Polycations as Aptamer-Binding Modulators for Sensitive Fluorescence Anisotropy Assay of Aflatoxin B1.

Fluorescence induced by the excitation of a fluorophore with plane-polarized light has a different polarization depending on the size of the fluorophore-containing reagent and the rate of its rotation. Based on this effect, many analytical systems have been implemented in which an analyte contained in a sample and labeled with a fluorophore (usually fluorescein) competes to bind to antibodies. Replacing antibodies in such assays with aptamers, low-cost and stable oligonucleotide receptors, is complicated because binding a fluorophore to them causes a less significant change in the polarization of emissions. This work proposes and characterizes the compounds of the reaction medium that improve analyte binding and reduce the mobility of the aptamer-fluorophore complex, providing a higher analytical signal and a lower detection limit. This study was conducted on aflatoxin B1 (AFB1), a ubiquitous toxicant contaminating foods of plant origins. Eight aptamers specific to AFB1 with the same binding site and different regions stabilizing their structures were compared for affinity, based on which the aptamer with 38 nucleotides in length was selected. The polymers that interact reversibly with oligonucleotides, such as poly-L-lysine and polyethylene glycol, were tested. It was found that they provide the desired reduction in the depolarization of emitted light as well as high concentrations of magnesium cations. In the selected optimal medium, AFB1 detection reached a limit of 1 ng/mL, which was 12 times lower than in the tris buffer commonly used for anti-AFB1 aptamers. The assay time was 30 min. This method is suitable for controlling almond samples according to the maximum permissible levels of their contamination by AFB1. The proposed approach could be applied to improve other aptamer-based analytical systems.

Kinetics of Human Serum Albumin Adsorption on Polycation Functionalized Silica.

The adsorption kinetics of human serum albumin (HSA) on bare and poly-L-arginine (PARG)-modified silica substrates were investigated using reflectometry and atomic force microscopy (AFM). Measurements were carried out at various pHs, flow rates and albumin concentrations in the 10 and 150 mM NaCl solutions. The mass transfer rate constants and the maximum protein coverages were determined for the bare silica at pH 4.0 and theoretically interpreted in terms of the hybrid random sequential adsorption model. These results were used as reference data for the analysis of adsorption kinetics at larger pHs. It was shown that the adsorption on bare silica rapidly decreased with pH and became negligible at pH 7.4. The albumin adsorption on PARG-functionalized silica showed an opposite trend, i.e., it was negligible at pH 4 and attained maximum values at pH 7.4 and 150 mM NaCl, the conditions corresponding to the blood serum environment. These results were interpreted as the evidence of a significant role of electrostatic interactions in the albumin adsorption on the bare and PARG-modified silica. It was also argued that our results can serve as useful reference data enabling a proper interpretation of protein adsorption on substrates functionalized by polyelectrolytes.

Supramolecular citrate poly allylamine hydrochloride nanoparticles for citrate delivery and calcium oxalate nanocrystal dissolution.

HYPOTHESIS: Renal calculi (kidney stones) are mainly made by calcium oxalate and can cause different complications including malfunction of the kidney. The most important urinary stone inhibitors are citrate molecules. Unfortunately, the amount of citrate reaching the kidney after oral ingestion is low. We hypothesized that nanoparticles of polyallylamine hydrochloride (CIT-PAH) carrying citrate ions could simultaneously deliver citrates while PAH would complex oxalate triggering dissolution and removal of CaOx nanocrystals. EXPERIMENTS: We successfully prepared nanoparticles of citrate ions with polyallylamine hydrochloride (CIT-PAH), PAH with oxalate (OX-PAH) and characterize them by Small Angle X ray Scattering (SAXS), Transmission Electron Microscopy (TEM), Dynamic Light Scattering (DLS) and NMR. Dissolution of CaOx nanocrystals in presence of CIT-PAH have been followed with Wide Angle Xray Scattering (WAXS), DLS and Confocal Raman Microscopy. Raman spectroscopy was used to study the dissolution of crystals in synthetic urine samples. The release of citrate from CIT-PAH was followed by diffusion NMR. Molecular dynamics (MD) simulations were carried out to study the interaction of CIT and OX ions with PAH. FINDINGS: CIT-PAH nanoparticles dissolves CaOx nanocrystals as shown by NMR, DLS, TEM and WAXS in water and by Raman spectroscopy in artificial human urine. WAXS and Raman show that the crystal structure of CaOx disappears in the presence of CIT-PAH. DLS shows that the time required for CaOX dissolution will depend on the concentration of CIT-PAH NPs. NMR proves that citrate ions are released from the CIT PAH NPs during CaOX dissolution, MD simulations showed that oxalates exhibit a stronger interaction for PAH than citrate, explaining the removal of oxalate ions and replacement of the citrate in the polymer nanoparticles.

In vitro and ex vivo protoscolicidal effect of poly(amidoamine) nanoemulsion against Echinococcus granulosus.

Hydatidosis causes a serious health hazard to humans and animals leading to significant economic and veterinary and public health concern worldwide. The present study aimed to evaluate the in vitro and ex vivo protoscolicidal effects of synthesized poly(amidoamine), PAMAM, nanoemulsion. In this study, PAMAM was characterized through dynamic light scattering technique to investigate the particle size and zeta potential of nanoemulsified polymer. For the in vitro and ex vivo assays, we used eosin dye exclusion test and scanning electron microscope (SEM) to evaluate the effects of the prepared and characterized PAMAM nanoemulsion against protoscoleces from Echinococcus granulosus sensu lato G6 (GenBank: OQ443068.1) isolated from livers of naturally infected camels. Various concentrations (0.5, 1, 1.5 and 2 mg/mL) of PAMAM nanoemulsion at different exposure times (5, 10, 20 and 30 min) were tested against protoscolices. Our findings showed that PAMAM nanoemulsion had considerable concentration- and time-dependent protoscolicidal effect at both in vitro and ex vivo experiments. Regarding in vitro assay, PAMAM nanoemulsion had a potent protoscolicidal effect when compared with the control group with a highest protoscolicidal activity observed at the concentration of 2 mg/mL at all exposure times, such that 100% of protoscolices were killed after 20 min of exposure. Also, the mortality of protoscolices was 100% after 30 min of exposure to 1 and 1.5 mg/mL of PAMAM nanoemulsion, in vitro. Concerning ex vivo assay PAMAM nanoemulsion recorded the highest mortality rates at the concentration of 2 mg/mL (55, 99.4 and 100% at 10, 20, 30 min, respectively). Ultrastructure examination of examined protoscolices after 20 min of exposure to PAMAM nanoemulsion showed a complete loss of rostellar hooks, disruption of suckers with disorganization of hooks with partial or complete loss of them, and damage of protoscolices tegument with loss of their integrity in the form of holes and contraction of the soma region were observed in 1.5 and 2 mg/mL of PAMAM, in vitro and ex vivo, showing more damage in the in vitro conditions. It can be concluded that PAMAM nanoemulsion is a promising protoscolicidal agent offering a high protoscolicidal effect at a short exposure time. Further in vivo studies and preclinical animal trials are required to evaluate its efficacy and clinical applications against hydatid cysts.

Covalently grafting polycation to bacterial cellulose for antibacterial and anti-cell adhesive wound dressings.

Hydrogel-based wound dressings are becoming increasingly important for wound healing. Bacterial cellulose (BC) has been commonly used as wound dressings due to its good in vitro and in vivo biocompatibility. However, pure BC does not possess antibacterial properties. In this regard, polycation gel was grafted onto the BC using a surface-initiated activator regenerated by electron transfer atom transfer radical polymerization (SI-ARGET ATRP) with subsequent quaternization for antibacterial wound dressing. Dimethylethyl methacrylate (DMAEMA) was successfully polymerized on the BC surface which was confirmed by Fourier transform infrared spectroscopy and elemental analysis. The morphology structure, specific surface area, pore size, and mechanical properties were also characterized. The quaternized PDMAEMA grafted on the BC endowed it with excellent antibacterial activity against E. coli (Gram-negative) and S. aureus (Gram-positive) with a killing rate of 89.2 % and 93.4 %, respectively. The number of cells was significantly reduced on QPD/BC hydrogel, demonstrating its good anti-adhesion ability. In vitro cellular evaluation revealed that the antibacterial wound dressing exhibited good biocompatibility. Overall, this study provides a feasible method to develop antibacterial and anti-cell adhesive hydrogel, which has a promising potential for wound healing.

Naturally biocompatible melanin based iron-complex nanoparticles for pH-responsive magnetic resonance imaging.

The sensitivity and diagnostic accuracy of magnetic resonance imaging mainly depend on the relaxation capacity of contrast agents (CAs) and their accumulated amount at the pathological region. Due to the better biocompatibility and high-spin capacity, Fe-complexes have been studied widely as an alternative to replace popular Gd-based CAs associated with potential biotoxicity. Compared with a variety of Fe complex-based CAs, such as small molecular, macrocyclic, multinuclear complexes, the form of nanoparticle exhibits outstanding longitudinal relaxation, but the clinical transformation was still limited by the inconspicuous difference of contrast between tumor and normal tissue. The enhanced effect of contrast is a positive relation as relaxation of CAs and their concentration in desired region. To specifically improve the amount of CAs accumulated in the tumor, pH-responsive polymer poly(2-ethyl-2-oxazoline) (PEOz) was modified on melanin, a ubiquitous natural pigment providing much active sites for chelating with Fe(III). The Fe(III)-Mel-PEOz we prepared could raise the tumor cell endocytosis efficiency via switching surface charge from anion to cation with the stimuli of the decreasing pH of tumor microenvironment. The change of pH has negligible effect on ther1of Fe(III)-Mel-PEOz, which is always maintained at around 1.0 mM-1s-1at 0.5 T. Moreover, Fe(III)-Mel-PEOz exhibited low cytotoxicity, and satisfactory enhancement of positive contrast effectin vivo. The excellent biocompatibility and stable relaxation demonstrate the high potential of Fe(III)-Mel-PEOz in the diagnosis of tumor.

Sensing and Microbiological Activity of a New Blue Fluorescence Polyamidoamine Dendrimer Modified with 1,8-Naphthalimide Units.

A novel second-generation blue fluorescent polyamidoamine dendrimer peripherally modified with sixteen 4-N,N-dimethylaninoethyloxy-1,8-naphthalimide units was synthesized. Its basic photophysical characteristics were investigated in organic solvents of different polarity. It was found that in these solvents, the dendrimer is colorless and emitted blue fluorescence with different intensities depending on their polarity. The effect of the pH of the medium on the fluorescence intensity was investigated and it was found that in the acidic medium, the fluorescence is intense and is quenched in the alkaline medium. The ability of the dendrimer to detect metal ions (Pb2+, Zn2+, Mg2+, Sn2+, Ba2+, Ni2+, Sn2+, Mn2+, Co2+, Fe3+, and Al3+) was also investigated, and it was found that in the presence of Fe3+, the fluorescent intensity was amplified more than 66 times. The antimicrobial activity of the new compound has been tested in vitro against Gram-positive B. cereus and Gram-negative P. aeruginosa. The tests were performed in the dark and after irradiation with visible light. The antimicrobial activity of the compound enhanced after light irradiation and B. cereus was found slightly more sensitive than P. aeruginosa. The increase in antimicrobial activity after light irradiation is due to the generation of singlet oxygen particles, which attack bacterial cell membranes.

Mitoribosome structure with cofactors and modifications reveals mechanism of ligand binding and interactions with L1 stalk.

The mitoribosome translates mitochondrial mRNAs and regulates energy conversion that is a signature of aerobic life forms. We present a 2.2 Å resolution structure of human mitoribosome together with validated mitoribosomal RNA (rRNA) modifications, including aminoacylated CP-tRNAVal. The structure shows how mitoribosomal proteins stabilise binding of mRNA and tRNA helping to align it in the decoding center, whereas the GDP-bound mS29 stabilizes intersubunit communication. Comparison between different states, with respect to tRNA position, allowed us to characterize a non-canonical L1 stalk, and molecular dynamics simulations revealed how it facilitates tRNA transitions in a way that does not require interactions with rRNA. We also report functionally important polyamines that are depleted when cells are subjected to an antibiotic treatment. The structural, biochemical, and computational data illuminate the principal functional components of the translation mechanism in mitochondria and provide a description of the structure and function of the human mitoribosome.

Targeting c-Myc with decoy oligodeoxynucleotide-loaded polycationic nanoparticles inhibits cell growth and induces apoptosis in cancer stem-like cells (NTERA-2).

BACKGROUND: An increase in cancer stem cell (CSC) populations and their resistance to common treatments could be a result of c-Myc dysregulations in certain cancer cells. In the current study, we investigated anticancer effects of c-Myc decoy ODNs loaded-poly (methacrylic acid-co-diallyl dimethyl ammonium chloride) (PMA-DDA)-coated silica nanoparticles as carriers on cancer-like stem cells (NTERA-2). METHODS AND RESULTS: The physicochemical characteristics of the synthesized nanocomposites (SiO2@PMA-DDA-DEC) were analyzed using FT-IR, DLS, and SEM techniques. UV-Vis spectrophotometer was applied to analyze the release pattern of decoy ODNs from the nanocomposite. Furthermore, uptake, cell viability, apoptosis, and cell cycle assays were used to investigate the anticancer effects of nanocomposites loaded with c-Myc decoy ODNs on NTERA-2 cancer cells. The results of physicochemical analytics demonstrated that SiO2@PMA-DDA-DEC nanocomposites were successfully synthesized. The prepared nanocomposites were taken up by NTERA-2 cells with high efficiency, and could effectively inhibit cell growth and increase apoptosis rate in the treated cells compared to the control group. Moreover, SiO2@PMA-DDA nanocomposites loaded with c-Myc decoy ODNs induced cell cycle arrest at the G0/G1 phase in the treated cells. CONCLUSIONS: The conclusion drawn from this study is that c-Myc decoy ODN-loaded SiO2@PMA-DDA nanocomposites can effectively inhibit cell growth and induce apoptosis in NTERA-2 cancer cells. Moreover, given that a metal core is incorporated into this synthetic nanocomposite, it could potentially be used in conjunction with irradiation as part of a decoy-radiotherapy combinational therapy in future investigations.

The selective extraction of dietary polyamines from chicken breast using the application of a lab-on-a-chip electromembrane and dispersive liquid-liquid microextraction followed by gas chromatography-mass spectrometry.

Excessive dietary polyamines (PAs), including putrescine (PUT), spermine (SPM), and spermidine (SPD), have become a worldwide concern due to their carcinogenicity and reduced shelf life. A modern miniaturized on-chip electromembrane extraction (EME) has been applied to extract these compounds from chicken breast samples. This method is based fundamentally on ionic compounds' electrostatic attraction, diffusion, and solubility in the acceptor phase. The chemical structure of polyamines enables their efficient extraction using an electric driving force on a microchip device. HCl solution (0.1 mol L-1) was applied as an aqueous acceptor solvent. Dispersive liquid-liquid microextraction was performed after EME to facilitate joining three-phase EME to GC-MS and improve the merit figures. The total ranges of 3.77-7.89 µg g-1, 3.48-7.02 µg g-1, and 0.78-2.20 µg g-1 were acquired as PUT, SPM and SPD concentrations in chicken breast, respectively. The results demonstrate that the level of PAs in fresh chicken breast samples is not concerning, but it may reduce the quality of chicken meat over time. This novel analytical technique has several advantages: high recovery, substantial quickness, remarkable selectivity, and good enrichment factors. This emerging method could be generalized to other studies to analyze different foodstuffs.

Chemical Versatility in Catalysis and Inhibition of the Class IIb Histone Deacetylases.

ConspectusThe zinc-dependent histone deacetylases (HDACs 1-11) belong to the arginase-deacetylase superfamily of proteins, members of which share a common α/ß fold and catalytic metal binding site. While several HDACs play a role in epigenetic regulation by catalyzing acetyllysine hydrolysis in histone proteins, the biological activities of HDACs extend far beyond histones. HDACs also deacetylate nonhistone proteins in the nucleus as well as the cytosol to regulate myriad cellular processes. The substrate pool is even more diverse in that certain HDACs can hydrolyze other covalent modifications. For example, HDAC6 is also a lysine decrotonylase, and HDAC11 is a lysine-fatty acid deacylase. Surprisingly, HDAC10 is not a lysine deacetylase but instead is a polyamine deacetylase. Thus, the HDACs are biologically and chemically versatile catalysts as they regulate the function of diverse protein and nonprotein substrates throughout the cell.Owing to their critical regulatory functions, HDACs serve as prominent targets for drug design. At present, four HDAC inhibitors are FDA-approved for cancer chemotherapy. However, these inhibitors are active against multiple HDAC isozymes, and a lack of selectivity is thought to contribute to undesirable side effects. Current medicinal chemistry campaigns focus on the development of isozyme-selective inhibitors, and many such studies largely focus on HDAC6 and HDAC10. HDAC6 is a target for therapeutic intervention due to its cellular role as a tubulin deacetylase and tau deacetylase, and selective inhibitors are being studied in cancer chemotherapy and the treatment of peripheral neuropathy. Crystal structures of enzyme-inhibitor complexes reveal how various features of inhibitor design, such as zinc-coordinating groups, bifurcated capping groups, and aromatic fluorination patterns, contribute to affinity and isozyme selectivity. The polyamine deacetylase HDAC10 is also an emerging target for cancer chemotherapy. Crystal structures of intact substrates trapped in the HDAC10 active site reveal the molecular basis of strikingly narrow substrate specificity for N8-acetylspermidine hydrolysis. Active site features responsible for substrate specificity have been successfully exploited in the design of potent and selective inhibitors.In this Account, I review the structural chemistry and inhibition of HDACs, highlighting recent X-ray crystallographic and functional studies of HDAC6 and HDAC10 in my laboratory. These studies have yielded fascinating snapshots of catalysis as well as novel chemical transformations involving bound inhibitors. The zinc-bound water molecule in the HDAC active site is the catalytic nucleophile in the deacetylation reaction, but this activated water molecule can also react with inhibitor C═O or C═N groups to yield unanticipated reaction products that bind exceptionally tightly. Versatile active site chemistry unleashes the full inhibitory potential of such compounds, and X-ray crystallography allows us to view this chemistry in action.

Trehalose-polyamine/DNA nanocomplexes: impact of vector architecture on cell and organ transfection selectivity.

A novel family of precision-engineered gene vectors with well-defined structures built on trehalose and trehalose-based macrocycles (cyclotrehalans) comprising linear or cyclic polyamine heads have been synthesized through procedures that exploit click chemistry reactions. The strategy was conceived to enable systematic structural variations and, at the same time, ensuring that enantiomerically pure vectors are obtained. Notably, changes in the molecular architecture translated into topological differences at the nanoscale upon co-assembly with plasmid DNA, especially regarding the presence of regions with short- or long-range internal order as observed by TEM. In vitro and in vivo experiments further evidenced a significant impact on cell and organ transfection selectivity. Altogether, the results highlight the potential of trehalose-polyamine/pDNA nanocomplex monoformulations to achieve targeting transfection without the need for any additional cell- or organ-sorting component.

Multifunctional Poly(2-ethyl-2-oxazoline) Copolymers Containing Dithiolane and Pentafluorophenyl Esters as Effective Reactive Linkers for Gold Surface Coatings.

Surface functionalization with biological macromolecules is an important task for the development of sensor materials, whereby the interaction with other biological materials should be suppressed. In this work, we developed a novel multifunctional poly(2-ethyl-2-oxazoline)-dithiolane conjugate as a versatile linker for gold surface immobilization of amine-containing biomolecules, containing poly(2-ethyl-2-oxazoline) as antifouling polymer, dithiolane for surface immobilization, and activated esters for protein conjugation. First, a well-defined carboxylic acid containing copoly(2-ethyl-2-oxazoline) was synthesized by cationic ring-opening copolymerization of 2-ethyl-2-oxazoline with a methyl ester-containing 2-oxazoline monomer, followed by postpolymerization modifications. The side-chain carboxylic groups were then converted to amine-reactive pentafluorophenyl (PFP) ester groups. Part of the PFP groups was used for the attachment of the dithiolane moiety, which can efficiently bind to gold surfaces. The final copolymer contained 1.4 mol% of dithiolane groups and 4.5 mol% of PFP groups. The copolymer structure was confirmed by several analytical techniques, including NMR spectroscopy and size-exclusion chromatography. The kinetics of the PFP ester aminolysis and hydrolysis demonstrated significantly faster amidation compared to hydrolysis, which is essential for subsequent protein conjugation. Successful coating of gold surfaces with the polymer was confirmed by spectroscopic ellipsometry, showing a polymer brush thickness of 4.77 nm. Subsequent modification of the coated surfaces was achieved using bovine serum albumin as a model protein. This study introduces a novel reactive polymer linker for gold surface functionalization and offers a versatile polymer platform for various applications including biosensing and surface functionalization.

Antimicrobial Properties of New Polyamines Conjugated with Oxygen-Containing Aromatic Functional Groups.

Antibiotic resistance is now a first-order health problem, which makes the development of new families of antimicrobials imperative. These compounds should ideally be inexpensive, readily available, highly active, and non-toxic. Here, we present the results of our investigation regarding the antimicrobial activity of a series of natural and synthetic polyamines with different architectures (linear, tripodal, and macrocyclic) and their derivatives with the oxygen-containing aromatic functional groups 1,3-benzodioxol, ortho/para phenol, or 2,3-dihydrobenzofuran. The new compounds were prepared through an inexpensive process, and their activity was tested against selected strains of yeast, as well as Gram-positive and Gram-negative bacteria. In all cases, the conjugated derivatives showed antimicrobial activity higher than the unsubstituted polyamines. Several factors, such as the overall charge at physiological pH, lipophilicity, and the topology of the polyamine scaffold were relevant to their activity. The nature of the lipophilic moiety was also a determinant of human cell toxicity. The lead compounds were found to be bactericidal and fungistatic, and they were synergic with the commercial antifungals fluconazole, cycloheximide, and amphotericin B against the yeast strains tested.

Unprecedented reactivity of polyamines with aldehydic DNA modifications: structural determinants of reactivity, characterization and enzymatic stability of adducts.

Apurinic/apyrimidinic (AP) sites, 5-formyluracil (fU) and 5-formylcytosine (fC) are abundant DNA modifications that share aldehyde-type reactivity. Here, we demonstrate that polyamines featuring at least one secondary 1,2-diamine fragment in combination with aromatic units form covalent DNA adducts upon reaction with AP sites (with concomitant cleavage of the AP strand), fU and, to a lesser extent, fC residues. Using small-molecule mimics of AP site and fU, we show that reaction of secondary 1,2-diamines with AP sites leads to the formation of unprecedented 3'-tetrahydrofuro[2,3,4-ef]-1,4-diazepane ('ribodiazepane') scaffold, whereas the reaction with fU produces cationic 2,3-dihydro-1,4-diazepinium adducts via uracil ring opening. The reactivity of polyamines towards AP sites versus fU and fC can be tuned by modulating their chemical structure and pH of the reaction medium, enabling up to 20-fold chemoselectivity for AP sites with respect to fU and fC. This reaction is efficient in near-physiological conditions at low-micromolar concentration of polyamines and tolerant to the presence of a large excess of unmodified DNA. Remarkably, 3'-ribodiazepane adducts are chemically stable and resistant to the action of apurinic/apyrimidinic endonuclease 1 (APE1) and tyrosyl-DNA phosphoesterase 1 (TDP1), two DNA repair enzymes known to cleanse a variety of 3' end-blocking DNA lesions.

Pyrene-Containing Polyamines as Fluorescent Receptors for Recognition of PFOA in Aqueous Media.

The globally widespread perfluorooctanoic acid (PFOA) is a concerning environmental contaminant, with a possible toxic long-term effects on the environment and human health The development of sensible, rapid, and low-cost detection systems is a current change in modern environmental chemistry. In this context, two triamine-based chemosensors, L1 and L2, containing a fluorescent pyrene unit, and their Zn(II) complexes are proposed as fluorescent probes for the detection of PFOA in aqueous media. Binding studies carried out by means of fluorescence and NMR titrations highlight that protonated forms of the receptors can interact with the carboxylate group of PFOA, thanks to salt bridge formation with the ammonium groups of the aliphatic chain. This interaction induces a decrease in the fluorescence emission of pyrene at neutral and slightly acidic pH values. Similarly, emission quenching has also been observed upon coordination of PFOA by the Zn(II) complexes of the receptors. These results evidence that simple polyamine-based molecular receptors can be employed for the optical recognition of harmful pollutant molecules, such as PFOA, in aqueous media.

Enhanced alpha-amylase inhibition activity of amine-terminated PAMAM dendrimer stabilized pure copper-doped magnesium oxide nanoparticles.

The present work aims to prepare copper-doped MgO nanoparticles via a sol-gel approach and study their antidiabetic alpha-amylase inhibition activity with undoped MgO nanoparticles. The ability of G5 amine-terminated polyamidoamine (PAMAM) dendrimer for the controlled release of copper-doped MgO nanoparticles to exhibit alpha-amylase inhibition activity was also evaluated. The synthesis of MgO nanoparticles via sol-gel approach and optimization of calcination temperature and time has led to the formation of nanoparticles with different shapes (spherical, hexagonal, and rod-shaped) and a polydispersity in size ranging from 10 to 100 nm with periclase crystalline phase. The presence of copper ions in the MgO nanoparticles has altered their crystallite size, eventually modifying their size, morphology, and surface charge. The efficiency of dendrimer to stabilize spherical copper-doped MgO nanoparticles (ca. 30 %) is higher than in other samples, which was confirmed by UV-Visible, DLS, FTIR, and TEM analysis. The amylase inhibition assay emphasized that the dendrimer nanoparticles stabilization has led to the prolonged enzyme inhibition ability of MgO and copper-doped MgO nanoparticles for up to 24 h.

Structural basis of amine odorant perception by a mammal olfactory receptor.

Odorants are detected as smell in the nasal epithelium of mammals by two G-protein-coupled receptor families, the odorant receptors and the trace amine-associated receptors1,2 (TAARs). TAARs emerged following the divergence of jawed and jawless fish, and comprise a large monophyletic family of receptors that recognize volatile amine odorants to elicit both intraspecific and interspecific innate behaviours such as attraction and aversion3-5. Here we report cryo-electron microscopy structures of mouse TAAR9 (mTAAR9) and mTAAR9-Gs or mTAAR9-Golf trimers in complex with ß-phenylethylamine, N,N-dimethylcyclohexylamine or spermidine. The mTAAR9 structures contain a deep and tight ligand-binding pocket decorated with a conserved D3.32W6.48Y7.43 motif, which is essential for amine odorant recognition. In the mTAAR9 structure, a unique disulfide bond connecting the N terminus to ECL2 is required for agonist-induced receptor activation. We identify key structural motifs of TAAR family members for detecting monoamines and polyamines and the shared sequence of different TAAR members that are responsible for recognition of the same odour chemical. We elucidate the molecular basis of mTAAR9 coupling to Gs and Golf by structural characterization and mutational analysis. Collectively, our results provide a structural basis for odorant detection, receptor activation and Golf coupling of an amine olfactory receptor.

PAMAM-Functionalized Paper as a New Substrate for the Paper Spray Mass Spectrometry Measurement of Proteins.

Paper surface functionalization with polyamidoamine (PAMAM) dendrimers has been developed for increased sensitivity analysis of proteins by paper spray mass spectrometry (PS-MS). PAMAM is a branched polymeric compound with an ethylenediamine core linked to repeating PAMAM units that generates an outer surface rich in primary amines. These positively charged amine groups can interact electrostatically with negatively charged residues (e.g., aspartate, glutamate) on the protein surface. PAMAM inner amide moieties can also promote hydrogen bonding with protein surface oxygens, making PAMAM a useful material for protein extraction. PAMAM-functionalized PS-MS paper strips were used to extract proteins from biofluids, dipped in acetonitrile to remove unbound constituents, dried, and then measured with PS-MS. The use of this strategy was optimized and compared with unmodified paper strips. PAMAM-functionalized paper substrates provided sixfold greater sensitivity for albumin, 11-fold for hemoglobin, sevenfold for insulin, and twofold for lysozyme. The analytical performance of the functionalized paper substrate was evaluated through the analysis of albumin in urine, achieving linearity with R2 > 0.99, LOD of 1.1 µg mL-1, LOQ of 3.8 µg mL-1, precision better than 10%, and relative recovery 70-83%. The method was applied to quantify urinary albumin from nine anonymous patient samples (concentrations ranged from 6.5 to 77.4 µg mL-1), illustrating its potential for the diagnosis of microalbuminuria. These data demonstrate the utility of paper modification with the PAMAM dendrimer for sensitive PS-MS analysis of proteins, opening a path for further applications in clinical diagnosis through the analysis of disease-related proteins.

Determination of polyamines in urine via electrospun nanofibers-based solid-phase extraction coupled with GC-MS and application to gastric cancer patients.

The simultaneous determination of polyamines and their metabolites in urine samples was achieved by gas chromatography-mass spectrometry in the selected ion monitoring mode. After conjugating with the ion-pair reagent bis-2-ethylhexylphosphate in the aqueous phase, the polyamines in the samples were extracted with polystyrene nanofiber-based packed-fiber solid-phase extraction followed by a derivatization step using pentafluoropropionyl anhydride. With optimal conditions, all analytes were separated well. For analytes of putrescine, cadaverine, N-acetylputrescine, and N-acetylcadaverine, the linearity was good in the range of 0.05-500 µmol/L (R2  ≥ 0.993). While for spermidine, spermine, acetylspermidine, N8 -acetylspermidine, and N-acetylspermine, the linearity was good in the range of 0.5-500 µmol/L (R2  ≥ 0.990). The recoveries of three spiked concentrations (0.5, 5, 300 µmol/L) were 85.6%-108.4%, and relative standard deviations for intra- and interday were in the range of 2.9%-13.4% and 4.5%-15.1%, respectively. The method was successfully applied to the analysis of urine samples of gastric cancer patients. The results showed that the levels of most polyamines and N-acetylated polyamines from the patient group were significantly higher than those from the control group. The altered concentrations of the above-mentioned metabolites suggest their role in the pathogenesis of gastric cancer, and they should be further evaluated as potential markers of gastric cancer.