Optimization Strategies for Mass Spectrometry-Based Untargeted Metabolomics Analysis of Small Polar Molecules in Human Plasma.

The untargeted approach to mass spectrometry-based metabolomics has a wide potential to investigate health and disease states, identify new biomarkers for diseases, and elucidate metabolic pathways. All this holds great promise for many applications in biological and chemical research. However, the complexity of instrumental parameters on advanced hybrid mass spectrometers can make the optimization of the analytical method immensely challenging. Here, we report a strategy to optimize the selected settings of a hydrophilic interaction liquid chromatography-tandem mass spectrometry method for untargeted metabolomics studies of human plasma, as a sample matrix. Specifically, we evaluated the effects of the reconstitution solvent in the sample preparation procedure, the injection volume employed, and different mass spectrometry-related operating parameters including mass range, the number of data-dependent fragmentation scans, collision energy mode, duration of dynamic exclusion time, and mass resolution settings on the metabolomics data quality and output. This study highlights key instrumental variables influencing the detection of metabolites along with suggested settings for the IQ-X tribrid system and proposes a new methodological framework to ensure increased metabolome coverage.

Improving the Mechanical Rigidity of Hyaluronic Acid by Integration of a Supramolecular Peptide Matrix.

Hyaluronic acid (HA), a major component of the extracellular matrix, is an attractive material for various medical applications. Yet, its low mechanical rigidity and fast in vivo degradation hinder its utilization. Here, we demonstrate the reinforcement of HA by its integration with a low-molecular-weight peptide hydrogelator to produce a composite hydrogel. The formulation of HA with the fluorenylmethoxycarbonyl diphenylalanine (FmocFF) peptide, one of the most studied self-assembling hydrogel-forming building blocks, showing notable mechanical properties, resulted in the formation of stable, homogeneous hydrogels. Electron microscopy analysis demonstrated a uniform distribution of the two matrices in the composite forms. The composite hydrogels showed improved mechanical properties and stability to enzymatic degradation while maintaining their biocompatibility. Moreover, the storage modulus of the FmocFF/HA composite hydrogels reached up to 25 kPa. The composite hydrogels allowed sustained release of curcumin, a hydrophobic polyphenol showing antioxidant, anti-inflammatory, and antitumor activities. Importantly, the rate of curcumin release was modulated as a function of the concentration of the FmocFF peptide within the hydrogel matrix. This work provides a new approach for conferring mechanical rigidity and stability to HA without the need of cross-linking, thus potentially facilitating its utilization in different clinical applications, such as sustained drug release.

Synthetic 9-cis-beta-carotene inhibits photoreceptor degeneration in cultures of eye cups from rpe65rd12 mouse model of retinoid cycle defect.

The retinoid cycle enzymes regenerate the visual chromophore 11-cis retinal to enable vision. Mutations in the genes encoding the proteins of the retinoid cycle are the leading cause for recessively inherited retinal dystrophies such as retinitis pigmentosa, Leber congenital amaurosis, congenital cone-rod dystrophy and fundus albipunctatus. Currently there is no treatment for these blinding diseases. In previous studies we demonstrated that oral treatment with the 9-cis-ß-carotene rich Dunaliella Bardawil algae powder significantly improved visual and retinal functions in patients with retinitis pigmentosa and fundus albipunctatus. Here we developed a convenient and economical synthetic route for biologically active 9-cis-ß-carotene from inexpensive building materials and demonstrated that the molecule is stable for at least one month. Synthetic 9-cis-ß-carotene rescued cone photoreceptors from degeneration in eye cup cultures of mice with a retinoid cycle genetic defect. This study suggests that synthetic 9-cis-ß-carotene may serve as an effective treatment for retinal dystrophies involving the retinoid cycle.

Functional Single-Chain Polymer Nanoparticles: Targeting and Imaging Pancreatic Tumors in Vivo.

The development of tools for the early diagnosis of pancreatic adenocarcinoma is an urgent need in order to increase treatment success rate and reduce patient mortality. Here, we present a modular nanosystem platform integrating soft nanoparticles with a targeting peptide and an active imaging agent for diagnostics. Biocompatible single-chain polymer nanoparticles (SCPNs) based on poly(methacrylic acid) were prepared and functionalized with the somatostatin analogue PTR86 as the targeting moiety, since somatostatin receptors are overexpressed in pancreatic cancer. The gamma emitter 67Ga was incorporated by chelation and allowed in vivo investigation of the pharmacokinetic properties of the nanoparticles using single photon emission computerized tomography (SPECT). The resulting engineered nanosystem was tested in a xenograph mouse model of human pancreatic adenocarcinoma. Imaging results demonstrate that accumulation of targeted SCPNs in the tumor is higher than that observed for nontargeted nanoparticles due to improved retention in this tissue.

Light-emitting self-assembled peptide nucleic acids exhibit both stacking interactions and Watson-Crick base pairing.

The two main branches of bionanotechnology involve the self-assembly of either peptides or DNA. Peptide scaffolds offer chemical versatility, architectural flexibility and structural complexity, but they lack the precise base pairing and molecular recognition available with nucleic acid assemblies. Here, inspired by the ability of aromatic dipeptides to form ordered nanostructures with unique physical properties, we explore the assembly of peptide nucleic acids (PNAs), which are short DNA mimics that have an amide backbone. All 16 combinations of the very short di-PNA building blocks were synthesized and assayed for their ability to self-associate. Only three guanine-containing di-PNAs-CG, GC and GG-could form ordered assemblies, as observed by electron microscopy, and these di-PNAs efficiently assembled into discrete architectures within a few minutes. The X-ray crystal structure of the GC di-PNA showed the occurrence of both stacking interactions and Watson-Crick base pairing. The assemblies were also found to exhibit optical properties including voltage-dependent electroluminescence and wide-range excitation-dependent fluorescence in the visible region.

The influence of chemical chaperones on enzymatic activity under thermal and chemical stresses: common features and variation among diverse chemical families.

Molecular and chemical chaperones are key components of the two main mechanisms that ensure structural stability and activity under environmental stresses. Yet, chemical chaperones are often regarded only as osmolytes and their role beyond osmotic regulation is not fully understood. Here, we systematically studied a large group of chemical chaperones, representatives of diverse chemical families, for their protective influence under either thermal or chemical stresses. Consistent with previous studies, we observed that in spite of the structural similarity between sugars and sugar alcohols, they have an apparent difference in their protective potential. Our results support the notion that the protective activity is mediated by the solvent and the presence of water is essential. In the current work we revealed that i) polyols and sugars have a completely different profile of protective activity toward trifluoroethanol and thermal stress; ii) minor changes in solvent composition that do not affect enzyme activity, yet have a great effect on the ability of osmolytes to act as protectants and iii) increasing the number of active groups of carbohydrates makes them better protectants while increasing the number of active groups of methylamines does not, as revealed by attempts to synthesize de novo designed methylamines with multiple functional groups.

Peptide-based hydrogel nanoparticles as effective drug delivery agents.

Peptide-based hydrogel nanoparticles represent a promising alternative to current drug delivery approaches. We have previously demonstrated that the Fmoc-FF aromatic dipeptide building block can self-assemble in aqueous solutions to form nano-scaled ordered hydrogels of remarkable mechanical rigidity. Here, we present a scalable process for the assembly of this peptide into hydrogel nanoparticles (HNPs) aimed to be utilized as potential drug delivery carriers. Fmoc-FF based HNPs were formulated via modified inverse-emulsion method using vitamin E-TPGS as an emulsion stabilizer and high speed homogenization. The formed HNPs exhibited two distinguishable populations with an average size of 21.5±1.3 and 225.9±0.8 nm. Gold nanoparticles were encapsulated within the hydrogel nanoparticles as contrast agents to monitor the formation of the assemblies and their ultrastructural properties. Next, we demonstrated a robust experimental procedure developed and optimized for the formulation, purification, storage and handling procedures of HNPs. Encapsulation of doxorubicin (Dox) and 5-flourouracil (5-Fu) within the HNPs matrix showed release kinetics of the drugs depending on their chemical structure, molecular weight and hydrophobicity. The results clearly indicate that Fmoc-FF based hydrogel nanoparticles have the potential to be used as encapsulation and delivery system of various drugs and bioactive molecules.

Quantitative structure-activity relationship analysis of ß-amyloid aggregation inhibitors.

Inhibiting the aggregation process of the ß-amyloid peptide is a promising strategy in treating Alzheimer's disease. In this work, we have collected a dataset of 80 small molecules with known inhibition levels and utilized them to develop two comprehensive quantitative structure-activity relationship models: a Bayesian model and a decision tree model. These models have exhibited high predictive accuracy: 87% of the training and test sets using the Bayesian model and 89 and 93% of the training and test sets, respectively, by the decision tree model. Subsequently these models were used to predict the activities of several new potential ß-amyloid aggregation inhibitors and these predictions were indeed validated by in vitro experiments. Key chemical features correlated with the inhibition ability were identified. These include the electro-topological state of carbonyl groups, AlogP and the number of hydrogen bond donor groups. The results demonstrate the feasibility of the developed models as tools for rapid screening, which could help in the design of novel potential drug candidates for Alzheimer's disease.

Self-assembled arrays of peptide nanotubes by vapour deposition.

The use of bionanostructures in real-world applications will require precise control over biomolecular self-assembly and the ability to scale up production of these materials. A significant challenge is to control the formation of large, homogeneous arrays of bionanostructures on macroscopic surfaces. Previously, bionanostructure formation has been based on the spontaneous growth of heterogenic populations in bulk solution. Here, we demonstrate the self-assembly of large arrays of aromatic peptide nanotubes using vapour deposition methods. This approach allows the length and density of the nanotubes to be fine-tuned by carefully controlling the supply of the building blocks from the gas phase. Furthermore, we show that the nanotube arrays can be used to develop high-surface-area electrodes for energy storage applications, highly hydrophobic self-cleaning surfaces and microfluidic chips.

Magnetically responsive carboxylated magnetite-polydipyrrole/polydicarbazole nanocomposites of core-shell morphology. Preparation, characterization, and use in DNA hybridization.

Novel bis-heterocyclic mono- and dicarboxylated dipyrrole and dicarbazole monomers have been synthesized in a modular manner. Their oxidative polymerization around magnetite nanosized particles has been investigated and optimized toward new magnetic magnetite-polydipyrrole/polydicarbazole nanocomposites (NCs) of a core-shell morphology. These NCs were thoroughly characterized by FT-IR, TGA (Thermal Gravimetric Analysis), low- and high-resolution TEM/HR-TEM microscopies, and Mössbauer spectroscopy along with magnetization studies. Exploiting the versatile COOH chemistry (activation by water-soluble diimides) introduced by the polymeric shell, DNA hybridization experiments have been conducted onto NC surfaces using an efficient blue-colored HRP-based enzymatic screening biological system. Highly parallel NC-supported DNA hybridization experimentations revealed that these NCs presented an interesting potential for DNA-based diagnostic applications.

New magnetically responsive polydicarbazole-magnetite nanoparticles.

Magnetically responsive COOH-polydicarbazole-magnetite nanocomposites have been prepared by chemical oxidation of three COOH-dicarbazole monomers and - in the presence of magnetite nanoparticles. These functionalized nanoparticles have been tested for DNA hybridization experiments.

A novel approach to a one-pot synthesis of unsubstituted oligo(alpha-thiophenes).

Oligo(alpha-thiophenes) alpha-4T and alpha-8T were prepared by the following one-pot sequential conversions: thiophene (T) --> alpha-2T --> alpha-4T --> alpha-8T. PdCl(2)-induced coupling of a mono-alpha-mercuration derivative of each of the n-mers T, alpha-2T, and alpha-4T was applied in these conversions.