Acid-base and metal ion binding properties of 2-thiocytidine in aqueous solution

The thionucleoside 2-thiocytidine (C2S) occurs in nature in transfer RNAs; it receives attention in diverse fields like drug research and nanotechnology. By potentiometric pH titrations we measured the acidity constants of H(C2S)+ and the stability constants of the M(C2S)2+ and M(C2S−H)+ complexes (M2+=Zn2+, Cd2+), and we compared these results with those obtained previously for its parent nucleoside, cytidine (Cyd). Replacement of the (C2)=O unit by (C2)=S facilitates the release of the proton from (N3)H+ in H(C2S)+ (pK a = 3.44) somewhat, compared with H(Cyd)+ (pK a = 4.24). This moderate effect of about 0.8 pK units contrasts with the strong acidification of about 4 pK units of the (C4)NH2 group in C2S (pK a = 12.65) compared with Cyd (pK a≈16.7); the reason for this result is that the amino-thione tautomer, which dominates for the neutral C2S molecule, is transformed upon deprotonation into the imino-thioate form with the negative charge largely located on the sulfur. In the M(C2S)2+ complexes the (C2)S group is the primary binding site rather than N3 as is the case in the M(Cyd)2+ complexes, though owing to chelate formation N3 is to some extent still involved in metal ion binding. Similarly, in the Zn(C2S−H)+ and Cd(C2S−H)+ complexes the main metal ion binding site is the (C2)S− unit (formation degree above 99.99% compared with that of N3). However, again a large degree of chelate formation with N3 must be surmised for the M(C2S−H)+ species in accord with previous solid-state studies of related ligands. Upon metal ion binding, the deprotonation of the (C4)NH2 group (pK a = 12.65) is dramatically acidified (pK a≈3), confirming the very high stability of the M(C2S−H)+ complexes. To conclude, the hydrogen-bonding and metal ion complex forming capabilities of C2S differ strongly from those of its parent Cyd; this must have consequences for the properties of those RNAs which contain this thionucleosid

The Methods of Digging for “Gold” within the Salt: Characterization of Halophilic Prokaryotes and Identification of Their Valuable Biological Products Using Sequencing and Genome Mining Tools

Halophiles, the salt-loving organisms, have been investigated for at least a hundred years. They are found in all three domains of life, namely Archaea, Bacteria, and Eukarya, and occur in saline and hypersaline environments worldwide. They are already a valuable source of various biomolecules for biotechnological, pharmaceutical, cosmetological and industrial applications. In the present era of multidrug-resistant bacteria, cancer expansion, and extreme environmental pollution, the demand for new, effective compounds is higher and more urgent than ever before. Thus, the unique metabolism of halophilic microorganisms, their low nutritional requirements and their ability to adapt to harsh conditions (high salinity, high pressure and UV radiation, low oxygen concentration, hydrophobic conditions, extreme temperatures and pH, toxic compounds and heavy metals) make them promising candidates as a fruitful source of bioactive compounds. The main aim of this review is to highlight the nucleic acid sequencing experimental strategies used in halophile studies in concert with the presentation of recent examples of bioproducts and functions discovered in silico in the halophile’s genomes. We point out methodological gaps and solutions based on in silico methods that are helpful in the identification of valuable bioproducts synthesized by halophiles. We also show the potential of an increasing number of publicly available genomic and metagenomic data for halophilic organisms that can be analysed to identify such new bioproducts and their producers.Publication’s printing cost was co-financed by the European Union from the European Social Fund under the "InterDOC-STARt" project (POWR.03.02.00-00-I033/16-00)

Pluronic® F-127 enhances antifungal activity of fluconazole against resistant Candida strains

Candida strains as the most frequent causes of infections, along with their increased drug resistance, pose significant clinical and financial challenges to the healthcare system. Some polymeric excipients were reported to interfere with the multidrug resistance mechanism. Bearing in mind that there are a limited number of marketed products with fluconazole (FLU) for the topical route of administration, Pluronic F-127 (PLX)/FLU formulations were investigated in this work. The aims of this study were to investigate (i) whether PLX-based formulations can increase the susceptibility of resistant Candida strains to FLU, (ii) whether there is a correlation between block polymer concentration and the antifungal efficacy of the FLU-loaded PLX formulations, and (iii) what the potential mode of action of PLX assisting FLU is. The yeast growth inhibition upon incubation with PLX formulations loaded with FLU was statistically significant. The highest efficacy of the azole agent was observed in the presence of 5.0 and 10.0% w/v of PLX. The upregulation of the CDR1/CDR2 genes was detected in the investigated Candida strains, indicating that the efflux of the drug from the fungal cell was the main mechanism of the resistance

Improved clinical investigation and evaluation of high-risk medical devices: the rationale and objectives of CORE-MD (Coordinating Research and Evidence for Medical Devices)

: In the European Union (EU) the delivery of health services is a national responsibility but there are concerted actions between member states to protect public health. Approval of pharmaceutical products is the responsibility of the European Medicines Agency, whereas authorizing the placing on the market of medical devices is decentralized to independent 'conformity assessment' organizations called notified bodies. The first legal basis for an EU system of evaluating medical devices and approving their market access was the medical device directives, from the 1990s. Uncertainties about clinical evidence requirements, among other reasons, led to the EU Medical Device Regulation (2017/745) that has applied since May 2021. It provides general principles for clinical investigations but few methodological details-which challenges responsible authorities to set appropriate balances between regulation and innovation, pre- and post-market studies, and clinical trials and real-world evidence. Scientific experts should advise on methods and standards for assessing and approving new high-risk devices, and safety, efficacy, and transparency of evidence should be paramount. The European Commission recently awarded a Horizon 2020 grant to a consortium led by the European Society of Cardiology and the European Federation of National Associations of Orthopaedics and Traumatology, that will review methodologies of clinical investigations, advise on study designs, and develop recommendations for aggregating clinical data from registries and other real-world sources. The CORE-MD project (Coordinating Research and Evidence for Medical Devices) will run until March 2024; here we describe how it may contribute to the development of regulatory science in Europe

The Degradation Product of Ramipril Is Potentially Carcinogenic, Genotoxic and Mutagenic

(1) Background: The aim of this study was to identify the degradation product of ramipril (RAM) formed under dry air and to verify its potential modes of carcinogenicity. We intended to check whether its formation and presence in final dosage forms could pose a cancer risk to humans who are treated with RAM due to cardiological indications. The carcinogenicity of this compound was evaluated with respect to two mechanisms: a potential direct DNA-damage and indirect toxicity, secondary to forming mutagenic N-nitroso metabolites. (2) Methods: Firstly, a forced ageing test under dry air was conducted for pure RAM in order to induce its degradation. The validated HPLC system was used to describe the kinetic order of this reaction. The emerging degradation impurity was identified by HPLC-MS. In the second stage, the cancer risk of the identified RAM degradant was predicted using a structure-based assessment by in silico QSAR model, employing three endpoints: carcinogenicity, genotoxicity and mutagenicity. In the third stage, the obtained QSAR results were experimentally verified. To verify genotoxicity prediction, in vitro micronucleus assay was employed. It enabled us to assess the potential direct DNA-damaging properties of RAM degradant at high concentrations (as screening series) and at concentrations usually observed in human blood (to mimic the clinical scenario). To verify the QSAR mutagenicity prediction, an in vitro Ames test was carried out. It was designed so as to detect two mechanisms of mutagenicity: a direct one (for pure degradant) and an indirect one (via N-nitroso-metabolites formation). N-nitroso-metabolites for mutagenicity assessment were obtained using NAP test. (3) Results: The kinetic mechanism of RAM degradation was first-order, the degradation rate constant was k = 1.396 ± 0.133 × 10−5 s−1 (T = 373 K), thus the formation of impurity was rapid. Energy of activation was 174.12 ± 46.2 kJ/mol, entropy was positive, thus reaction was bimolecular and favored; enthalpy was 171.65 ± 48.7 kJ/mol, thus reaction was endothermic. Only one degradation impurity was formed, and it was identified as RAM diketopiperazine derivative (DKP). QSAR simulation predicted that DKP could be carcinogenic and genotoxic, but this result had only moderate reliability. DKP was also predicted to be non-mutagenic and this prediction was strong (endpoint score 0.2). The confirmatory micronucleus experiment for genotoxicity prediction suggested that DKP was cytotoxic and it could be also aneugenic at a high concentration (0.22 mg/mL), evidenced by a three-fold increase in micronuclei relative to the control (11.86:33.33%, p = 0.0184). At physiologic concentrations, its cytotoxicity and genotoxicity did not occur. This means that the genotoxicity of DKP was limited by a threshold mechanism. In the mutagenicity in vitro assessment, pure DKP was not mutagenic, but its nitrosation product induced base substitutions mutations in test bacteria TA100 following metabolic activation at a concentration of 4.5 mg/mL, confirming its mutagenicity. (4) Conclusions: RAM rapidly cyclizes to diketopiperazine derivative under dry air. This impurity resides in drugs administered to patients. DKP is potentially aneugenic and cytotoxic at high concentrations, yet at concentrations typically occurring in human blood, this effect is unlikely. The exposure of patients to high concentrations of DKP, exceeding the typical blood level and standard RAM dosing, could lead to cancer development, thus the safe threshold for human exposure to DKP must be verified in follow-up in vivo experiments. Based on our results, it is impossible to establish the maximum safe dose of pure DKP to humans. Furthermore, DKP itself is not mutagenic, but it is liable to the formation of mutagenic nitroso-metabolites in vivo. Nitroso-derivatives of DKP are in vitro mutagens and their real-life impact on humans must be further evaluated in in vivo studies. Until this is carried out, RAM should not be formulated by manufacturers using dry procedures to minimize DKP formation and reduce risk of human carcinogenesis, since DKP could cause cancer via two independent mechanisms: direct genotoxicity when the exposure over standard RAM dosing occurs, and indirect mutagenicity via in vivo N-nitrosamine formation

Acid–base and metal ion-binding properties of thiopyrimidine derivatives

The thionucleoside 2-thiocytidine (C2S) as well as the thiouridines (US) occur in Nature, especially in transfer RNAs, and they also receive attention in diverse fields like nanotechnology and drug research. If (C2)O in cytidine (Cyd) is replaced by (C2)S to give the thio analogue C2S, the release of H + from (N3)H in H(C2S) + (p K a = 3.44) is facilitated somewhat [H(Cyd) + ; p K a = 4.24], yet, the deprotonation of the (C4)NH 2 group is much more affected: the p K a decreases from ca. 16.7 in Cyd to 12.65 in C2S. This is because the amino-thione tautomer dominating in the neutral C2S, transfers into the imino-thioate form, which has the charge largely localized on (C2)S − . As a consequence, the M(C2S) 2+ species (M 2+ = Zn 2+ or Cd 2+ ) transfer very easily into their deprotonated M(C2S − H) + forms. This reaction is extremely facilitated by M 2+ coordination at (C2)S − and occurs already at a pH slightly above 3. It is shown that the (C2)S M 2+ coordination dominates to more than 99% in both the M(C2S) 2+ and the M(C2S − H) + complexes; their structures, including chelate formation with the participation of N3, are evaluated. In 2-thiouridine (U2S), 4-thiouridine (U4S), and 2,4-dithiouridine (U2S4S), the release of H + from (N3)H, compared to Urd (p K a = 9.18), is facilitated by ca. 1 to 2 p K units, the charge being largely localized on the (C)S sites; this leads with (U4S − H) − and (U2S4S − H) − to the reduction of Cu(II) to Cu(I), transforming the thiolate into a disulfide. In Cu(U2S − H) + Cu(II) is stable, most likely due to steric constraints inhibiting disulfide formation. The stability of the M(US − H) + complexes with Ni 2+ , Cu 2+ or Cd 2+ is enhanced by about 1.3 to 2 log units compared to the corresponding uridinate complexes. The properties of the biologically relevant Zn(US − H) + are expected to be between those with Ni 2+ and Cd 2+ . The relatively high affinity of the (C)S sites for these M 2+ is reflected in the 2-thiouridine 5′-monophosphate (U2SMP 2− ) and 4-thiouridine 5′-monophosphate (U4SMP 2− ) complexes, M 2+ being located to more than 99% at the thiouracil residue and only traces are coordinated at the phosphate group. In the N3-deprotonated Cu[(U2S − H)MP] − species the anti conformer is partly turned into the syn one allowing thus a formation degree of about 60% of the macrochelate formed by (C2)S − and the phosphate group. The corresponding coordination pattern also seems to hold for Cd[(U2S − H)MP] − , though the formation degree of the macrochelate is lower. No macrochelate formation is detected for Ni[(U2S − H)MP] − , as well as for Ni[(U4S − H)MP] − and Cd[(U4S − H)MP] − . The reasons for the indicated coordination patterns are discussed, as well as the biological implications of the summarized results, especially with regard to tRNAs

Antimicrobial Peptide – Metal Interactions – Relationship between Coordination Chemistry, Structure, Thermodynamics and Mode of Action

Increasing bacterial and fungal drug resistance makes novel, effective antimicrobial treatments actively sought. Because of the general lack of resistance towards AMPs, they are being relied on as a novel class of therapeutics aimed to conquer drug-resistant bacteria and fungi [1]. Biologically indispensable metal ions have a dual effect on the activity of antimicrobial peptides: (i) AMPs bind them, so that microbes cannot get enough metals essential for their life and virulence (withdrawal of metal ions, nutritional immunity) or (ii) AMPs need the given metal ion as a booster of their antimicrobial activity (metal ions affect the AMP charge and/or structure) [2]. The presence of Zn(II) and Cu(II) significantly enhances the antimicrobial activity of calcitermin, an antimicrobial peptide from the fluid of the human airways (a C-terminal cleavage fragment of calgranulin C), SAAPs – anionic peptides from sheep and clavanins – His-rich, cationic peptides from tunicates. MIC breakpoints of several of these complexes are much lower than the ones for commonly used antibiotics and antifungal agents. We discuss the details of the coordination mode, structure and stability of the studied complexes, in order to understand the relationship between their bioinorganic chemistry and mode of action. Financial support by the National Science Centre (UMO-2017/26/A/ST5/00364, SONATA BIS grant to MRZ) is gratefully acknowledged. References [1] J. M. Ageitos, A. Sanchez-Perez, P. Calo-Mata, T. G. Villa, Biochem. Pharmacol., 2017, 133, 117-138. [2] D. Łoboda, H. Kozłowski, M. Rowińska-Żyrek, New J. Chem., 2018, 42, 7560-7568

Short-chain oligopeptides with copper(II) binding properties: the impact of specific structural modifications on the copper(II) coordination abilities

A series of linear tetrapeptides containing two histidyl residues in position 2 and 4, namely DHGH, DHGDH, KHGH, KHGdH, Ac-DHGH-NH2, Ac-DHGdH-NH2, Ac-KHGH-NH2, and Ac-KHGdH-NH2, were syn- thesized and characterised. Their copper(II) binding properties were investigated in depth through a vari- ety of physicochemical methods. Potentiometric titrations were first carried out to establish the stoichiometry and the stability of the resulting copper(II)–peptide complexes. The copper(II) chromoph- ores that are formed in the various cases in dependence of pH were subsequently characterised by exten- sive spectroscopic analysis (UV–Vis, EPR, CD) in strict correlation with potentiometric data. The effects of the nature of the first amino acid (Lys versus Asp) and of N-terminal amino group protection on copper(II) binding were specifically addressed. On turn, the careful comparison of the copper(II) coordination abil- ities of the linear peptides with those of their cyclic analogs provided insight into the effects of cyclization on the overall metal binding properties

Polityczna kreacja ładu ekonomicznego. Teoria i praktyka

Władza polityczna posiada współcześnie ogromny wpływ na kreowanie wielu aspektów rzeczywistości gospodarczej. Niniejsze opracowanie stanowi głos w dyskusji na temat teoretycznych, historycznych oraz współczesnych powiązań systemu ekonomicznego z polityką. Jego celem jest przybliżenie czytelnikowi relacji i mechanizmów zachodzących pomiędzy politycznym systemem opartym na demokracji, a gospodarką w wymiarze krajowym i międzynarodowym. Większość szkół ekonomicznych na ogół przyjmuje, że rynek jest najlepszym sposobem organizowania procesów gospodarczych, a system demokracji parlamentarnej najlepszą metodą sprawowania władzy. Wielość systemów gospodarczych opartych o tę zasadę jest faktem, jak i faktem jest różna ich sprawność, czy efektywność działania. Faktem także jest zróżnicowanie gospodarek ze względu na uwarunkowania kulturowo-historyczne. Po prostu demokracja i rynek nie jedno ma imię. Badanie tej różnorodności i trwałości systemów, określenie czynników wywołujących ich zmianę stanowi ważki temat badań ekonomicznych o dużych walorach praktycznych. Monografia zawiera wartościowy, interdyscyplinarny ładunek wiedzy.Udostępnienie publikacji Wydawnictwa Uniwersytetu Łódzkiego finansowane w ramach projektu „Doskonałość naukowa kluczem do doskonałości kształcenia”. Projekt realizowany jest ze środków Europejskiego Funduszu Społecznego w ramach Programu Operacyjnego Wiedza Edukacja Rozwój; nr umowy: POWER.03.05.00-00-Z092/17-00