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Secondary polyphenol metabolites, urolithins (UROs), have anti-oxidative, anti-inflammatory, and antidiabetic properties. Therefore, their biological activity relies on blood transport via human serum albumin (HSA) and tissue distribution. The main goal we set was to investigate the interaction between HSA and different URO (URO A, URO B, URO C, URO D, and glucuronidated URO A and B) using a combination of multi-spectroscopic instrumental and in silico approaches. The fluorescence spectroscopy revealed that URO can quench the naturally occurring fluorescence of HSA in a concentration-dependent manner. The HSA fluorescence was quenched by both a static and dynamic mechanism. The results showed that free UROs bind to HSA with higher affinity than their conjugated forms. CD spectroscopy and FTIR revealed that the alpha-helical structure of HSA is preserved. The calculated Gibbs free energy change indicates that the URO-HSA complex forms spontaneously. There is a single binding site on the HSA surface. The molecular docking results indicated that unconjugated Uro binds to Sudlow I, while their conjugation affects this binding site, so in the conjugated form, they bind to the cleft. Docking experiments indicate that all UROs are capable of binding to both thyroxine recognition sites of ligand-bound HSA proteins. Examining interactions under the following conditions (298 K, 303 K, and 310 K, pH 7.4) is of great importance for determining the pharmacokinetics of these bioactive compounds, as the obtained results can be used as a basis for modulating the potential dosing regimen.

The significant role of papain-like cysteine proteases, including papain, cathepsin L and SARS-CoV-2 PLpro, in biomedicine and biotechnology makes them interesting model systems for sensor development. These enzymes have a free thiol group that is suitable for many sensor designs including strong binding to gold nanoparticles or low-molecular-weight inhibitors. Focusing on the importance of the preservation of native protein structure for inhibitor-binding and molecular-imprinting, which has been applied in some efficient examples of sensor development, the aim of this work was to examine the effects of the free-thiol-group's reversible blocking on papain denaturation that is the basis of its activity loss and aggregation. To utilize biophysical methods common in protein structural transitions characterization, such as fluorimetry and high-resolution infrared spectroscopy, low-molecular-weight electrophilic thiol blocking reagent S-Methyl methanethiosulfonate (MMTS) was used in solution. MMTS binding led to a two-fold increase in 8-Anilinonaphthalene-1-sulfonic acid fluorescence, indicating increased hydrophobic residue exposure. A more in-depth analysis showed significant transitions on the secondary structure level upon MMTS binding, mostly characterized by the lowered content of α-helices and unordered structures (either for approximately one third), and the increase in aggregation-specific β-sheets (from 25 to 52%) in a dose-dependant manner. The recovery of this inhibited protein showed that reversibility of inhibition is accompanied by reversibility of protein denaturation. Nevertheless, a 100-fold molar excess of the inhibitor led to the incomplete recovery of proteolytic activity, which can be explained by irreversible denaturation. The structural stability of the C-terminal β-sheet rich domain of the papain-like cysteine protease family opens up an interesting possibility to use its foldamers as a strategy for sensor development and other multiple potential applications that rely on the great commercial value of papain-like cysteine proteases.

Varroa destructor is a parasitic mite responsible for the loss of honey bee ( Apis mellifera ) colonies. This study aimed to find a promising marker in honey for the bee colony infestation level using fluorescence spectroscopy and biochemical analyses. We examined whether the parameters of the honey samples' fluorescence spectra and biochemical parameters, both related to proteins and phenolics, may be connected with the level of honey bee colonies' infestation. The infestation level was highly positively correlated with the catalase activity in honey (r = 0.936). Additionally, the infestation level was positively correlated with the phenolic spectral component (r = 0.656), which was tentatively related to the phenolics in honey. No correlation was found between the diastase activity in honey and the colonies' infestation level. The results indicate that the catalase activity in honey and the PFC1 spectral component may be reliable markers for the V. destructor infestation level of the colonies. The obtained data may be related to the honey yield obtained from the apiaries.

The surface of microorganisms is covered with carbohydrates, which makes them unique, self-sustaining glycan probes. Lectins are able to bind to these probes, and this interaction can be exploited for selecting microorganisms or novel lectins. To examine lectin-microorganism interactions, we have previously developed an enzyme-linked lectin sorbent assay (ELLSA) with whole bacterial cells. To further test the validity of this methodology, here we compare it with flow cytometry. For this purpose, we used biotinylated recombinantly produced lectin from Musa acuminata (BanLec), this lectin's recombinantly produced chimera with green fluorescent protein (BanLec-eGFP) and a lectin from Ricinus communis (RCA 120 ), both biotinylated and FITC labeled. Parallel testing showed equivalent results for the two methods, in terms of the presence or absence of binding, with signal intensity yielding high Pearson correlation coefficient of 0.8 for BanLec and 0.95 for RCA 120 . The ELLSA method demonstrated multiple advantages, such as reliability and convenience for high-throughput analysis; it also required less lectin and yielded more consistent results. As such, ELLSA proved to be a useful tool for profiling microbial glycan structures or testing novel lectins., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Trypsin is a serine protease with important applications such as protein sequencing and tissue dissociation. Preserving protein structure and its activity during freeze-thawing and prolonging its shelf life is one of the most interesting tasks in biochemistry. In the present study, trypsin cryoprotection was achieved by altering buffer composition. Sodium phosphate buffer at pH 8.0 led to pH shift-induced destabilization of trypsin and formation of a molten globule, followed by significant activity loss (about 70%). Potassium phosphate and ammonium bicarbonate buffers at pH 8.0 were used with up to 90% activity recovery rate after 7 freeze-thaw cycles. The addition of non-ionic surfactants Tween 20 and Tween 80 led to up to 99% activity recovery rate. Amide I region changes, corresponding to specific secondary structures in the Fourier transform infrared (FTIR) spectrum, were modest in the case of Tween 20 and Tween 80. On the other hand, the addition of Triton X-100 led to the destabilization of α-helicoidal segments of trypsin structure after 7 freeze-thaw cycles but also increased protein substrate availability., (Copyright © 2020 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Oligomeric intermediates on the pathway of amyloid fibrillation are suspected as the main cytotoxins responsible for amyloid-related pathogenicity. As they appear to be a part of the lag phase of amyloid fibrillation when analyzed using standard methods such as Thioflavin T (ThT) fluorescence, a more sensitive method is needed for their detection. Here we apply Fourier transform infrared spectroscopy (FTIR) in attenuated total reflectance (ATR) mode for fast and cheap analysis of destabilized hen-egg-white lysozyme solution and detection of oligomer intermediates of amyloid fibrillation. Standard methods of protein aggregation analysis- Thioflavin T (ThT) fluorescence, atomic force microscopy (AFM), and 8-anilinonaphthalene-1-sulphonic acid (ANS) fluorescence were applied and compared to FTIR spectroscopy data. Results show the great potential of FTIR for both, qualitative and quantitative monitoring of oligomer formation based on the secondary structure changes. While oligomer intermediates do not induce significant changes in ThT fluorescence, their secondary structure changes were very prominent. Normalization of specific Amide I region peak intensities by using Amide II peak intensity as an internal standard provides an opportunity to use FTIR spectroscopy for both qualitative and quantitative analysis of biological samples and detection of potentially toxic oligomers, as well as for screening of efficiency of fibrillation procedures.

Measurement of antithyroglobulin antibodies (TgAb) is an inevitable laboratory tool in the management of thyroid gland diseases. Currently available immunoassays still have limitations underlying the necessity of the introduction of fast, sensitive, and label-free technologies. Our aim was to develop a method for TgAb measurement in human serum based on the quartz crystal microbalance (QCM) technology. We immobilized thyroglobulin on the surface of Attana LNB Carboxyl sensor chip®, prepared standard curve covering the range of 1-50000 kIU/L, and established optimal measurement conditions. The validation included determination of the detection limit (LOD), functional sensitivity, linearity, precision, as well as the comparison with the results of the radioimmunoassay (RIA). The LOD and functional sensitivity were 4.2 kIU/L and 4.7 kIU/L, respectively. The method was linear in the range of 20-10000 kIU/L. The regression equation for comparison with RIA was C QCM = 1.0056 • C RIA - 24.2778, whereby no significant proportional or systematic difference was present. There was a good agreement with RIA in the classification of patients according to the clinical significance of the results. The developed method has advantages over currently available assays in terms of better LOQ, a higher upper limit of linearity, and precision. The characteristics of the developed method unambiguously show that the application of the QCM biosensors offers a highly reliable novel approach for the measurement of TgAb in human serum., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Numerous applications of proteolytic enzymes include dissociation of fermented meat products for the enumeration of `foodborne pathogenic bacteria. The use of trypsin for this cause is abandoned due to the high concentration of the enzyme affecting released bacteria. Papain, as a suggested replacement, and fig latex preparation with high extent of papain-like enzymes have the potential to be applied for bacteria enumeration. Both enzymatic preparations, originating from papaya and fig, showed a broader range of substrate specificities including gelatinolytic activity, especially prominent in the case of ficin and attributed to both, cysteine protease ficin and serine protease by the analysis of 2D zymography with specific inhibitors. The activity towards native collagen, mild in the case of papain, and extensive in the case of fig latex was proved by structural analysis of digested collagen by infrared spectroscopy. Further exploration of their potential for dissociation of fermented meat products showed that both papain and fig latex enzymes are stable in the presence of detergents Tween 20 and Triton X-100 and effective in the enumeration of Listeria monocytogenes. Gelatenolytic activity, and at least partial collagenolytic activity and stability in procedure conditions make papaya and fig latex proteases potent for this application in significantly lower concentrations than previously used enzymes. As a mixture of proteolytic enzymes with divergent characteristics, fig latex preparation shows higher efficiency in Listeria monocytogenes release than papain, conserved even in the presence of stronger non-ionic detergent Triton X-100., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Burkholderia cepacia is well known as the causative agent of infections in humans where often shares niche with other pathogens, like Pseudomonas aeruginosa. Clinical isolate Burkholderia sp. BCC4135 was selected due to its strong quorum quenching (QQ) activity. Whole genome sequencing unveiled this isolate as B. cepacia with unique sequence type ST1485 and a myriad of genes belonging to resistome and virulome. Two QQ lactonases YtnP and Y2-aiiA originated from B. cepacia BCC4135 were cloned, expressed, and functionally characterized. They were active against a broad substrate spectrum of the N-acyl-homoserine lactones (AHLs). The YtnP lactonase was inactive, while Y2-aiiA was active against N-tetradecanoyl-dl-homoserine lactone (C14-HSL) which could imply the difference in their biological roles from the aspect of its quorum sensing (QS) autoregulation and interference with the QS systems of bacteria residing within the same niche. Both YtnP and Y2-aiiA were able to attenuate virulence potential of P. aeruginosa MMA83 clinical isolate declining its biofilm formation and virulence factors production. B. cepacia BCC4135 lactonases interfered with the las, rhl, and even pqs QS circuit of P. aeruginosa MMA83 transcription and the effect of combined enzymes was even more prominent. B. cepacia BCC4135 also employs the CepI/R QS system for governing its own virulence traits and possibly self-regulates the QQ/QS network through the different expression and activity of YtnP and/or Y2-aiiA. Our findings pointed out that BCC4135 lactonases could be exploited as an effective antivirulence drugs against P. aeruginosa and gave us a new insight into B. cepacia QQ/QS machinery., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Amyloid fibrils are highly ordered self-assembled (poly)peptide aggregates with cross-β structural pattern. Ovalbumin was used as a model for exploring the potential of infrared spectroscopy in detecting structural transitions and quantitative monitoring of amyloid fibrillation. Low pH (pH 2) and high temperature (90 °C) over the course of 24 h were conditions applied for amyloid formation. Fibrillation of ovalbumin was monitored by ThT and ANS fluorescence, and SDS PAGE. A significant increase in ThT fluorescence with a plateau reached after 4 h of incubation, without the lag phase, was detected. Structural transitions leading to amyloid fibrillation were analysed using all three Amide regions in ATR-FTIR spectra. Significant changes were detected in Amide I and Amide III region (decrease of α-helix and increase of β-sheet peaks). To establish a fast, precise and simple method for quantitative monitoring of amyloid fibrillation, the Amide I/Amide II ratios of aggregation specific β-sheets (1625 and 1695 cm -1 , respectively) with 1540 cm -1 as internal standard were used, resulting in good correlation (R 2  = 0.93 and 0.95) with the data observed by monitoring ThT fluorescence. On the other hand, assessing aggregation specific β-sheet contents by self-deconvolution showed lower correlation with ThT fluorescence (R 2  = 0.75 and 0.64). Here we examined structural transitions during ovalbumin fibrillation in a qualitative and quantitative manner by exploiting the full potential of Amide regions simultaneously. Secondary structure distribution was monitored using second derivative spectra in Amide I region. A novel, simple mathematical calculation for quantitative monitoring of fibrils formation was presented employing that the increase in low and high frequency aggregation specific β-sheet in Amide I region compared to the internal standard in Amide II region is suitable for fibril formation monitoring., 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 © 2018. Published by Elsevier B.V.)

Pseudomonas aeruginosa is one of the most common opportunistic pathogens that use quorum sensing (QS) system to regulate virulence factors expression and biofilm development. Delftia sp. 11304 was selected among 663 Gram-negative clinical isolates based on its QS inhibitory activity against P. aeruginosa MMA83 clinical isolate. Whole genome sequencing identified this isolate as D. tsuruhatensis and revealed genetic armamentarium of virulence factors and antibiotic resistance determinants. Ethyl acetate extract of D. tsuruhatensis 11304 culture supernatant (QSI extract) prevented biofilm formation of P. aeruginosa MMA83, but was unable to cause biofilm decomposition. QSI extract showed a synergistic effect in combination with meropenem and gentamycin, against P. aeruginosa MMA83. A dose-dependent reduction of the virulence factors: elastase, rhamnolipid and pyocyanin production by P. aeruginosa MMA83 and significant downregulation of lasI, lasR, rhlI, rhlR, pqs and mvfR expression were observed. Matrix-assisted Laser Desorption Ionization (MALDI) mass spectrometry of D. tsuruhatensis 11304 QSI extract revealed the presence of N-acyl homoserine lactones (AHL) with chain lengths of C12 to C18. The main ion peak was identified as N-octadecanoylhomoserine lactone (C 18 -HSL). Commercial C 18 -HSL (20 µM) reduced pyocyanin production as well as mRNA level of the lasI gene. A novel AHL species, dihydroxy-N-octadecanoylhomoserine lactone, was also described.

Proteolytic enzymes are used for proteolysis and peptide synthesis which can be run in various conditions including low pH value and the presence of ethanol. The most common cysteine protease applied in acidic-alcoholic conditions is well-characterized papain. Ficin, which is closely related to papain in terms of proteolytic activity and substrate specificity, could potentially be applied in the alcoholic beverage industry and peptide synthesis. The aim of this study was to compare papain and ficin stability in process conditions. Comparative stability study showed that ficin as a mixture of different isoforms has a broader range of stability in respect of pH and cold storage stability, in comparison to papain. It retains about 70% of initial activity after 3-week cold storage at low pH and in the presence of ethanol. Unlike ficin, papain loses about 70% of initial activity in the same incubation period as it is more prone to non-native aggregation that was confirmed by FTIR analysis. The presence of multiple isoforms of ficin stabilizes the protease against cold denaturation and aggregation, making it more suitable for biotechnological and laboratory usage than single papain isoform. It is more cold-stable in alcoholic-acidic and acidic conditions suggesting possible replacement of papain with even lower enzyme concentration.

Collagenolytic proteases have many potential applications in different areas of science, industry, and medicine. The determination of the activity of such proteins is paramount to their application. Here, we describe methods which can be applied to determine the activity and some basic characteristics of potential collagenases.

Papain is a cysteine protease with wide substrate specificity and many applications. Despite its widespread applications, cold stability of papain has never been studied. Here, we used differential spectroscopy to monitor thermal denaturation process. Papain was the most stabile from 45 °C to 60 °C with ΔG°321 of 13.9±0.3 kJ/mol and Tm value of 84±1 °C. After cold storage, papain lost parts of its native secondary structures elements which gave an increase of 40% of intermolecular β-sheet content (band maximum detected at frequency of 1621 cm(-1) in Fourier transform infrared (FT-IR) spectrum) indicating the presence of secondary structures necessary for aggregation. The presence of protein aggregates after cold storage was also proven by analytical size exclusion chromatography. After six freeze-thaw cycles around 75% of starting enzyme activity of papain was lost due to cold denaturation and aggregation of unfolded protein. Autoproteolysis of papain did not cause significant loss of the protein activity. Upon the cold storage, papain underwent structural rearrangements and aggregation that correspond to other cold denatured proteins, rather than autoproteolysis which could have the commercial importance for the growing polypeptide based industry., (Copyright © 2015 Elsevier B.V. All rights reserved.)

In search for novel biologically active metal based compounds, an evaluation of in vitro cytotoxic, antioxidant, and antimicrobial activity of new Pt(II) complex and its Zn(II), Cu(II), and Co(III) analogues, with NNO tridentately coordinated N-heteroaromatic Schiff base ligand (E)-2-[N'-(1-pyridin-2-yl-ethylidene)hydrazino]acetate, was performed. Investigation of antioxidative properties showed that all of the compounds have strong radical scavenging potencies. The Zn(II) complex showed potent inhibition of DNA cleavage by hydroxyl radical. A cytotoxic action of investigated compounds was evaluated on cultures of human promyelocitic leukaemia (HL-60), human glioma (U251), rat glioma (C6), and mouse melanoma (B16) cell lines. It was shown that binuclear pentacoordinated Zn(II) complex possesses a strong dose-dependent cytotoxic activity, of the same order of magnitude as cisplatin on B16, C6, and U251 cells. Furthermore, Zn(II) complex causes oxidative stress-induced apoptotic death of HL-60 leukemic cells, associated with caspase activation, phosphatidylserine externalization, and DNA fragmentation., (© 2013 Wiley Periodicals, Inc.)