Facile Synthesis of Dual-Functional Cross-Linked Membranes with Contact-Killing Antimicrobial Properties and Humidity-Response.

Poly(2-hydroxyethylmethacrylate-co-2-(dimethylamino)ethyl methacrylate), P(HEMA-co-DMAEMAx), copolymers were quaternized through the reaction of a part of (dimethylamino)ethyl moieties of DMAEMA units with 1-bromohexadecane. Antimicrobial coatings were further prepared through the cross-linking reaction between the remaining DMAEMA units of these copolymers and the epoxide ring of poly(N,N-dimethylacrylamide-co-glycidyl methacrylate), P(DMAm-co-GMAx), copolymers. The combination of P(HEMA-co-DMAEMAx)/P(DMAm-co-GMAx) copolymers not only enabled control over quaternization and cross-linking for coating stabilization but also allowed the optimization of the processing routes towards a more facile cost-effective methodology and the use of environmentally friendly solvents like ethanol. Careful consideration was given to achieve the right content of quaternized units, qDMAEMA, to ensure antimicrobial efficacy through an appropriate amphiphilic balance and sufficient free DMAEMA groups to react with GMA for coating stabilization. Optimal synthesis conditions were achieved by membranes consisting of cross-linked P(HEMA78-co-DMAEMA9-co-qDMAEMA13)/P(DMAm-co-GMA42) membranes. The obtained membranes were multifunctional as they were self-standing and antimicrobial, while they demonstrated a distinct fast response to changes in humidity levels, widening the opportunities for the construction of "smart" antimicrobial actuators, such as non-contact antimicrobial switches.

Rational Design, Synthesis and Binding Affinity Studies of Anthraquinone Derivatives Conjugated to Gonadotropin-Releasing Hormone (GnRH) Analogues towards Selective Immunosuppression of Hormone-Dependent Cancer.

Gonadotropin-releasing hormone (GnRH) is pivotal in regulating human reproduction and fertility through its specific receptors. Among these, gonadotropin-releasing hormone receptor type I (GnRHR I), which is a member of the G-protein-coupled receptor family, is expressed on the surface of both healthy and malignant cells. Its presence in cancer cells has positioned this receptor as a primary target for the development of novel anti-cancer agents. Moreover, the extensive regulatory functions of GnRH have underscored decapeptide as a prominent vehicle for targeted drug delivery, which is accomplished through the design of appropriate conjugates. On this basis, a rationally designed series of anthraquinone/mitoxantrone-GnRH conjugates (con1-con8) has been synthesized herein. Their in vitro binding affinities range from 0.06 to 3.42 nM, with six of them (con2-con7) demonstrating higher affinities for GnRH than the established drug leuprolide (0.64 nM). Among the mitoxantrone based GnRH conjugates, con3 and con7 show the highest affinities at 0.07 and 0.06 nM, respectively, while the disulfide bond present in the conjugates is found to be readily reduced by the thioredoxin (Trx) system. These findings are promising for further pharmacological evaluation of the synthesized conjugates with the prospect of performing future clinical studies.

Preparation of Antimicrobial Coatings from Cross-Linked Copolymers Containing Quaternary Dodecyl-Ammonium Compounds.

One of the concerns today's societies face is the development of resistant pathogenic microorganisms. The need to tackle this problem has driven the development of innovative antimicrobial materials capable of killing or inhibiting the growth of microorganisms. The present study investigates the dependence of the antimicrobial activity and solubility properties on the hydrophilicity/hydrophobicity ratio of antimicrobial coatings based on quaternary ammonium compounds. In this line, suitable hydrophilic and hydrophobic structural units were selected for synthesizing the antimicrobial copolymers poly(4-vinylbenzyl dimethyldodecylammonium chloride-co-acrylic acid), P(VBCDDA-co-AA20) and poly(dodecyltrimethylammonium 4-styrene sulfonate-co-glycidyl methacrylate), P(SSAmC12-co-GMA20), bearing an alkyl chain of 12 carbons either through covalent bonding or through electrostatic interaction. The cross-linking reaction of the carboxylic group of acrylic acid (AA) with the epoxide group of glycidyl methacrylate (GMA) of these two series of reactive antimicrobial copolymers was explored in blends, obtained through solution casting after curing at various temperatures. The release of the final products in pure water and NaCl 1 M solutions (as analyzed by gravimetry and total organic carbon, TOC/total nitrogen, TN analyses), could be controlled by the coating composition. The cross-linked polymeric membranes of composition 60/40 w/w % ratios led to 97.8 and 99.7% mortality for Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), respectively, whereas the coating 20/80 w/w % resulted in 96.6 and 99.8% cell reduction. Despite the decrease in hydrophobicity (from a 16- to a 12-carbon alkyl chain), the new materials maintained the killing efficacy, while at the same time resulting in increased release to the aqueous solution.

Recovery and quantification of a myelin oligodendrocyte glycoprotein peptide from rat plasma after protein precipitation.

The recovery of high molecular weight peptides from complex biological samples is a challenging task. Herein, a reliable, cost effective and rapid methodology was developed for the recovery and quantification of a myelin oligodendrocyte glycoprotein epitope namely (LysGly)5MOG35-55, from rat plasma. Removal of plasma proteins before quantification of the peptide was achieved after precipitation by an acetonitrile/water/formic acid solution. Using the developed protocol, average recoveries of the peptide from plasma ranged between 83.3 and 90.3%.

Identification of the zinc, copper and cadmium metalloproteome of the protozoon Tetrahymena thermophila by systematic bioinformatics.

Tetrahymena thermophila (T. thermophila) is a ciliated protozoon that can detect freshwater pollution by heavy metals ("whole-cell biosensor"). This work employed a systematic bioinformatics approach to predict and analyze the metalloproteome of T. thermophila for the essential Zn, Cu and the non-essential Cd. 3784 metal-binding proteins were identified compared to the 456 annotated so far in UniProt. The localization, functional classification, and the functionally enriched network of the newly identified metalloproteome are presented. Cd toxicity could be explained in terms of the metal replacing Cu and especially Zn in MAPKs, transporters and antioxidant enzymes. The predicted results for Cd toxicity and responses reflect those observed experimentally in different organisms after their exposure to Cd.

Conjugation of a peptide to mannan and its confirmation by tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

The conjugation of polysaccharides to peptides is essential for antigen delivery and vaccine development. Herein, we show that tricine SDS-PAGE in combination with Coomassie Blue staining was adequate to determine the conjugation efficacy of a peptide (epitope 35-55 of myelin oligodendrocyte glycoprotein) to mannan. In addition, tricine SDS-PAGE and periodic acid-Schiff stains were able to monitor the redox state of mannan. Using the described protocol, more than 99.9% of a peptide containing five lysines at its N-terminus was confirmed conjugated to mannan.

Inhibition of bacterial thioredoxin reductase: an antibiotic mechanism targeting bacteria lacking glutathione.

Increasing antibiotic resistance makes the identification of new antibacterial principles an urgent task. The thioredoxin system including thioredoxin reductase (TrxR), thioredoxin (Trx), and NADPH plays critical roles in cellular DNA synthesis and defense against oxidative stress. Notably, TrxR is very different in structure and mechanism in mammals and bacteria. Ebselen [2-phenyl-1,2 benzisoselenazol-3(2H)-one], a well-known antioxidant and a substrate for mammalian TrxR and Trx, is rapidly bacteriocidal for methicillin-resistant Staphylococcus aureus by an unknown mechanism. We have discovered that ebselen is a competitive inhibitor of Escherichia coli TrxR with a Ki of 0.52 ± 0.13 µM, through reaction with the active site dithiol of the enzyme. Bacteria lacking glutathione (GSH) and glutaredoxin, in which TrxR and Trx are essential for DNA synthesis, were particularly sensitive to ebselen. In growth-inhibited E. coli strains, Trx1 and Trx2 were oxidized, demonstrating that electron transfer via thioredoxin was blocked. Ebselen and its sulfur analog ebsulfur were bactericidal for GSH-negative pathogens. Ebsulfur inhibited a clinically isolated Helicobacter pylori strain with a minimum inhibitory concentration value as low as 0.39 µg/ml. These results demonstrate that bacterial Trx and TrxR are viable antibacterial drug targets using benzisoselenazol and benzisothiazol derivates.

Solution structure and biophysical properties of MqsA, a Zn-containing antitoxin from Escherichia coli.

The gene ygiT (mqsA) of Escherichia coli encodes MqsA, the antitoxin of the motility quorum sensing regulator (MqsR). Both proteins are considered to form a DNA binding complex and to be involved in the formation of biofilms and persisters. We have determined the three-dimensional solution structure of MqsA by high-resolution NMR. The protein comprises a well-defined N-terminal domain with a Zn finger motif usually found in eukaryotes, and a defined C-terminal domain with a typical prokaryotic DNA binding helix-turn-helix motif. The two well-defined domains of MqsA have almost identical structure in solution and in the two published crystal structures of dimeric MqsA bound to either MqsR or DNA. However, the connection of the two domains with a flexible linker yields a large variety of possible conformations in solution, which is not reflected in the crystal structures. MqsA binds Zn with all four cysteines, a stoichiometry of 1:1 and a femtomolar affinity (K(a)≥ 10¹7M⁻¹ at 23°C, pH 7.0).

Distinct or Overlapping Areas of Mitochondrial Thioredoxin 2 May Be Used for Its Covalent and Strong Non-Covalent Interactions with Protein Ligands.

In silico approaches were employed to examine the characteristics of interactions between human mitochondrial thioredoxin 2 (HsTrx2) and its 38 previously identified mitochondrial protein ligands. All interactions appeared driven mainly by electrostatic forces. The statistically significant residues of HsTrx2 for interactions were characterized as "contact hot spots". Since these were identical/adjacent to putative thermodynamic hot spots, an energy network approach identified their neighbors to highlight possible contact interfaces. Three distinct areas for binding emerged: (i) one around the active site for covalent interactions, (ii) another antipodal to the active site for strong non-covalent interactions, and (iii) a third area involved in both kinds of interactions. The contact interfaces of HsTrx2 were projected as respective interfaces for Escherichia coli Trx1 (EcoTrx1), 2, and HsTrx1. Comparison of the interfaces and contact hot spots of HsTrx2 to the contact residues of EcoTx1 and HsTrx1 from existing crystal complexes with protein ligands supported the hypothesis, except for a part of the cleft/groove adjacent to Trp30 preceding the active site. The outcomes of this study raise the possibility for the rational design of selective inhibitors for the interactions of HsTrx2 with specific protein ligands without affecting the entirety of the functions of the Trx system.

Humidity-Responsive Antimicrobial Membranes Based on Cross-Linked Copolymers Functionalized with Ionic Liquid Moieties.

Humidity-responsive materials have attracted increasing attention for their potential use in various applications, e.g., sensors, soft robotics, and human-machine interfaces. Much effort has been focused on the use of ionic liquids for the construction of humidity-responsive sensors; yet, not enough attention has been paid on the susceptibility of the used poly(ionic liquid)s to microorganisms. This is especially relevant to the wide use of the polymers for biomedical applications, e.g., wearable body-condition sensors or healthcare control systems. We herein describe the development of dual functional, self-standing, monolayer antimicrobial membranes derived from cross-linked copolymers functionalized with ionic liquids. In a first step, random copolymers of poly(4-vinylbenzyl N-alkyl imidazolium chloride-co-acrylic acid), P(VBCImCn-co-AA20), were synthesized bearing aliphatic chains of different lengths (where n = 1, 4, 8, 12, 16 carbon atoms) to investigate the effect of hydrophobicity/hydrophilicity on the humidity-responsive properties of the copolymer and its antimicrobial activity. The aforementioned copolymers were later blended with the complementary reactive copolymers of poly(cetyl trimethylammonium 4-styrene sulfonate-co-glycidyl methacrylate), P(SSAmC16-co-GMA20), to provide highly stable films and coatings through thermal cross-linking. The membrane P(VBCImC12-co-AA20)/P(SSAmC16-co-GMA20) with a molar ratio of 3:1 (mol AA/mol GMA) exhibited immediate and high response to moisture through folding or flipping motions when placed on a wet filter paper or on the palm of a hand. The inhibition of growth for selected bacterial species (Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus) on the copolymer membranes was dependent on the length of the imidazolium alkyl chain and the species. Additionally, in the case of the cross-linked P(VBCImCn-co-AA20)/P(SSAmC16-co-GMA20) membranes, the overall efficacy was very high against all microorganisms tested, which, combined with their high humidity responsiveness, enables their potential application.

Bacterial Wars-a tool for the prediction of bacterial predominance based on network analysis measures.

Bacterial Wars (BW) is a network-based tool that applies a two-step pipeline to display information on the competition of bacterial species found in the same microbiome. It utilizes antimicrobial peptide (AMP) sequence similarities to obtain a relationship between species. The working hypothesis (putative AMP defense) is that friendly species share sequence similarity among the putative AMPs of their proteomes and are therefore immune to their AMPs. This may not happen in competing bacterial species with dissimilar putative AMPs. Similarities in the putative AMPs of bacterial proteomes may be thus used to predict predominance. The tool provides insights as to which bacterial species are more likely to 'die' in a competing environmental niche.

The -Cys-X1-X2-Cys- motif of reduced glutaredoxins adopts a consensus structure that explains the low pK(a) of its catalytic cysteine.

The -Cys-X1-X2-Cys- active site motif is central to the function of enzymes of the thioredoxin superfamily, including glutaredoxins. Their chemistry depends on the lowered pK(a) of the N-terminal thiolate cysteine of the -Cys-X1-X2-Cys- sequence; therefore its structure, dynamics, and electrostatics matter. Much information about the glutaredoxin structures was obtained by nuclear magnetic resonance (NMR), yet these various NMR structures produced heterogeneous and discordant views of the -Cys-X1-X2-Cys- motifs. This study addresses these inconsistencies by a computational and experimental investigation of three diverse reduced -Cys-X1-X2-Cys- motifs, from human glutaredoxin 1 (hGrx1), Escherichia coli glutaredoxin 2 (EcGrx2), and T4 virus glutaredoxin (T4Grx). The NMR models do not account for the low pK(a) of the N-terminal cysteine. However, extensive investigations of the NMR conformers by simulations yielded consensus structures for the -Cys-X1-X2-Cys- motif, with well-defined orientations for the cysteines. pK(a) calculations indicated that the consensus structure stabilizes the thiolate by local hydrogen bonds. The consensus structures of EcGrx2 and T4Grx formed the basis for predicting low pK(a) values for their N-terminal cysteines. Subsequent experimental titrations showed that these pK(a) values are <5, supporting the validity of the consensus structure. The simulations also revisited the conformational dynamics of side chains around the -Cys-X1-X2-Cys- motif, which allowed reconciliation of calculated and measured pK(a) values for important hGrx1 mutants. The conformational spread of these side chains, which differs between NMR and molecular dynamics models, is likely to be relevant to substrate recognition. The new structural models determined in this work should prove to be valuable in future molecular studies of the glutaredoxins.

Development of Environmentally Friendly Biocidal Coatings Based on Water-soluble Copolymers for Air-cleaning Filters.

Air pollution by pathogens has posed serious concern on global health during the last decades, especially since the breakout of the last pandemic. Therefore, advanced high-efficiency techniques for air purification are highly on demand. However, in air-filtering devices, the prevention of secondary pollution that may occur on the filters remains a challenge. Toward this goal, in the present work, we demonstrate a facile and eco-friendly process for the biocidal treatment of commercial high-efficiency particulate air filters. The antibacterial filters were successfully prepared through spray coating of aqueous solutions based on biocidal water-soluble polymers, poly(sodium 4-styrene sulfonate-co-cetyl trimethylammonium 4-styrene sulfonate-co-glycidyl methacrylate) [P(SSNa24-co-SSAmC1656-co-GMA20)] and poly(2-dimethylaminoethyl)methacrylate. Significantly, an optimized green route was developed for the synthesis of the used polymers in aqueous conditions and their stabilization through cross-linking reaction, leading to biocidal air filters with long-lasting activity. The developed coatings presented strong and rapid antibacterial activity against Staphylococcus aureus (in 5 min) and Escherichia coli (in 15 min). Moreover, the cytotoxicity test of the polymeric materials toward Α549 lung adenocarcinoma cells indicated very low toxicity as they did not affect either the cell growth or cell morphology. The above-mentioned results together with the scalable and easy-to-produce green methodology suggest that these materials can be promising candidates as filter coatings for use on air-purification devices.

Putative Antimicrobial Peptides Within Bacterial Proteomes Affect Bacterial Predominance: A Network Analysis Perspective.

The predominance of bacterial taxa in the gut, was examined in view of the putative antimicrobial peptide sequences (AMPs) within their proteomes. The working assumption was that compatible bacteria would share homology and thus immunity to their putative AMPs, while competing taxa would have dissimilarities in their proteome-hidden AMPs. A network-based method ("Bacterial Wars") was developed to handle sequence similarities of predicted AMPs among UniProt-derived protein sequences from different bacterial taxa, while a resulting parameter ("Die" score) suggested which taxa would prevail in a defined microbiome. T he working hypothesis was examined by correlating the calculated Die scores, to the abundance of bacterial taxa from gut microbiomes from different states of health and disease. Eleven publicly available 16S rRNA datasets and a dataset from a full shotgun metagenomics served for the analysis. The overall conclusion was that AMPs encrypted within bacterial proteomes affected the predominance of bacterial taxa in chemospheres.

Purification and biophysical characterization of the core protease domain of anthrax lethal factor.

Anthrax lethal toxin (LeTx) stands for the major virulence factor of the anthrax disease. It comprises a 90kDa highly specific metalloprotease, the anthrax lethal factor (LF). LF possesses a catalytic Zn(2+) binding site and is highly specific against MAPK kinases, thus representing the most potent native biomolecule to alter and inactivate MKK [MAPK (mitogen-activated protein kinase) kinases] signalling pathways. Given the importance of the interaction between LF and substrate for the development of anti-anthrax agents as well as the potential treatment of nascent tumours, the analysis of the structure and dynamic properties of the LF catalytic site are essential to elucidate its enzymatic properties. Here we report the recombinant expression and purification of a C-terminal part of LF (LF(672-776)) that harbours the enzyme's core protease domain. The biophysical characterization and backbone assignments ((1)H, (13)C, (15)N) of the polypeptide revealed a stable, well folded structure even in the absence of Zn(2+), suitable for high resolution structural analysis by NMR.

The multiple functions of the thiol-based electron flow pathways of Escherichia coli: Eternal concepts revisited.

Electron flow via thiols is a theme with many variations in all kingdoms of life. The favourable physichochemical properties of the redox active couple of two cysteines placed in the optimised environment of the thioredoxin fold allow for two electron transfers in between top biological reductants and ultimate oxidants. The reduction of ribonucleotide reductases by thioredoxin and thioredoxin reductase of Escherichia coli (E. coli) was one of the first pathways to be elucidated. Diverse functions such as protein folding in the periplasm, maturation of respiratory enzymes, detoxification of hydrogen peroxide and prevention of oxidative damage may be based on two electron transfers via thiols. A growing field is the relation of thiol reducing pathways and the interaction of E. coli with different organisms. This concept combined with the sequencing of the genomes of different bacteria may allow for the identification of fine differences in the systems employing thiols for electron flow between pathogens and their corresponding mammalian hosts. The emerging possibility is the development of novel antibiotics.

Implications of the mitochondrial interactome of mammalian thioredoxin 2 for normal cellular function and disease.

Multiple thioredoxin isoforms exist in all living cells. To explore the possible functions of mammalian mitochondrial thioredoxin 2 (Trx2), an interactome of mouse Trx2 was initially created using (i) a monothiol mouse Trx2 species for capturing protein partners from different organs and (ii) yeast two hybrid screens on human liver and rat brain cDNA libraries. The resulting interactome consisted of 195 proteins (Trx2 included) plus the mitochondrial 16S RNA. 48 of these proteins were classified as mitochondrial (MitoCarta2.0 human inventory). In a second step, the mouse interactome was combined with the current four-membered mitochondrial sub-network of human Trx2 (BioGRID) to give a 53-membered human Trx2 mitochondrial interactome (52 interactor proteins plus the mitochondrial 16S RNA). Although thioredoxins are thiol-employing disulfide oxidoreductases, approximately half of the detected interactions were not due to covalent disulfide bonds. This finding reinstates the extended role of thioredoxins as moderators of protein function by specific non-covalent, protein-protein interactions. Analysis of the mitochondrial interactome suggested that human Trx2 was involved potentially in mitochondrial integrity, formation of iron sulfur clusters, detoxification of aldehydes, mitoribosome assembly and protein synthesis, protein folding, ADP ribosylation, amino acid and lipid metabolism, glycolysis, the TCA cycle and the electron transport chain. The oxidoreductase functions of Trx2 were verified by its detected interactions with mitochondrial peroxiredoxins and methionine sulfoxide reductase. Parkinson's disease, triosephosphate isomerase deficiency, combined oxidative phosphorylation deficiency, and lactate dehydrogenase b deficiency are some of the diseases where the proposed mitochondrial network of Trx2 may be implicated.

Low molecular weight inhibitors of the protease anthrax lethal factor.

Anthrax Lethal Factor (LF) is a zinc-dependent metalloprotease that together with the protective antigen constitute the anthrax lethal toxin, the most prominent virulence factor of the disease anthrax. This review summarizes the current knowledge on anthrax toxicity and defense in relation to LF. Particular emphasis is placed on the structural aspects of LF, the properties of its substrates and the achievements in the design of low molecular weight inhibitors of the catalytic activity of the metalloenzyme.

Dendrin expression in glomerulogenesis and in human minimal change nephrotic syndrome.

BACKGROUND: Dendrin is an 81-kD cytosolic protein hitherto described in the brain, where it is associated with the actin cytoskeleton. Recently, we found dendrin in foot processes of mouse glomerular podocytes. Here we describe its expression both during mouse glomerulogenesis and in the normal and diseased human kidney for the first time. METHODS: Dendrin expression was characterized using RT-PCR and immunohistochemistry and semi-quantified using immunoelectron microscopy. RESULTS: In glomerulogenesis, dendrin mRNA and protein appeared first at the early capillary loop stage. It was concentrated to the pre-podocytes on the basal side of podocalyxin, an apical cell membrane marker. In human tissue, dendrin transcripts were detected in the brain and kidney. In the mature kidney dendrin localized solely in the podocytes, close to the filtration slit diaphragms. A comparison with the slit-associated protein zonula occludens-1 (ZO-1) was done in minimal change nephrotic syndrome (MCNS). Dendrin and ZO-1 were re-distributed from slit regions to the podocyte cytoplasm in areas with foot process effacement (FPE). In areas without FPE, dendrin and ZO-1 distributions were unchanged compared to controls. The total amounts of dendrin or ZO-1 markers were unchanged. This differs from nephrin that, according to our previous results, is also decreased in non-effaced areas. CONCLUSIONS: The expression of dendrin during glomerulogenesis and in the normal human kidney is similar to that previously shown for nephrin, which suggests that dendrin associates with the slit diaphragm complex. In MCNS patients, dendrin and ZO-1 are re-distributed within the podocytes. Whether this is a cause or a consequence of FPE remains unclear.