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Cyclic peptides present a robust platform for drug design, offering high specificity and stability due to their conformationally constrained structures. In this study, we introduce an updated version of the Cyclic Peptide Matching program (cPEPmatch) tailored for the identification of cyclic peptides capable of mimicking protein-glycosaminoglycan (GAG) binding sites. We focused on engineering cyclic peptides to replicate the GAG-binding affinity of antithrombin III (ATIII), a protein that plays a crucial role in modulating anticoagulation through interaction with the GAG heparin. By integrating computational and experimental methods, we successfully identified a cyclic peptide binder with promising potential for future optimization. MD simulations and MM-GBSA calculations were used to assess binding efficacy, supplemented by umbrella sampling to approximate free energy landscapes. The binding specificity was further validated through NMR and ITC experiments. Our findings demonstrate that the computationally designed cyclic peptides effectively target GAGs, suggesting their potential as novel therapeutic agents. This study advances our understanding of peptide-GAG interactions and lays the groundwork for future development of cyclic peptide-based therapeutics.

Acridinium esters, due to their capability for chemiluminescence (CL), are employed as indicators and labels in biomedical diagnostics and other fields. In this work, the influence of ionic surfactants, hexadecyltrimethylammonium chloride and bromide (CTAC and CTAB, cationic) and sodium dodecyl sulphate (SDS, anionic) on the CL parameters and mechanism of representative emitter, 10-methyl-9-[(2-methylphenoxy)carbonyl]acridinium trifluoromethanesulphonate (2MeX) in a H2O2/NaOH environment, is studied. Our investigations revealed that the type of surfactant and its form in solution have an impact on the CL kinetic constants and integral efficiencies, while changes in those emission properties resulting from the type of ion (Cl− vs. Br−) are negligible. The major changes were recorded for systems containing surfactants at concentrations higher than the critical micelle concentration. The cationic surfactants (CTAC, CTAB) cause a substantial increase in CL emission kinetics and a moderate increase in its integral efficiency. At the same time, the opposite effect is observed in the case of SDS. Molecular dynamics simulations suggest that changes in emission parameters are likely due to differences in the binding strength of 2MeX substrate with surfactant molecules, which is higher for SDS than for CTAC. The results can help in rational designing of optimal acridinium CL systems and demonstrate their usefulness in distinguishing the pre- and post-micellar environment and the charge of surfactants.

IntroductionIn the era of increasing bacterial resistance to antibiotics, new bactericidal substances are sought, and lysins derived from extremophilic organisms have the undoubted advantage of being stable under harsh environmental conditions. The PhiKo endolysin is derived from the phiKo bacteriophage infecting Gram-negative extremophilic bacterium Thermus thermophilus HB27. This enzyme shows similarity to two previously investigated thermostable type-2 amidases, the Ts2631 and Ph2119 from Thermus scotoductus bacteriophages, that revealed high lytic activity not only against thermophiles but also against Gram-negative mesophilic bacteria. Therefore, antibacterial potential of the PhiKo endolysin was investigated in the study presented here.MethodsEnzyme activity was assessed using turbidity reduction assays (TRAs) and antibacterial tests. Differential scanning calorimetry was applied to evaluate protein stability. The Collection of Anti-Microbial Peptides (CAMP) and Antimicrobial Peptide Calculator and Predictor (APD3) were used to predict regions with antimicrobial potential in the PhiKo primary sequence. The minimum inhibitory concentration (MIC) of the RAP-29 synthetic peptide was determined against Gram-positive and Gram-negative selected strains, and mechanism of action was investigated with use of membrane potential sensitive fluorescent dye 3,3′-Dipropylthiacarbocyanine iodide (DiSC3(5)).Results and discussionThe PhiKo endolysin is highly thermostable with melting temperature of 91.70°C. However, despite its lytic effect against such extremophiles as: T. thermophilus, Thermus flavus, Thermus parvatiensis, Thermus scotoductus, and Deinococcus radiodurans, PhiKo showed moderate antibacterial activity against mesophiles. Consequently, its protein sequence was searched for regions with potential antibacterial activity. A highly positively charged region was identified and synthetized (PhiKo105-133). The novel RAP-29 peptide lysed mesophilic strains of staphylococci and Gram-negative bacteria, reducing the number of cells by 3.7–7.1 log units and reaching the minimum inhibitory concentration values in the range of 2–31 μM. This peptide is unstructured in an aqueous solution but forms an α-helix in the presence of detergents. Moreover, it binds lipoteichoic acid and lipopolysaccharide, and causes depolarization of bacterial membranes. The RAP-29 peptide is a promising candidate for combating bacterial pathogens. The existence of this cryptic peptide testifies to a much wider panel of antimicrobial peptides than thought previously.

The crystal structures of two newly synthesized nitrilotriacetate oxidovanadium(IV) salts, namely [QH][VO(nta)(H2O)](H2O)2 (I) and [(acr)H][VO(nta)(H2O)](H2O)2 (II), were determined. Additionally, the cytotoxic effects of four N-heterocyclic nitrilotriacetate oxidovanadium(IV) salts—1,10-phenanthrolinium, [(phen)H][VO(nta)(H2O)](H2O)0.5 (III), 2,2′-bipyridinium [(bpy)H][VO(nta)(H2O)](H2O) (IV), and two newly synthesized compounds (I) and (II)—were evaluated against prostate cancer (PC3) and breast cancer (MCF-7) cells. All the compounds exhibited strong cytotoxic effects on cancer cells and normal cells (HaCaT human keratinocytes). The structure–activity relationship analysis revealed that the number and arrangement of conjugated aromatic rings in the counterion had an impact on the antitumor effect. The compound (III), the 1,10-phenanthrolinium analogue, exhibited the greatest activity, whereas the acridinium salt (II), with a different arrangement of three conjugated aromatic rings, showed the lowest toxicity. The increased concentrations of the compounds resulted in alterations to the cell cycle distribution with different effects in MCF-7 and PC3 cells. In MCF-7 cells, compounds I and II were observed to block the G2/M phase, while compounds III and IV were found to arrest the cell cycle in the G0/G1 phase. In PC3 cells, all compounds increased the rates of cells in the G0/G1 phase.

ABSTRACT This work reports detailed characteristics of the antimicrobial peptide Intestinalin (P30), which is derived from the LysC enzyme of Clostridium intestinale strain URNW. The peptide shows a broader antibacterial spectrum than the parental enzyme, showing potent antimicrobial activity against clinical strains of Gram-positive staphylococci and Gram-negative pathogens and causing between 3.04 ± 0.12 log kill for Pseudomonas aeruginosa PAO1 and 7.10 ± 0.05 log kill for multidrug-resistant Acinetobacter baumannii KPD 581 at a 5 μM concentration. Moreover, Intestinalin (P30) prevents biofilm formation and destroys 24-h and 72-h biofilms formed by Acinetobacter baumannii CRAB KPD 205 (reduction levels of 4.28 and 2.62 log CFU/mL, respectively). The activity of Intestinalin is combined with both no cytotoxicity and little hemolytic effect against mammalian cells. The nuclear magnetic resonance and molecular dynamics (MD) data show a high tendency of Intestinalin to interact with the bacterial phospholipid cell membrane. Although positively charged, Intestinalin resides in the membrane and aggregates into small oligomers. Negatively charged phospholipids stabilize peptide oligomers to form water- and ion-permeable pores, disrupting the integrity of bacterial cell membranes. Experimental data showed that Intestinalin interacts with negatively charged lipoteichoic acid (logK based on isothermal titration calorimetry, 7.45 ± 0.44), causes membrane depolarization, and affects membrane integrity by forming large pores, all of which result in loss of bacterial viability. IMPORTANCE Antibiotic resistance is rising rapidly among pathogenic bacteria, becoming a global public health problem that threatens the effectiveness of therapies for many infectious diseases. In this respect, antimicrobial peptides appear to be an interesting alternative to combat bacterial pathogens. Here, we report the characteristics of an antimicrobial peptide (of 30 amino acids) derived from the clostridial LysC enzyme. The peptide showed killing activity against clinical strains of Gram-positive and Gram-negative pathogens. Experimental data and computational modeling showed that this peptide forms transmembrane pores, directly engaging the negatively charged phospholipids of the bacterial cell membrane. Consequently, dissipation of the electrochemical gradient across cell membranes affects many vital processes, such as ATP synthesis, motility, and transport of nutrients. This kind of dysfunction leads to the loss of bacterial viability. Our firm conviction is that the presented study will be a helpful resource in searching for novel antimicrobial peptides that could have the potential to replace conventional antibiotics.

In this paper, we describe the chemical synthesis, preliminary evaluation of antimicrobial properties and mechanisms of action of a novel group of lipidated derivatives of three naturally occurring α-helical antimicrobial peptides, LL-I (VNWKKVLGKIIKVAK-NH2), LK6 (IKKILSKILLKKL-NH2), ATRA-1 (KRFKKFFKKLK-NH2). The obtained results showed that biological properties of the final compounds were defined both by the length of the fatty acid and by the structural and physico-chemical properties of the initial peptide. We consider C8–C12 length of the hydrocarbon chain as the optimal for antimicrobial activity improvement. However, the most active analogues exerted relatively high cytotoxicity toward keratinocytes, with the exception of the ATRA-1 derivatives, which had a higher selectivity for microbial cells. The ATRA-1 derivatives had relatively low cytotoxicity against healthy human keratinocytes but high cytotoxicity against human breast cancer cells. Taking into account that ATRA-1 analogues carry the highest positive net charge, it can be assumed that this feature contributes to cell selectivity. As expected, the studied lipopeptides showed a strong tendency to self-assembly into fibrils and/or elongated and spherical micelles, with the least cytotoxic ATRA-1 derivatives forming apparently smaller assemblies. The results of the study also confirmed that the bacterial cell membrane is the target for the studied compounds.

This study investigates short cationic antimicrobial lipopeptides composed of 2–4 amino acid residues and C12-C18 fatty acids attached to the N-terminal part of the peptides. The findings were discussed in the context of the relationship among biological activity, self-assembly, stability, and membrane interactions. All the lipopeptides showed the ability to self-assemble in PBS solution. In most cases, the critical aggregation concentration (CAC) much surpassed the minimal inhibitory concentration (MIC) values, suggesting that monomers are the main active form of lipopeptides. The introduction of β-alanine into the peptide sequence resulted in a compound with a high propensity to fibrillate, which increased the peptide stability and activity against S. epidermidis and C. albicans and reduced the cytotoxicity against human keratinocytes. The results of our study indicated that the target of action of lipopeptides is the bacterial membrane. Interestingly, the type of peptide counterion may affect the degree of penetration of the lipid bilayer. In addition, the binding of the lipopeptide to the membrane of Gram-negative bacteria may lead to the release of calcium ions necessary for stabilization of the lipopolysaccharide layer.

With the rapid and continuous emergence of antimicrobial resistance, bacterial infections became a significant global healthcare concern. One of the proposed strategies to combat multidrug-resistant pathogens is to use additional compounds, such as natural biologically active substances, as adjuvants for existing antibiotics. In this study, we investigated the potential of caffeine, the widely consumed alkaloid, to modulate the antibacterial effects of antibiotics commonly used in clinical practice. We used disc diffusion assay to evaluate the effects of caffeine on 40 antibiotics in two Staphylococcus aureus strains (methicillin-resistant and methicillin-sensitive). Based on the results of this step, we selected five antibiotics for which the greatest caffeine-induced improvements in antibacterial activity were observed, and further analyzed their interactions with caffeine using a checkerboard approach. Caffeine at concentrations of 250 µg/mL or higher halved the MIC values of ticarcillin, cefepime, gentamycin, azithromycin, and novobiocin for all gram-negative species investigated (Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii). At the highest caffeine concentrations tested (up to 16 mg/mL), decreases in MIC values were 8- to 16-fold. The obtained results prove that caffeine modulates the activity of structurally diverse antibiotics, with the most promising synergistic effects observed for cefepime and azithromycin toward gram-negative pathogens.

Due to the fact that surfactant molecules are known to alter the structure (and consequently the function) of a protein, protein–surfactant interactions are very important in the biological, pharmaceutical, and cosmetic industries. Although there are numerous studies on the interactions of albumins with surfactants, the investigations are often performed at fixed environmental conditions and limited to separate surface-active agents and consequently do not present an appropriate comparison between their different types and structures. In the present paper, the interactions between selected cationic, anionic, and nonionic surfactants, namely hexadecylpyridinium chloride (CPC), hexadecyltrimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS), polyethylene glycol sorbitan monolaurate, monopalmitate, and monooleate (TWEEN 20, TWEEN 40, and TWEEN 80, respectively) with bovine serum albumin (BSA) were studied qualitatively and quantitatively in an aqueous solution (10 mM cacodylate buffer; pH 5.0 and 7.0) by steady-state fluorescence spectroscopy supported by UV spectrophotometry and CD spectroscopy. Since in the case of all studied systems, the fluorescence intensity of BSA decreased regularly and significantly under the action of the surfactants added, the fluorescence quenching mechanism was analyzed thoroughly with the use of the Stern–Volmer equation (and its modification) and attributed to the formation of BSA–surfactant complexes. The binding efficiency and mode of interactions were evaluated among others by the determination, comparison, and discussion of the values of binding (association) constants of the newly formed complexes and the corresponding thermodynamic parameters (ΔG, ΔH, ΔS). Furthermore, the influence of the structure of the chosen surfactants (charge of hydrophilic head and length of hydrophobic chain) as well as different environmental conditions (pH, temperature) on the binding mode and the strength of the interaction has been investigated and elucidated.

The binding interactions of bovine serum albumin (BSA) with tetraphenylborate ions ([B(Ph)4]−) have been investigated by a set of experimental methods (isothermal titration calorimetry, steady-state fluorescence spectroscopy, differential scanning calorimetry and circular dichroism spectroscopy) and molecular dynamics-based computational approaches. Two sets of structurally distinctive binding sites in BSA were found under the experimental conditions (10 mM cacodylate buffer, pH 7, 298.15 K). The obtained results, supported by the competitive interactions experiments of SDS with [B(Ph)4]− for BSA, enabled us to find the potential binding sites in BSA. The first site is located in the subdomain I A of the protein and binds two [B(Ph)4]− ions (logK(ITC)1 = 7.09 ± 0.10; ΔG(ITC)1 = −9.67 ± 0.14 kcal mol−1; ΔH(ITC)1 = −3.14 ± 0.12 kcal mol−1; TΔS(ITC)1 = −6.53 kcal mol−1), whereas the second site is localized in the subdomain III A and binds five ions (logK(ITC)2 = 5.39 ± 0.06; ΔG(ITC)2 = −7.35 ± 0.09 kcal mol−1; ΔH(ITC)2 = 4.00 ± 0.14 kcal mol−1; TΔS(ITC)2 = 11.3 kcal mol−1). The formation of the {[B(Ph)4]−}–BSA complex results in an increase in the thermal stability of the alfa-helical content, correlating with the saturation of the particular BSA binding sites, thus hindering its thermal unfolding.

Clostridium botulinum is a Gram-positive, anaerobic, spore-forming bacterium capable of producing botulinum toxin and responsible for botulism of humans and animals. Phage-encoded enzymes called endolysins, which can lyse bacteria when exposed externally, have potential as agents to combat bacteria of the genus Clostridium. Bioinformatics analysis revealed in the genomes of several Clostridium species genes encoding putative N-acetylmuramoyl-l-alanine amidases with anti-clostridial potential. One such enzyme, designated as LysB (224-aa), from the prophage of C. botulinum E3 strain Alaska E43 was chosen for further analysis. The recombinant 27,726 Da protein was expressed and purified from E. coli Tuner(DE3) with a yield of 37.5 mg per 1 L of cell culture. Size-exclusion chromatography and analytical ultracentrifugation experiments showed that the protein is dimeric in solution. Bioinformatics analysis and results of site-directed mutagenesis studies imply that five residues, namely H25, Y54, H126, S132, and C134, form the catalytic center of the enzyme. Twelve other residues, namely M13, H43, N47, G48, W49, A50, L73, A75, H76, Q78, N81, and Y182, were predicted to be involved in anchoring the protein to the lipoteichoic acid, a significant component of the Gram-positive bacterial cell wall. The LysB enzyme demonstrated lytic activity against bacteria belonging to the genera Clostridium, Bacillus, Staphylococcus, and Deinococcus, but did not lyse Gram-negative bacteria. Optimal lytic activity of LysB occurred between pH 4.0 and 7.5 in the absence of NaCl. This work presents the first characterization of an endolysin derived from a C. botulinum Group II prophage, which can potentially be used to control this important pathogen.

Antimicrobial resistance is a major healthcare threat globally. Xanthines, including caffeine and pentoxifylline, are attractive candidates for drug repurposing, given their well-established safety and pharmacological profiles. This study aimed to analyze potential interactions between xanthines and aromatic antibiotics (i.e., tetracycline and ciprofloxacin), and their impact on antibiotic antibacterial activity. UV-vis spectroscopy, statistical-thermodynamical modeling, and isothermal titration calorimetry were used to quantitatively evaluate xanthine-antibiotic interactions. The antibacterial profiles of xanthines, and xanthine-antibiotic mixtures, towards important human pathogens Staphylococcus aureus, Enterococcus faecium, Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, and Enterobacter cloacae were examined. Caffeine and pentoxifylline directly interact with ciprofloxacin and tetracycline, with neighborhood association constant values of 15.8–45.6 M−1 and enthalpy change values up to −4 kJ·M−1. Caffeine, used in mixtures with tested antibiotics, enhanced their antibacterial activity in most pathogens tested. However, antagonistic effects of caffeine were also observed, but only with ciprofloxacin toward Gram-positive pathogens. Xanthines interact with aromatic antibiotics at the molecular and in vitro antibacterial activity level. Given considerable exposure to caffeine and pentoxifylline, these interactions might be relevant for the effectiveness of antibacterial pharmacotherapy, and may help to identify optimal treatment regimens in the era of multidrug resistance.

The alarming raise of multi-drug resistance among human microbial pathogens makes the development of novel therapeutics a priority task. In contrast to conventional antibiotics, antimicrobial peptides (AMPs), besides evoking a broad spectrum of activity against microorganisms, could offer additional benefits, such as the ability to neutralize toxins, modulate inflammatory response, eradicate bacterial and fungal biofilms or prevent their development. The latter properties are of special interest, as most antibiotics available on the market have limited ability to diffuse through rigid structures of biofilms. Lipidation of AMPs is considered as an effective approach for enhancement of their antimicrobial potential and in vivo stability; however, it could also have undesired impact on selectivity, solubility or the aggregation state of the modified peptides. In the present work, we describe the results of structural modifications of compounds designed based on cationic antimicrobial peptides DK5 and CAR-PEG-DK5, derivatized at their N-terminal part with fatty acids with different lengths of carbon chain. The proposed modifications substantially improved antimicrobial properties of the final compounds and their effectiveness in inhibition of biofilm development as well as eradication of pre-formed 24 h old biofilms of Candida albicans and Staphylococcus aureus. The most active compounds (C5-DK5, C12-DK5 and C12-CAR-PEG-DK5) were also potent against multi-drug resistant Staphylococcus aureus USA300 strain and clinical isolates of Pseudomonas aeruginosa. Both experimental and in silico methods revealed strong correlation between the length of fatty acid attached to the peptides and their final membranolytic properties, tendency to self-assemble and cytotoxicity.

Antimicrobial peptides (AMPs) constitute a promising tool in the development of novel therapeutic agents useful in a wide range of bacterial and fungal infections. Among the modifications improving pharmacokinetic and pharmacodynamic characteristics of natural AMPs, an important role is played by lipidation. This study focuses on the newly designed and synthesized lipopeptides containing multiple Lys residues or their shorter homologues with palmitic acid (C16) attached to the side chain of a residue located in the center of the peptide sequence. The approach resulted in the development of lipopeptides representing a model of surfactants with two polar headgroups. The aim of this study is to explain how variations in the length of the peptide chain or the hydrocarbon side chain of an amino acid residue modified with C16, affect biological functions of lipopeptides, their self-assembling propensity, and their mode of action.

Matrix metalloproteinases (MMPs) are extracellular matrix degradation factors, promoting cancer progression. Hence, they could provide an enzyme-assisted delivery of doxorubicin (DOX) in cancer treatment. In the current study, the intercalation process of DOX and tetrapeptide–DOX, the product of the MMPs’ cleavage of carrier-linked DOX, into dsDNA was investigated using stationary and time-resolved fluorescence spectroscopy, UV-Vis spectrophotometry and isothermal titration calorimetry (ITC). The molecular dynamics (MD) simulations on the same tetrapeptide–DOX…DNA and DOX…DNA systems were also performed. The undertaken studies indicate that DOX and tetrapeptide–DOX can effectively bond with dsDNA through the intercalation mode; however, tetrapeptide–DOX forms less stable complexes than free DOX. Moreover, the obtained results demonstrate that the differences in DNA affinity of both forms of DOX can be attributed to different intercalation modes. Tetrapeptide–DOX shows a preference to intercalate into DNA through the major groove, whereas DOX does it through the minor one. In summary, we can conclude that the tetrapeptide–DOX intercalation to DNA is significant and that even the lack of non-specific proteases releasing DOX from the tetrapeptide conjugate, the presence of which is suggested by the literature for the efficient release of DOX, should not prevent the cytostatic action of the anthracycline.

In the following paper, we present the results of our studies on the interactions of the Aβ1-42 peptide and its three short fragments, namely Aβ5-16 (RHDSGYEVHHQK; HZ1), Aβ8-13 (SGYEVH; HZ2), and Aβ8-12 (SGYEV; HZ3) with selected painkillers (ibuprofen and aspirin) and compounds of natural origin (anabasine and epinephrine). Steady-state fluorescence spectroscopy was used to study the binding properties of the selected systems. Additionally, based on molecular dynamics (MD) calculations supported by NMR-derived restrains, we have proposed the most likely area of the interactions of Aβ1-42 and Aβ5-16 peptides with the investigated compounds. The influence of symmetrically oriented side chains of amino acid residues present in the first part of the Aβ1-42 sequence on the stability of the resulting complexes has been discussed. Finally, the changes in the peptide structures on account of complex formation were analyzed.

Discovering that metals are essential for the structure and function of biomolecules has given a completely new perspective on the role of metal ions in living organisms. Nowadays, the design and synthesis of new metal-based compounds, as well as metal ion binding components, for the treatment of human diseases is one of the main aims of bioinorganic chemistry. One of the areas in vanadium-based compound research is their potential anticancer activity. In this review, we summarize recent molecular and cellular mechanisms in the cytotoxic activity of many different synthetic vanadium complexes as well as inorganic salts. Such mechanisms shall include DNA binding, oxidative stress, cell cycle regulation and programed cell death. We focus mainly on cellular studies involving many type of cancer cell lines trying to highlight some new significant advances.

Herein, we have investigated principally with the use of UV and fluorescence (steady-state and time-resolved) spectroscopy the interactions between selected pentapeptides with tyrosine residue (EYHHQ, EHYHQ, EHHQY, and KYHHE) and various metal ions (Cu2+, Mn2+, Co2+, Ni2+, Zn2+, Cr3+, Cd2+, Ag+, Pb2+, Sr2+, Ba2+, Ca2+, Mg2+, Al3+, Fe2+, and Ga3+) in order to establish the relationship between the position of a tyrosine residue in the peptide sequence and the metal ion-binding properties. Among the peptides studied, EHYHQ was evaluated as an efficient and selective ligand for developing a chemosensor for the detection of copper(II) ions. While significant fluorescence emission quenching was observed for that peptide in the presence of Cu2+ cations, other metal cations used at the same and at considerably higher concentrations caused a negligible change of the fluorescence emission spectrum, indicating a high selectivity of EHYHQ for Cu2+ ions. Under optimum conditions, fluorescence intensity was inversely proportional to the concentration of Cu2+ ions. The limit of detection of Cu2+ ions with the use of EHYHQ was determined at the level of 26.6 nM. The binding stoichiometry of the complexes of the studied peptides with Cu2+ ions was evaluated spectrophotometrically and fluorimetrically (as in the case of EHYHQ confirmed by mass spectrometry) and found to be 1:2 (Cu2+-peptide) for all the investigated systems. Furthermore, the stability constant (K) values of these complexes were determined. The reversibility of the proposed Cu2+ ions sensor was confirmed, the pH range where the sensor acts was determined, while its analytical performance was compared with some other reported recently fluorescent sensors. The mechanism of the interactions between EHYHQ and Cu2+ was proposed on the basis of NMR spectroscopy investigations.

The purpose of this study was to examine the application of the coordinated cis-[Cr(C2O4)(pm)(OH)2]+ cation where pm denotes pyridoxamine, as a specific sensing ion for the detection of hydrogen peroxide (H2O2). The proposed method for H2O2 detection includes two key steps. The first step is based on the nonenzymatic decarboxylation of pyruvate upon reaction with H2O2, while the second step is based on the interaction of cis-[Cr(C2O4)(pm)(OH2)2]+ with the CO2 released in the previous step. Using this method H2O2 generated during glutamate-induced oxidative stress was detected in HT22 hippocampal cells. The coordination ion cis-[Cr(C2O4)(pm)(OH2)2]+ and the spectrophotometric stopped-flow technique were applied to determine the CO2 concentration in cell lysates, supernatants and cell-free culture medium. Prior to CO2 assessment pyruvate was added to all samples studied. Pyruvate reacts with H2O2 with 1:1 stoichiometry, and consequently the amount of CO2 released in this reaction is equivalent to the amount of H2O2.

It is shown that probably three residues: His6, His14 and His16 in the original sequence Aβ(1−42) serve as metal-binding sites for Cu2+ ions. On the other hand, there is a possibility that only one of them plays a crucial role in the formation of the {Aβ(1−42) --Cu2+} complex. The isothermal titration calorimetry (ITC) measurements supported by molecular dynamic simulation (MD) with the NMR derived restrains were used to investigate the interactions of Cu2+ with Aβ(5−16), a fragment of the Aβ(1−42) protein, with the following sequence: Ac--Arg--His--Asp--Ser--Gly--Tyr--Glu--Val--His--His--Gln--Lys--NH2, termed HZ1. The conditional thermodynamic parameters suggest that the formation of the Cu2+--HZ1 complex is both an enthalpy and entropy driven process under the experimental conditions. The studies presented here (after comparison with our previous results) show that the affinity of peptides to copper metal ions depends on two factors: the primary structure (amino acid composition) and the shape of the peptide conformation adopted.

This paper reports on dihydroxycoumarins as fluorescent probes suitable for the detection and determination of the nitroxide radical, namely 4-amino-TEMPO. Since 4-amino-TEMPO is used as a spin label for the detection of various radicals and damage caused by these species, its determination under physiological conditions might help us to understand the mechanism of the oxidative stress. Among different coumarins studied, only dihydroxy-substituted derivatives show high sensitivity, specificity, and selectivity for the nitroxide radical. In this assay, dihydroxy-substituted coumarins under the action of 4-amino-TEMPO show a very fast and significant increase in fluorescence intensity and lifetime. Among them 6,7-dihydroxycoumarin (esculetin) exhibits the strongest fluorescence enhancement (up to 40 times), with an estimated limit of detection equal to 16.7 nM—a significantly lower value when compared with UV-Vis or electron paramagnetic resonance (EPR) spectroscopy. The method is characterized by an easy procedure of sample preparation and very short time of analysis. The mechanism of the interaction between 6,7-dihydroxycoumarin and 4-amino-TEMPO has been examined with the use of a series of complementary techniques, such as steady-state and time-resolved fluorescence spectroscopy, UV-Vis spectroscopy, electron paramagnetic resonance spectroscopy, potentiometric titration, and high-performance liquid chromatography. It has been proven that the only route of the reaction in the system studied is a proton transfer from the molecule of esculetin to the amino group of the nitroxide. Biological studies performed on prostate cancer cells, breast cancer cells, and normal skin fibroblasts revealed significant anticancer properties of 6,7-dihydroxycoumarin, which caused a considerable decrease in the viability and number of cancer cells, and affected their morphology, contrary to normal fibroblasts. Furthermore, the experiment performed on prostate cancer cells showed that fluorescence emission of esculetin is closely related to intracellular pH—the higher pH, the higher observed fluorescence intensity (in accordance with a chemical experiment). On the other hand, the studies performed in different pH levels revealed that when pH of the solution increases, the observed fluorescence intensity enhancement under the action of 4-amino-TEMPO decreases (better sensing properties of esculetin towards the nitroxide in lower pH).

Among metal-based nanoparticles, silver nanoparticles (AgNPs) are particularly appealing because of their stability, functionality, and documented antimicrobial properties. AgNPs also offer the possibility of different surface modifications. In this work, we functionalized AgNPs with thiobarbituric acid or 11-mercaptoundecanoic acid residues to improve the nanoparticles’ biological activities. Subsequently, we assessed the physicochemical properties of newly synthesized AgNPs using a wide range of biophysical methodologies, including UV/vis and fluorescence spectroscopy, atomic force and scanning electron microscopy, and dynamic light scattering and isothermal titration calorimetry. Next, we examined the effect of nanoparticles functionalization on AgNPs mutagenicity and toxicity. Our study revealed that AgNPs’ surface modification affects nanoparticles aggregation, and also impacts nanoparticles’ interaction with model acridine mutagen ICR-191. AgNPs coated with MUA showed the most interesting interactions with tested ICR-191, slightly modulating its toxicity properties by decreasing the viability in treated cells.

Pancreatic cancer is characterized by one of the lowest five-year survival rates. In search for new treatments, some studies explored several metal complexes as potential anticancer drugs. Therefore, we investigated three newly synthesized oxidovanadium(IV) complexes with 2-methylnitrilotriacetate (bcma3−), N-(2-carbamoylethyl)iminodiacetate (ceida3−) and N-(phosphonomethyl)-iminodiacetate (pmida4−) ligands as potential anticancer compounds using pancreatic cancer cell lines. We measured: Cytotoxicity using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), neutral red (NR) and lactate dehydrogenase (LDH) assay; antiproliferative activity by bromodeoxyuridine BrdU assay; reactive oxygen species (ROS) generation and cell cycle analysis by flow cytometry; protein level by Western blot and cellular morphology by confocal laser scanning microscopy. The results showed that these oxidovanadium(IV) complexes were cytotoxic on pancreatic cancer cells (PANC-1 and MIA PaCa2), but not on non-tumor human immortalized pancreas duct epithelial cells (hTERT-HPNE) over the concentration range of 10–25 μM, following 48 h incubation. Furthermore, molecular mechanisms of cytotoxicity of [4-NH2-2-Me(Q)H][VO(bcma)(H2O)]2H2O (T1) were dependent on antiproliterative activity, increased ROS generation, cell cycle arrest in G2/M phase with simultaneous triggering of the p53/p21 pathway, binucleation, and induction of autophagy. Our study indicates that oxidovanadium(IV) coordination complexes containing 2-methylnitrilotriacetate ligand are good candidates for preclinical development of novel anticancer drugs targeting pancreatic cancer.

The direct and accurate estimation of nitric dioxide levels is an extremely laborious and technically demanding procedure in the molecular diagnostics of inflammatory processes. The aim of this work is to demonstrate that a stop-flow technique utilizing a specific spectroscopic biosensor can be used for detection of nanomolar quantities of NO2 in biological milieu. The use of novel compound cis-[Cr(C2O4)(AaraNH2)(OH2)2]+ increases NO2 estimation accuracy by slowing down the rate of NO2 uptake. In this study, an animal model of pancreatitis, where nitrosative stress is induced by either 3g/kg bw or 1.5 g/kg bw dose of L-arginine, was used. Biochemical parameters and morphological characteristics of acute pancreatitis were monitored, specifically assessing pancreatic acinar cell death mode, NO2 generation and cellular glutathione level. The severity of the process correlated positively with NO2 levels in pancreatic acinar cell cytosol samples, and negatively with cellular glutathione levels.

The paper presents a new convenient, inexpensive, and reagent-saving general methodology for the determination of pKa values for components of the mixture of diverse chemical classes weak organic acids and bases in water solution, without the need to separate individual analytes. The data obtained from simple pH-metric microtitrations are numerically processed into reliable pKa values for each component of the mixture. Excellent agreement has been obtained between the determined pKa values and the reference literature data for compounds studied.