)ethyl]-2-furamide (S29434 or NMDPEF; IC 50 = 5-16 nM) of QR2 at different organizational levels. We provide full detailed syntheses, describe its cocrystallization with and behavior at QR2 on a millisecond timeline, show that it penetrates cell membranes and inhibits QR2-mediated reactive oxygen species (ROS) production within the 100 nM range, and describe its actions in several in vivo models and lack of actions in various ROS-producing systems. The inhibitor is fairly stable in vivo, penetrates cells, specifically inhibits QR2, and shows activities that suggest a key role for this enzyme in different pathologic conditions, including neurodegenerative diseases., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

Epithelium mammary carcinoma is a cancer with a high death rate among women. One factor having a significant impact on metastasis is cell migration. The aim of this study was to compare migration rate inhibition of caffeic acid (CA) and its phenethyl ester (CAPE) on MCF-7 breast cancer cells. Microscopic evaluation was used to determine the morphology of carcinoma cells, before and after 24-hour treatment with CA and CAPE using a dose of 50 µM. The cytotoxic effect was measured by XTT-NR-SRB assay (tetrazolium hydroxide-neutral red-Sulforhodamine B) for 24-hour and 48-hour periods, using CA and CAPE, with doses of 50 and 100 µM. These doses were used to determine cell migration inhibition using a wound closure assay for 0-hour, 8-hour, 16-hour, and 24-hour periods. Both CA and CAPE treatments displayed cytotoxic activity in a dose- and time-dependent trend. CAPE displayed IC 50 values more than twice as low as CA. IC 50 values for the XTT assay were as follows: CA was 102.98 µM for 24 hours and 59.12 µM for 48 hours, while CAPE was 56.39 µM for 24 hours and 28.10 µM for 48 hours. For the NR assay: CA was 84.87 µM at 24 hours and 65.05 µM at 48 hours, while CAPE was 69.05 µM at 24 hours and 29.05 µM at 48 hours. For the SRB assay: At 24 hours, CA was 83.47 µM and 53.46 µM at 48 hours, while CAPE was 38.53 µM at 24 hours and 20.15 µM at 48 hours. Both polyphenols induced migration inhibition, resulting in practically halting the wound closure. CAPE produced better results than CA with the same doses and experiment times, though both CA and CAPE displayed cytotoxic activity against MCF-7 cells, as well as inhibited migration.

The dietary supplement market in Poland has been growing rapidly, and the number of registered products and their consumption increases steadily. Among the most popular and the easiest to get are herbal supplements, available in any supermarket. The aim of this paper was to investigate the mercury content in the herbal supplements. The dietary supplements that have been examined (24) are available on the Polish market and contain one or more herbal ingredients. Supplements were pulverized in porcelain mortar and identified by AMA 254 atomic absorption spectrometer. The range of variations for all tested supplements was within 0.02-4293.07 μg/kg. The arithmetic mean of the total result was 193.77 μg/kg. A higher mercury content then this mean was found in preparations-bamboo shoots and alga Chlorella pyrenoidosa. The studies have shown that mercury is present in every examined herbal supplement, and its content exceeds in two preparations (with bamboo and alga) the permissible limit of 0.10 mg/kg. There were statistically significant differences in the occurrence of mercury depending on the herbal ingredient in the supplement. The lowest content was found in the preparation with Tanacetum parthenium and the highest with bamboo shoots. The mercury content in the tested herbal supplements was statistically significant in the form of a supplement-a tablet and a capsule. Daily, weekly, monthly, and yearly consumption of mercury with examined supplements was calculated-the results did not exceed the PTWI-provisional tolerable weekly intake of mercury. To increase consumer safety, it is imperative to conduct further research on dietary supplements and implement a stricter quality control of the dietary supplements.

Breast cancer is one of the most common causes of mortality in women. Flavonoids, among other compounds, are bioactive constituents of propolis. In this comparative study, we investigated the effects of flavonoids apigenin (API), genistein (GEN), hesperidin (HES), naringin (NAR) and quercetin (QUE) on the proliferation, apoptosis, and cell cycle of two different human cancer cells - MDA-MB-231, estrogen-negative, and MCF-7, estrogen-positive receptor breast carcinoma cells. Many cytotoxic reports of flavonoids were performed by MTT assay. However, it's reported that MTT is reduced in metabolically active cells and yields an insoluble purple formazan, which indicates that obtained cytotoxic results of flavonoids could be inconsistent. Cell viability was measured by NR, neutral red assay, while the percentage of apoptotic cells and cell cycle arrest were determined by flow cytometry and Muse cell cycle assay, respectively. The results showed a high dose-dependent effect in cell viability tests. IC50 values were as follows (MCF-7/MDA-MB-231, for 48 h, in µM): 9.39/50.83 for HES, 25.19/88.17 for API, 40.26/333.51 for NAR, 49.49/47.50 for GEN and 95.12/130.10 for QUE. Flavonoid-induced apoptosis was dose- and time-dependent, for both cancer cell lines, though flavonoids were more active on MCF-7 cells. The flavonoids also induced cell cycle arrest in cancer cells.

The aim of this study was to verify whether L-lactide/DL-lactide copolymer 80/20 (PLDLLA) and platelet-rich plasma (PRP) trigger bone formation within critical-sized calvarial defects in adult sheep ( n = 6). Two craniectomies, each ca. 3 cm in diameter, were created in each animal. The first craniectomy was protected with an inner polylactide membrane, filled with PRP-polylactide granules, and covered with outer polylactide membrane. The second control craniectomy was left untreated. The animals were euthanized at 6, 7, 17, 19, 33, and 34 weeks after surgery, and the quality and the rate of reossification were assessed histomorphometrically and microtomographically. The study demonstrated that application of implants made of PLDLLA 80/20 combined with an osteopromotive substance (e.g., PRP) may promote bone healing in large calvarial defect in sheep. These promising proof-of-concept studies need to be verified in the future on a larger cohort of animals and over a longer period of time in order to draw definitive conclusions.

We have studied a preventive effect of polyphenol-rich bee pollen ethanol extract (EEP) against histological changes in the liver and cardiac blood vessels, abnormalities of lipid profile, and the levels of oxidized low density lipoproteins (ox-LDL), asymmetric dimethylarginine (ADMA), angiotensin-converting enzyme (ACE), and angiotensin II (ANG II) caused by a high-fat diet in C 57 BL₆ mice. Supplementing the diet with EEP in the doses of 0.1 g/kg body mass (BM) and 1 g/kg BM resulted in a decrease of total cholesterol by 31% and 35%, respectively. It also decreased the level of low density lipoproteins by 67% and 90%, respectively. No differences in the levels of high density lipoprotein and triacylglycerols were observed. EEP reduced the level of ox-LDL by 33% and 47%, ADMA by 13% and 51%, ACE by 17% and 30%, as well as ANG II by 11% and 15% in a dose-dependent manner, which proves a protective effect of EEP in a high-fat diet. EEP reduces and/or prevents hepatic steatosis and degenerative changes caused by a high-fat diet in C 57 BL₆ mice, which indicates its hepatoprotective effect. EEP used with standard feed does not disturb a normal concentration of the assayed parameters., Competing Interests: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

The aim of this study was to determine the effect of polyphenol-rich ethanol extract of bee pollen (EEP) on atherosclerosis induced by a high-fat diet in ApoE-knockout mice. EEP was given with feed in two doses of 0.1 and 1 g/kg body mass (BM). The studies have been conducted in a period of 16 weeks. The following factors were estimated: total cholesterol (TC), oxidized low density lipoproteins (ox-LDL), asymmetric dimethylarginine (ADMA), angiotensin-converting enzyme (ACE) and angiotensin II (ANG II) in the 5th, 10th, 12th, 14th, and 16th week of the experiment. In the last, i.e., 16th week of the studies the development of coronary artery disease (CAD) was also estimated histopathologically. Supplementing diet with EEP resulted in decreasing TC level. EEP reduced oxidative stress by lowering the levels of ox-LDL, ADMA, ANG II and ACE. EEP protected coronary arteries by significantly limiting the development of atherosclerosis (the dose of 0.1 g/kg BM) or completely preventing its occurrence (the dose of 1 g/kg BM). The obtained results demonstrate that EEP may be useful as a potential anti-atherogenic agent., Competing Interests: The authors declare no conflict of interest. The founding sponsor had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, nor in the decision to publish the results.

Heavy metals polluting the natural environment are absorbed by plants. The use of herbs as components of cosmetics may pose a health risk for humans. The aim of the study was to determine the concentrations of Pb, Cd and Hg in selected species of herbs (horsetail Equisetum arvense , nettle Urtica dioica , St. John's wort Hypericum perforatum , wormwood Artemisia absinthium , yarrow Achillea millefolium , cottonwood Solidago virgaurea ) self-collected from the natural environment in two different locations, and purchased in stores on the territory of Poland. The concentration of the metals studied was: 4.67-23.8 mg/kg Pb, 0.01-1.51 mg/kg Cd, 0.005-0.028 mg/kg Hg. Different concentrations of metals, depending on species and origin of plants, were found. The mean concentration of all studied metals was the lowest in St. John's wort, and the highest in nettle. In herbs purchased in Polish stores, the concentration of Pb was higher than in plants self-collected in the natural environment., Competing Interests: The authors declare no conflict of interest.

One of the deadliest cancers among women is a breast cancer. Research has shown that two natural substances occurring in propolis, caffeic acid (CA) and caffeic acid phenethyl ester (CAPE), have significant anticancer effects. The purpose of our in vitro study was to compare cytotoxic activity and migration rate inhibition using CA and CAPE (doses of 50 and 100 µm) against triple-negative, MDA-MB-231 breast adenocarcinoma line cells, drawn from Caucasian women. Viability was measured by XTT-NR-SRB assay (Tetrazolium hydroxide-Neutral Red-Sulforhodamine B) for 24 h and 48 h periods. Cell migration for wound healing assay was taken for 0 h, 8 h, 16 h, and 24 h periods. CAPE displayed more than two times higher cytotoxicity against MDA-MB-231 cells. IC 50 values for the XTT assay were as follows: CA for 24 h and 48 h were 150.94 µM and 108.42 µM, respectively, while CAPE was 68.82 µM for 24 h and 55.79 µM for 48 h. For the NR assay: CA was 135.85 µM at 24 h and 103.23 µM at 48 h, while CAPE was 64.04 µM at 24 h and 53.25 µM at 48 h. For the SRB assay: CA at 24 h was 139.80 µM and at 48 h 103.98 µM, while CAPE was 66.86 µM at 24 h and 47.73 µM at 48 h. Both agents suspended the migration rate; however, CAPE displayed better activity. Notably, for the 100 µM CAPE dose, motility of the tested breast carcinoma cells was halted., Competing Interests: The authors declare no conflict of interest. The founding sponsor had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

New transcripts generated by RNA polymerase II (RNAPII) are generally processed in order to form mature mRNAs. Two key processing steps include a precise cleavage within the 3' end of the pre-mRNA, and the subsequent polymerization of adenosines to produce the poly(A) tail. In yeast, these two functions are performed by a large multi-subunit complex that includes the Cleavage Factor IA (CF IA). The four proteins Pcf11, Clp1, Rna14 and Rna15 constitute the yeast CF IA, and of these, Pcf11 is structurally the least characterized. Here, we provide evidence for the binding of two Zn2+ atoms to Pcf11, bound to separate zinc-binding domains located on each side of the Clp1 recognition region. Additional structural characterization of the second zinc-binding domain shows that it forms an unusual zinc finger fold. We further demonstrate that the two domains are not mandatory for CF IA assembly nor RNA polymerase II transcription termination, but are rather involved to different extents in the pre-mRNA 3'-end processing mechanism. Our data thus contribute to a more complete understanding of the architecture and function of Pcf11 and its role within the yeast CF IA complex., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Studies show that caffeic acid (CA) and caffeic acid phenethyl ester (CAPE) are compounds with potent chemopreventive effects. Breast cancer is a common form of aggressive cancer among women worldwide. This study shows a comparison of CA and CAPE activity on triple-negative human caucasian breast adenocarcinoma line cells (MDA-MB-231). MDA-MB-231 cells were treated by CA and CAPE with doses of from 10 to 100 µM, for periods of 24 h and 48 h. Cytotoxicity MTT tests, apoptosis by Annexin V, and cell cycle with Dead Cell Assays were performed. Cytotoxic activity was greater for CAPE compared to CA (both incubation times, same dosage). IC 50 values for CAPE were 27.84 µM (24 h) and 15.83 µM (48 h) and for CA > 10,000 µM (24 h) and > 1000 µM (48 h). Polyphenols induced apoptosis, while CAPE (dose dependently), induced a higher apoptotic effect. CAPE also induced cell cycle arrest in S phase (time and dose dependently), CA did it only for 50 and 100 µM. A dose dependent decline was seen for the G0/G1 phase (CAPE, 48 h), as well as elimination of phase G2/M by 100 µM of CAPE (only mild effect for CA). Comparing CA and CAPE activity on MDA-MB-231, CAPE clearly showed better activity for the same dosages and experiment times., Competing Interests: The authors declare no conflict of interest. The founding sponsor had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, nor in the decision to publish the results.

For the efficient pathogenesis of Shigella, the causative agent of bacillary dysentery, full functionality of tRNA-guanine transglycosylase (TGT) is mandatory. TGT performs post-transcriptional modifications of tRNAs in the anticodon loop taking impact on virulence development. This suggests TGT as a putative target for selective anti-shigellosis drug therapy. Since bacterial TGT is only functional as homodimer, its activity can be inhibited either by blocking its active site or by preventing dimerization. Recently, we discovered that in some crystal structures obtained by soaking the full conformational adaptation most likely induced in solution upon ligand binding is not displayed. Thus, soaked structures may be misleading and suggest irrelevant binding modes. Accordingly, we re-investigated these complexes by co-crystallization. The obtained structures revealed large conformational rearrangements not visible in the soaked complexes. They result from spatial perturbations in the ribose-34/phosphate-35 recognition pocket and, consequently, an extended loop-helix motif required to prevent access of water molecules into the dimer interface loses its geometric integrity. Thermodynamic profiles of ligand binding in solution indicate favorable entropic contributions to complex formation when large conformational adaptations in the dimer interface are involved. Native MS titration experiments reveal the extent to which the homodimer is destabilized in the presence of each inhibitor. Unexpectedly, one ligand causes a complete rearrangement of subunit packing within the homodimer, never observed in any other TGT crystal structure before. Likely, this novel twisted dimer is catalytically inactive and, therefore, suggests that stabilizing this non-productive subunit arrangement may be used as a further strategy for TGT inhibition.

Background: Cellobiose dehydrogenase (CDH) is a fungal extracellular oxidoreductase which fuels lytic polysaccharide monooxygenase with electrons during cellulose degradation. Interdomain electron transfer between the flavin and cytochrome domain in CDH, preceding the electron flow to lytic polysaccharide monooxygenase, is known to be pH dependent, but the exact mechanism of this regulation has not been experimentally proven so far., Methods: To investigate the structural aspects underlying the domain interaction in CDH, hydrogen/deuterium exchange (HDX-MS) with improved proteolytic setup (combination of nepenthesin-1 with rhizopuspepsin), native mass spectrometry with ion mobility and electrostatics calculations were used., Results: HDX-MS revealed pH-dependent changes in solvent accessibility and hydrogen bonding at the interdomain interface. Electrostatics calculations identified these differences to result from charge neutralization by protonation and together with ion mobility pointed at higher electrostatic repulsion between CDH domains at neutral pH. In addition, we uncovered extensive O-glycosylation in the linker region and identified the long-unknown exact cleavage point in papain-mediated domain separation., Conclusions: Transition of CDH between its inactive (open) and interdomain electron transfer-capable (closed) state is shown to be governed by changes in the protein surface electrostatics at the domain interface. Our study confirms that the interdomain electrostatic repulsion is the key factor modulating the functioning of CDH., General Significance: The results presented in this paper provide experimental evidence for the role of charge repulsion in the interdomain electron transfer in cellobiose dehydrogenases, which is relevant for exploiting their biotechnological potential in biosensors and biofuel cells., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Translationally Controlled Tumor Protein (TCTP) is anti-apoptotic, key in development and cancer, however without the typical Bcl2 family members' structure. Here we report that TCTP contains a BH3-like domain and forms heterocomplexes with Bcl-xL. The crystal structure of a Bcl-xL deletion variant-TCTP11-31 complex reveals that TCTP refolds in a helical conformation upon binding the BH3-groove of Bcl-xL, although lacking the h1-subregion interaction. Experiments using in vitro-vivo reconstituted systems and TCTP(+/-) mice indicate that TCTP activates the anti-apoptotic function of Bcl-xL, in contrast to all other BH3-proteins. Replacing the non-conserved h1 of TCTP by that of Bax drastically increases the affinity of this hybrid for Bcl-xL, modifying its biological properties. This work reveals a novel class of BH3-proteins potentiating the anti-apoptotic function of Bcl-xL.

The aim of the present study was to visualize the benefits and advantages derived from preparations based on extracts of bee pollen as compared to pharmaceuticals commonly used in the treatment of burns. The bee pollen ointment was applied for the first time in topical burn treatment. Experimental burn wounds were inflicted on two white, domestic pigs. Clinical, histopathological, and microbiological assessment of specimens from burn wounds, inflicted on polish domestic pigs, treated with silver sulfadiazine or bee pollen ointment, was done. The comparative material was constituted by either tissues obtained from wounds treated with physiological saline or tissues obtained from wounds which were untreated. Clinical and histopathological evaluation showed that applied apitherapeutic agent reduces the healing time of burn wounds and positively affects the general condition of the animals. Moreover the used natural preparation proved to be highly effective antimicrobial agent, which was reflected in a reduction of the number of microorganisms in quantitative research and bactericidal activity of isolated strains. On the basis of the obtained bacteriological analysis, it may be concluded that the applied bee pollen ointment may affect the wound healing process of burn wounds, preventing infection of the newly formed tissue.

Bee pollen constitutes a natural source of antioxidants such as phenolic acids and flavonoids, which are responsible for its biological activity. Research has indicated the correlation between dietary polyphenols and cardioprotective, hepatoprotective, anti-inflammatory, antibacterial, anticancerogenic, immunostimulating, antianaemic effects, as well as their beneficial influence on osseous tissue. The beneficial effects of bee pollen on health result from the presence of phenolic acids and flavonoids which possess anti-inflammatory properties, phytosterol and linolenic acid which play an anticancerogenic role, and polysaccharides which stimulate immunological activity. Polyphenols are absorbed in the alimentary tract, metabolised by CYP450 enzymes, and excreted with urine and faeces. Flavonoids and phenolic acids are characterised by high antioxidative potential, which is closely related to their chemical structure. The high antioxidant potential of phenolic acids is due to the presence and location of hydroxyl groups, a carboxyl group in the immediate vicinity of ortho-diphenolic substituents, and the ethylene group between the phenyl ring and the carboxyl group. As regards flavonoids, essential structural elements are hydroxyl groups at the C5 and C7 positions in the A ring, and at the C3' and C4' positions in the B ring, and a hydroxyl group at the C3 position in the C ring. Furthermore, both, the double bond between C2 and C3, and a ketone group at the C4 position in the C ring enhance the antioxidative potential of these compounds. Polyphenols have an ideal chemical structure for scavenging free radicals and for creating chelates with metal ions, which makes them effective antioxidants in vivo.

We evaluate the potential of native mass spectrometry (MS) and ion mobility (IM-MS) for the screening of protein : ligand complexes when very subtle conformational changes are involved. As a proof of concept, we investigate the interactions between a peptide deformylase (PDF1B), a promising target for the development of new antibiotics, and three of its specific inhibitors that bind in different modes. First, real-time native MS reveals two types of ligands, both interacting in a 1 : 1 stoichiometry with PDF1B but with different affinities and gas phase stabilities. Conformational IM-MS screening then highlights two very close but significantly distinct ligand-induced conformations with collision cross sections that differ by less than 1%. Real-time IM-MS is used to monitor not only the dynamics of ligand binding to apoPDF1B but also the switching between holo conformations. This study provides additional evidence that the most potent ligands inhibit peptide deformylases through a slow-tight binding mechanism, in agreement with previous structural and enzymology studies. Furthermore, this approach, wherein the characteristics obtained by native MS are combined with IM-MS conformational screening, prove valuable in characterizing extremely subtle dynamic conformational changes induced when ligands bind to protein assemblies. We discuss the promise and limitations of IM-MS in the context of detection of very small conformational changes induced upon ligand binding.

Thiols are among the most frequently used functional groups in the field of bioconjugation. While there exists a variety of heterobifunctional reagents that allow for coupling thiols to other functions (e.g., amines, carboxylic acids), there is no specific reagent for creating heteroconjugates using two different thiols. In response to the ever-increasing demand for bioconjugation tools, we have developed p-(maleimide)-phenylpropionitrile (MAPN)-an efficient reagent for kinetically resolved thiol-to-thiol coupling. In a comparative study with its closest commercially available analogue, p-phenylenedimaleimide, MAPN has shown substantial advantages for the preparation of thiol-thiol heteroconjugates. Namely, an antibody-drug conjugate (ADC) with mertansine (DM1), conjugated to the cysteine residues of Trastuzumab, was prepared for the first time.

PRMT6 is a protein arginine methyltransferase involved in transcriptional regulation, human immunodeficiency virus pathogenesis, DNA base excision repair, and cell cycle progression. Like other PRMTs, PRMT6 is overexpressed in several cancer types and is therefore considered as a potential anti-cancer drug target. In the present study, we described six crystal structures of PRMT6 from Mus musculus, solved and refined at 1.34 Å for the highest resolution structure. The crystal structures revealed that the folding of the helix αX is required to stabilize a productive active site before methylation of the bound peptide can occur. In the absence of cofactor, metal cations can be found in the catalytic pocket at the expected position of the guanidinium moiety of the target arginine substrate. Using mass spectrometry under native conditions, we show that PRMT6 dimer binds two cofactor and a single H4 peptide molecules. Finally, we characterized a new site of in vitro automethylation of mouse PRMT6 at position 7., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Chemotherapy of breast cancer could be improved by bioactive natural substances, which may potentially sensitize the carcinoma cells' susceptibility to drugs. Numerous phytochemicals, including propolis, have been reported to interfere with the viability of carcinoma cells. We evaluated the in vitro cytotoxic activity of ethanol extract of propolis (EEP) and its derivative caffeic acid phenethyl ester (CAPE) towards two triple-negative breast cancer (TNBC) cell lines, MDA-MB-231 and Hs578T, by implementation of the MTT and lactate dehydrogenase (LDH) assays. The morphological changes of breast carcinoma cells were observed following exposure to EEP and CAPE. The IC50 of EEP was 48.35 µg∙mL-1 for MDA-MB-23 cells and 33.68 µg∙mL-1 for Hs578T cells, whereas the CAPE IC50 was 14.08 µM and 8.01 µM for the MDA-MB-231 and Hs578T cell line, respectively. Here, we report that propolis and CAPE inhibited the growth of the MDA-MB-231 and Hs578T lines in a dose-dependent and exposure time-dependent manner. EEP showed less cytotoxic activity against both types of TNBC cells. EEP and, particularly, CAPE may markedly affect the viability of breast cancer cells, suggesting the potential role of bioactive compounds in chemoprevention/chemotherapy by potentiating the action of standard anti-cancer drugs.

The aim of the study was to assess the propolis effect on fibronectin metabolism in the course of burn wounds healing process. A model of burn wound healing of pig skin was applied. The amount of the released glycoprotein was assessed by a surface plasmon resonance. The profile of extracted fibronectin components was also assessed by an electrophoresis in polyacrylamide gel, with a subsequent immunodetection by Western Blotting. Propolis burn treatment decreased the release of fibronectin components from healing wounds in relation to damages treated with silver sulfadiazine. The main reason of decreased extraction of fibronectin components from wounds treated with propolis was a substantial decrease of degradation product release of the mentioned glycoprotein, which was observed particularly from the 3rd to 5th day of the repair. Wounds treatment with propolis demonstrated, especially in relation to damages treated with silver sulfadiazine, the decreased release of synthesized fibronectin molecules. The obtained results suggest that propolis modifies fibronectin metabolism in the course of wound healing process. The influence of propolis is reflected in prevention of fibronectin biosynthesis as well as its degradation in the wound area. The above-mentioned metabolic changes may decrease the risk of complications in the repair wounds process.

Propolis is a potential source of natural antioxidants such as phenolic acids and flavonoids. Its wide biological effects have been known and used since antiquity. In the modern world natural substances are sought which would be able to counteract the effects of antioxidative stress, which underlies many diseases, such as cancer, diabetes and atherosclerosis. This paper aims to present the antioxidative activity of phenolic acids and flavonoids present in Polish propolis and the relationship between their chemical structure and antioxidative activity influencing its medicinal properties. Data concerning the biological activity of propolis are summarized here, including its antibacterial, anti-inflammatory, anticarcinogenic, antiatherogenic, estrogenic effects, as well as AIDS- counteracting and reparative-regenerative function.

Medicines of biogenic origin with micro-organic, regenerative and analgesic properties are becoming more and more significant in the treatment of burn wounds. These properties are found in apitherapeutics such as propolis and honey--products collected and processed by a honey bee. Their effect on the course of the healing processes is multidirectional. The aim of the study was a histopathological and biochemical analysis of the processes of scar formation in experimentally evoked burn wounds in white pigs treated with the 1% and 3% Sepropol balms containing standardized extracts of propolis and honey. The results were compared with the therapeutic effects obtained with dermazin cream (1% silver sulfadiazine). The level of collagen was determined in the wounds treated with 1% and 3% Sepropol and compared with the collagen level in healthy skin and wounds treated with dermazin. Granulation and regenerated epithelium formation times were compared, with the 3% Sepropol being by far the most effective. The 3% Sepropol also increased the collagen level to 116% with the control sub-groups scoring between 80% and 98%. The results show the healing process of burn wounds in pigs treated with the Sepropol balm starts earlier and has a faster course than the standard dermazin therapy.