BACKGROUND:Cholera is one of the most deadly diarrheal diseases that require new treatments. We investigated the neutralization of cholera toxin by five plant extracts obtained from the Rosaceae family that have been traditionally used in Poland to treat diarrhea (of unknown origin). METHODS:Hot water extracts were prepared from the dried plant materials and lyophilized before phytochemical analysis and assessment of antimicrobial activity using microdilution assays. The ability of the plant extracts to neutralize cholera toxin was analyzed by measurement of cAMP levels in cell cultures, enzyme-linked immunosorbent assay and electrophoresis, as well as flow cytometry and fluorescence microscopy studies of fluorescent-labeled cholera toxins with cultured human fibroblasts. RESULTS:The antimicrobial assays displayed modest bacteriostatic potentials. We found that the plant extracts modulate the effects of cholera toxin on intracellular cAMP levels. Three plant extracts (Agrimonia eupatoria L., Rubus fruticosus L., Fragaria vesca L.) suppressed the binding of subunit B of cholera toxin to the cell surface and immobilized ganglioside GM1 while two others (Rubus idaeus L., Rosa.canina L.) interfered with the toxin internalization process. CONCLUSIONS:The traditional application of the Rosaceae plant infusions for diarrhea appears relevant to cholera, slowing the growth of pathogenic bacteria and either inhibiting the binding of cholera toxin to receptors or blocking toxin internalization. The analyzed plant extracts are potential complements to standard antibiotic treatment and Oral Rehydration Therapy for the treatment of cholera.

Diffusion ordered spectroscopy (DOSY) is a well established NMR method that reports diffusion coefficients for individual resonances in NMR spectra. DOSY is primarily used to analyse mixtures of small molecules and the oligomeric state of biomolecules. DOSY has also been used to analyse polymers and investigate micellization properties but different acquisition and processing parameters are recommended for polymers. In particular, the molecular weight dispersion of polymers and micelles are at odds with the physical limits of DOSY. Confidence in the quality of published DOSY data is lowered when critical parameters are poorly optimized or not reported. This tutorial provides a 'top ten' of DOSY parameters, an explanation of their source and importance, as well as suggested starting parameters and optimization for polymer/micelle samples. By following these guidelines, DOSY can emerge from being an occasional method to confirm data obtained from other experimental techniques, to one that provides strong physical evidence in its own right as the originators of DOSY intended.

Human cystatin C (hCC) is a small protein belonging to the cystatin family of papain‐like cysteine proteinase inhibitors. We review the recent literature concerning structural aspects of hCC related to disease. We focus on the mechanisms of hCC dimerization, oligomerization, and amyloid formation. Amyloid formation is associated with a number of neurodegenerative diseases that affect the independence and quality of life of aging populations. hCC is one of the second‐wave proteins that have been found to undergo amyloidosis associated with disease. For hCC, this includes cerebral amyloid angiopathy, as well as a disorder resulting in reduced male fertility.

Protein quality control mechanisms protect cells from defective, mislocalized or supernumerary proteins. The clearance of those proteins is achieved by intracellular degradation pathways, among which the ubiquitin-proteasome system is a major player. In this system, proteins to be degraded are labeled through the covalent attachment of ubiquitin. This is achieved through a complex enzymatic network that comprises in human cells ~30 ubiquitin conjugating enzymes (E2s) and more than 500 ubiquitin ligases (E3s). We used bimolecular fluorescence complementation to systematically probe the E2-E3 interaction network in living yeast. This enabled us to identify the E2s Ubc6 and Ubc7 as interaction partners of Asi1 and Asi3, two putative E3s that were previously described to form a complex at the inner nuclear membrane (INM) and to inhibit the activity of the transcription factors Stp1 and Stp2. We demonstrated that Ubc6 and Ubc7 directly interact with the RING domains of both Asi1 and Asi3 and that they are involved in the ubiquitylation of nuclear Stp2. Furthermore, an unbiased microscopy screen revealed that multiple transmembrane proteins of the yeast endomembrane system are stabilized and accumulate at the nuclear rim when the Asi ubiquitin ligase is inactivated. This suggests that the Asi complex primarily targets transmembrane proteins that should normally not be at the INM. We propose that this INM associated degradation (INMAD) is a quality control mechanism which clears proteins mislocalized in the nucleus due to their fortuitous passage through nuclear pores. L9.2

One of the main factors causing bacterial diarrhea are AB₅ enterotoxins. This group is divided into four families: pertussis toxin, cholera toxin, shiga toxin and subtilase cytotoxin. In this review we will describe the activity, structure and function of the cholera and shiga toxin families. The AB₅ enterotoxins contain a catalytic subunit A and pentameric subunit B, which binds to the cell surface within lipid rafts. The cholera toxin family cause the constitutive activation of Gsa protein, which results in cAMP production, an opening of the chloride channels and releases chloride ions into the lumen of the small intestine. In contrast, the shiga toxin family has a cytotoxic effect on epithelial cells. It can inhibit protein synthesis leading to cell death. Although AB₅ has a toxic activity, the B₅ subunits have a significant potential as a transporter for proteins with anticancer activity and as a tool for the visualization of lipid rafts and cancer cells.

This chapter reviews articles on NMR and lipids published between June 2011 and May 2012. The number of papers devoted to NMR utilization to investigate lipids, their structures, behavior in native and artificial membranes, interactions with proteins and peptides, as well as with low molecular weight compounds, and biomedical applications is quite large (over 1100 articles in Pub-Med) although we included here only a selection of those papers that were accessible and peer-reviewed. The reviewed material has been arranged in sections devoted to the structure and function of lipids in membranes, their roles in membrane-related processes including membrane fusion and lipid-mediated signal transduction, interactions of lipids with membrane and soluble proteins, peptides and antibiotics, lipid metabolomics, visualization of lipid related processes in biomedicine, diagnosis and therapy, as well as methodological approaches.

Background Osteosarcoma (OS) is a highly aggressive bone cancer affecting children and young adults. Growing evidence connects the invasive potential of OS cells with their ability to form invadopodia (structures specialized in extracellular matrix proteolysis). Results In this study, we tested the hypothesis that commonly used in vitro stimulators of mineralization limit the invadopodia formation in OS cells. Here we examined the invasive potential of human osteoblast-like cells (Saos-2) and osteolytic-like (143B) OS cells treated with the stimulators of mineralization (ascorbic acid and B-glycerophosphate) and observed a significant difference in response of the tested cells to the treatment. In contrast to 143B cells, osteoblast-like cells developed a mineralization phenotype that was accompanied by a decreased proliferation rate, prolongation of the cell cycle progression and apoptosis. On the other hand, stimulators of mineralization limited osteolytic-like OS cell invasiveness into collagen matrix. We are the first to evidence the ability of 143B cells to degrade extracellular matrix to be driven by invadopodia. Herein, we show that this ability of osteolytic-like cells in vitro is limited by stimulators of mineralization. Conclusions Our study demonstrates that mineralization competency determines the invasive potential of cancer cells. A better understanding of the molecular mechanisms by which stimulators of mineralization regulate and execute invadopodia formation would reveal novel clinical targets for treating osteosarcoma.

Cancer cells degrade the extracellular matrix (ECM) in the basement membrane and blood vessel walls to emigrate and invade from original to peripheral tissues. This invasion of cells through ECM layers is a key step not only in tumor metastasis but also in other processes such as inflammation and development. All of them seem to be facilitated by the formation of small cellular protrusions of localized protease activity, termed podosomes in non-malignant cells and invadopodia in cancer cells. Understanding the mechanisms that lead to functional invadopodia is nowadays a subject of intense study. Herein, a brief overview of the molecular components and regulators of invadopodia will be provided. In this review we will summarize recent achievements and the latest methods of visualizing invadopodia formation and functions, with a strong emphasis on advanced microscopy approaches.

This chapter reviews articles on NMR and lipids published between June 2012 and May 2013. The number of papers devoted to NMR utilization to investigate lipids, their structures, behavior in native and artificial membranes, interactions with proteins and peptides, as well as with low molecular weight molecules, biomedical applications and new methods is growing (over 1300 articles in Pub-Med) although we included here only a selection of those papers that were accessible and peer-reviewed. The reviewed material has been arranged in chapters devoted to the structure and function of lipids in membranes, their roles in membrane-related processes including lipid-mediated signal transduction, interactions of lipids with membrane and soluble proteins, peptides and various low molecular weight compounds, lipid metabolomics, visualization of lipid related processes in biomedicine, lipid-based diagnosis, and methodological approaches.

Plants contain a broad spectrum of small molecules with potential antimicrobial properties. Here, we review the antimicrobial activities of plant extracts against enterotoxic bacteria encoding AB5 toxins, including Vibrio cholerae, Shigella dysenteriae and enterotoxic Escherichia coli strains. Several plant extracts have strong antimicrobial effects and the potential to boost Oral Rehydration Therapy, which is the first line of treatment for acute diarrhea.

Fluorescent analogues provide important tools for biochemical/biophysical research. However, the analogues contain chemical modifications much larger than those known to affect ligand-binding, such as the inversion of a carbon centre or substitution of an atom. We lack experimental tools and protocols to select the most appropriate fluorescent analogue. Herein, we use several NMR spectroscopy methods, including Saturation Transfer Difference (STD), STD competition and transferred nuclear Overhauser effect spectroscopy (Tr-NOESY), as tools to select appropriate fluorescent probes. Annexin A6 (AnxA6) is a ubiquitous protein that forms in vitro GTP-induced ion channels. We used this protein as a model and screened guanosine triphosphate (GTP) and four fluorescent analogues against AnxA6. STD reported that the GTP moiety of all ligands made similar contacts with the protein, despite additional interactions between the fluorescent tags and AnxA6. Competition STD experiments verified that the analogues and GTP bind to the same site. Tr-NOESY indicated that the bound conformation of the base relative to ribose is altered for some analogues compared to GTP. MANT-GTP or the BODIPY thioester of guanosine 5'-O-(3-thiotriphosphate) are the most suitable fluorescent analogues for AnxA6, according to NMR. These results reveal NMR as a useful technique to select and design proper fluorescent tags for biochemical/biophysical assays.

The osteogenic differentiation and new bone formation are commonly delayed by bacterial infection of orthopedic implants, which is urgent to be resolved quickly in the clinic. The current paper prepared a strontium-doped electrospinning fiber membrane with antibacterial and osteogenic properties by pulse electrochemical method. Polylactic acid/hydroxyapatite (PLLA/HA) composite fiber substrate was fabricated by electrospinning technology, and strontium doped SrHA/Cu/Polypyrrole (PPy) composite coating was constructed with pulse electrodeposition method on its surface. The strontium doping technique, degradation of Sr2+ and Cu2+, cellular compatibility, and the antibacterial activity of the fiber membrane were examined. The results revealed that the deposition of phosphorus and calcium on composite fiber was the highest, indicating good biological activity. The release of Sr2+ and Cu2+ was stable and gradual due to the modulation of PPy. The composite fiber presented excellent antibacterial performance and the antibacterial rate was close to 100% against Staphylococcus aureus and Escherichia coli. Furthermore, it is conducive to the adhesion, spread, and proliferation of vascular endothelial cells and osteoblasts, namely outstanding osteogenesis and angiogenesis abilities. In conclusion, the multifunctional PLLA/[email protected]/Cu/PPy composite fiber membrane with good antibacterial and osteogenic activity by electrospinning technology and pulsed electrochemical deposition method provides an effective strategy for the poor bone healing of infected bone defects.

Calcium ions are essential factors controlling the balance between cell survival, growth, differentiation and metabolism. Ca2+ acts as a global second messenger involved in the regulation of all aspects of cell function. Fluctuations in the intra- and extracellular Ca2+ concentration [Ca2+] in response to different environmental stimuli drive most cellular functions. Therefore, sustenance of calcium homeostasis requires perfect organization in time and space that is achieved by calcium binding proteins (CaBPs). These proteins are involved in sensing and transforming calcium signals to downstream cellular responses. Growing number of evidence suggests than many human disorders, including cancer progression, are related to deregulation of cellular calcium homeostasis and defects in CaBPs functions. In this review we will focus on the roles of S100A proteins in intracellular and extracellular calcium signalling and homeostasis. The S100A subfamily is among the most distinctive of EF-hand CaBPs and are found exclusively in vertebrates. They are believed to have evolved to enable activation of specific biochemical pathways in parallel to the activity of Ca2+ sensors such as calmodulin and/or annexins. The importance of S100 proteins is underscored by their deregulated expression in neurodegenerative and inflammatory disorders, myopathies and cancer. In addition, S100 proteins serve as diagnostic markers in the clinic and are under constant investigation. Their roles and the roles of the S100A protein partners in normal and pathology will be also discussed.

A new heteroditopic macrobicyclic compound (t(2)pN(5)O(3)) containing two separate polyoxa and polyaza compartments was synthesized in good yield through a [1 + 1] "tripod-tripod coupling" strategy. The X-ray crystal structure of H(3)t(2)pN(5)O(3)(3+) revealed the presence of one encapsulated water molecule accepting two hydrogen bonds from two protonated secondary amines and donating a hydrogen bond to one amino group. The acid-base behavior of the compound was studied by potentiometry at 298.2 K in aqueous solution and at ionic strength 0.10 M in KCl. The results revealed unusual protonation behavior, namely a surprisingly low fourth protonation constant contrary to what was expected for the compound. (1)H NMR and DOSY experiments, as well as molecular modeling studies, showed that the water encapsulation and the conformation observed in the solid state are retained in solution. The strong binding of the encapsulated water molecule, reinforced by the cooperative occurrence of a trifurcated hydrogen bond at the polyether compartment of the macrobicycle, account for the very low log K(4)(H) value obtained.

The precipitation of barium sulfate (barite) in aqueous solutions of ionic liquids (ILs) has been used as a model system to derive fundamental relationships between crystal nucleation phenomena and some properties of those liquid salts. A systematic dependence between the size of the precipitating crystalline particles and the limiting molar conductivity of ILs has been found. The unraveled correlation has been interpreted in the light of the effect of ILs on water structure dynamics in hydration shells of barite building units and in the bulk solution and respective consequences for crystal nucleation. The response of the crystal formation process to the presence of specific ILs has been used to extract information about some particular properties of ILs (ion association and hydration) that modify the crystal hydration environment.

The steroid binding mechanism of a DNA aptamer was studied using isothermal titration calorimetry (ITC), NMR spectroscopy, quasi-elastic light scattering (QELS), and small-angle X-ray spectroscopy (SAXS). Binding affinity determination of a series of steroid-binding aptamers derived from a parent cocaine-binding aptamer demonstrates that substituting a GA base pair with a GC base pair governs the switch in binding specificity from cocaine to the steroid deoxycholic acid (DCA). Binding of DCA to all aptamers is an enthalpically driven process with an unfavorable binding entropy. We engineered into the steroid-binding aptamer a ligand-induced folding mechanism by shortening the terminal stem by two base pairs. NMR methods were used to demonstrate that there is a transition from a state where base pairs are formed in one stem of the free aptamer, to where three stems are formed in the DCA-bound aptamer. The ability to generate a ligand-induced folding mechanism into a DNA aptamer architecture based on the three-way junction of the cocaine-binding aptamer opens the door to obtaining a series of aptamers all with ligand-induced folding mechanisms but triggered by different ligands. Hydrodynamic data from diffusion NMR spectroscopy, QELS, and SAXS show that for the aptamer with the full-length terminal stem there is a small amount of structure compaction with DCA binding. For ligand binding by the short terminal stem aptamer, we propose a binding mechanism where secondary structure forms upon DCA binding starting from a free structure where the aptamer exists in a compact form.

Matrix vesicles (MVs) are cell-derived membranous entities crucial for mineral formation in the extracellular matrix. One of the dominant groups of constitutive proteins present in MVs, recognised as regulators of mineralization in norm and pathology, are annexins. In this report, besides the annexins already described (AnxA2 and AnxA6), we identified AnxA1 and AnxA7, but not AnxA4, to become selectively enriched in MVs of Saos-2 cells upon stimulation for mineralization. Among them, AnxA6 was found to be almost EGTA-non extractable from matrix vesicles. Moreover, our report provides the first evidence of annexin-binding S100 proteins to be present in MVs of mineralizing cells. We observed that S100A10 and S100A6, but not S100A11, were selectively translocated to the MVs of Saos-2 cells upon mineralization. This observation provides the rationale for more detailed studies on the role of annexin-S100 interactions in MV-mediated mineralization.