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.

Perspectives on recent advancements in energy harvesting, sensing and bio-medical applications of piezoelectric gels.

The development of next-generation bioelectronics, as well as the powering of consumer and medical devices, require power sources that are soft, flexible, extensible, and even biocompatible. Traditional energy storage devices (typically, batteries and supercapacitors) are rigid, unrecyclable, offer short-lifetime, contain hazardous chemicals and possess poor biocompatibility, hindering their utilization in wearable electronics. Therefore, there is a genuine unmet need for a new generation of innovative energy-harvesting materials that are soft, flexible, bio-compatible, and bio-degradable. Piezoelectric gels or PiezoGels are a smart crystalline form of gels with polar ordered structures that belongs to the broader family of piezoelectric material, which generate electricity in response to mechanical stress or deformation. Given that PiezoGels are structurally similar to hydrogels, they offer several advantages including intrinsic chirality, crystallinity, degree of ordered structures, mechanical flexibility, biocompatibility, and biodegradability, emphasizing their potential applications ranging from power generation to bio-medical applications. Herein, we describe recent examples of new functional PiezoGel materials employed for energy harvesting, sensing, and wound dressing applications. First, this review focuses on the principles of piezoelectric generators (PEGs) and the advantages of using hydrogels as PiezoGels in energy and biomedical applications. Next, we provide a detailed discussion on the preparation, functionalization, and fabrication of PiezoGel-PEGs (P-PEGs) for the applications of energy harvesting, sensing and wound healing/dressing. Finally, this review concludes with a discussion of the current challenges and future directions of P-PEGs.

Metal Organic Spin Transistor.

Organic molecules and specifically bio-organic systems are attractive for applications due to their low cost, variability, environmental friendliness, and facile manufacturing in a bottom-up fashion. However, due to their relatively low conductivity, their actual application is very limited. Chiral metallo-bio-organic crystals, on the other hand, have improved conduction and in addition interesting magnetic properties. We developed a spin transistor using these crystals and based on the chiral-induced spin selectivity effect. This device features a memristor type behavior, which depend on trapping both charges and spins. The spin properties are monitored by Hall signal and by an external magnetic field. The spin transistor exhibits nonlinear drain-source currents, with multilevel controlled states generated by the magnetization of the source. Varying the source magnetization enables a six-level readout for the two-terminal device. The simplicity of the device paves the way for its technological application in organic electronics and bioelectronics.

Long-Range Spin-Selective Transport in Chiral Metal-Organic Crystals with Temperature-Activated Magnetization.

Room-temperature, long-range (300 nm), chirality-induced spin-selective electron conduction is found in chiral metal-organic Cu(II) phenylalanine crystals, using magnetic conductive-probe atomic force microscopy. These crystals are found to be also weakly ferromagnetic and ferroelectric. Notably, the observed ferromagnetism is thermally activated, so that the crystals are antiferromagnetic at low temperatures and become ferromagnetic above ∼50 K. Electron paramagnetic resonance measurements and density functional theory calculations suggest that these unusual magnetic properties result from indirect exchange interaction of the Cu(II) ions through the chiral lattice.

Bioinspired Supramolecular Packing Enables High Thermo-Sustainability.

Bottom-up self-assembled bioinspired materials have attracted increasing interest in a variety fields. The use of peptide supramolecular semiconductors for optoelectronic applications is especially intriguing. However, the characteristic thermal unsustainability limits their practical application. Here, we report the thermal sustainability of cyclo-ditryptophan assemblies up to 680 K. Non-covalent interactions underlie the stability mechanism, generating a low exciton-binding energy of only 0.29 eV and a high thermal-quenching-activation energy of up to 0.11 eV. The contributing forces comprise predominantly of aromatic interactions, followed by hydrogen bonding between peptide molecules, and, to a lesser extent, water-mediated associations. This thermal sustainability results in a temperature-dependent conductivity of the supramolecular semiconductors, showing 93 % reduction of the resistance from 320 K to 440 K. Our results establish thermo-sustainable peptide self-assembly for heat-sensitive applications.

Enhanced Fluorescence for Bioassembly by Environment-Switching Doping of Metal Ions.

The self-assembly of cyclodipeptides composed of natural aromatic amino acids into supramolecular structures of diverse morphologies with intrinsic emissions in the visible light region is demonstrated. The assembly process can be halted at the initial oligomerization by coordination with zinc ions, with the most prominent effect observed for cyclo-dihistidine (cyclo-HH). This process is mediated by attracting and pulling of the metal ions from the solvent into the peptide environment, rather than by direct interaction in the solvent as commonly accepted, thus forming an "environment-switching" doping mechanism. The doping induces a change of cyclo-HH molecular configurations and leads to the formation of pseudo "core/shell" clusters, comprising peptides and zinc ions organized in ordered conformations partially surrounded by relatively amorphous layers, thus significantly enhancing the emissions and allowing the application of the assemblies for ecofriendly color-converted light emitting diodes. These findings shed light into the very initial coordination procedure and elucidate an alternative mechanism of metal ions doping on biomolecules, thus presenting a promising avenue for integration of the bioorganic world and the optoelectronic field.

High-Efficiency Fluorescence through Bioinspired Supramolecular Self-Assembly.

Peptide self-assembly has attracted extensive interest in the field of eco-friendly optoelectronics and bioimaging due to its inherent biocompatibility, intrinsic fluorescence, and flexible modulation. However, the practical application of such materials was hindered by the relatively low quantum yield of such assemblies. Here, inspired by the molecular structure of BFPms1, we explored the "self-assembly locking strategy" to design and manipulate the assembly of metal-stabilized cyclic(l-histidine-d-histidine) into peptide material with the high-fluorescence efficiency. We used this bioorganic material as an emissive layer in photo- and electroluminescent prototypes, demonstrating the feasibility of utilizing self-assembling peptides to fabricate a biointegrated microchip that incorporates eco-friendly and tailored optoelectronic properties. We further employed a "self-encapsulation" strategy for constructing an advanced nanocarrier with integrated in situ monitoring. The strategy of the supramolecular capture of functional components exemplifies the use of bioinspired organic chemistry to provide frontiers of smart materials, potentially allowing a better interface between sustainable optoelectronics and biomedical applications.

Rigid Tightly Packed Amino Acid Crystals as Functional Supramolecular Materials.

The formation of ordered nanostructures by metabolites is gaining increased interest due to the simplicity of the building blocks and their natural occurrence. Specifically, aromatic amino acids possess the ability to form ordered supramolecular interactions due to their limited solubility in aqueous solution. Unexpectedly, l-tyrosine (l-Tyr) is almost 2 orders of magnitude less soluble in water compared to l-phenylalanine (l-Phe). However, the underlying mechanism is not fully understood as l-Tyr is more polar. Here, we explore the utilization of insoluble tyrosine assemblies for technological applications and their molecular basis by manipulating the basic building blocks of tightly packed dimers. We show that the addition of an amyloid inhibition agent increases l-Tyr solubility due to the disruption of the dimer formation. The molecular organization grants the l-Tyr crystal higher thermal stability and mechanical properties between three amino acids. Additionally, l-Tyr crystals are shown to generate high and stable piezoelectric power outputs under mechanical pressure in a sandwich device. By incorporating the rigid l-Tyr crystals into a soft polymer, a mechano-responsive bending composite was fabricated. Furthermore, the l-Tyr crystalline needles exhibit an active photowaveguiding property, making them promising candidates for the generation of photonic biomaterial-based devices. The present work exemplifies a feasible strategy to explore physical properties of supramolecular self-assemblies comprises minimalistic naturally occurring building blocks and their applications in energy harvesting, photonic devices, stretchable electronics, and soft robotics.

Metal-Ion Modulated Structural Transformation of Amyloid-Like Dipeptide Supramolecular Self-Assembly.

The misfolding of proteins and peptides potentially leads to a conformation transition from an α-helix or random coil to ß-sheet-rich fibril structures, which are associated with various amyloid degenerative disorders. Inhibition of the ß-sheet aggregate formation and control of the structural transition could therefore attenuate the development of amyloid-associated diseases. However, the structural transitions of proteins and peptides are extraordinarily complex processes that are still not fully understood and thus challenging to manipulate. To simplify this complexity, herein, the effect of metal ions on the inhibition of amyloid-like ß-sheet dipeptide self-assembly is investigated. By changing the type and ratio of the metal ion/dipeptide mixture, structural transformation is achieved from a ß-sheet to a superhelix or random coil, as confirmed by experimental results and computational studies. Furthermore, the obtained supramolecular metallogel exhibits excellent in vitro DNA binding and diffusion capability due to the positive charge of the metal/dipeptide complex. This work may facilitate the understanding of the role of metal ions in inhibiting amyloid formation and broaden the future applications of supramolecular metallogels in three-dimensional (3D) DNA biochip, cell culture, and drug delivery.

Microfluidics for real-time direct monitoring of self- and co-assembly biomolecular processes.

Molecular self-assembly is a major approach for the fabrication of functional supramolecular nanomaterials. This dynamic, straightforward, bottom-up procedure may result in the formation of various architectures at the nano-scale, with remarkable physical and chemical characteristics. Biological and bio-inspired building blocks are especially attractive due to their intrinsic tendency to assemble into well-organized structures, as well as their inherent biocompatibility. To further expand the morphological diversity, co-assembly methods have been developed, allowing to produce alternative unique architectures, enhanced properties, and improved structural control. However, in many cases, mechanistic understanding of the self- and co-assembly processes is still lacking. Microfluidic techniques offer a set of exclusive tools for real-time monitoring of biomolecular self-organization, which is crucial for the study of such dynamic processes. Assembled nuclei, confined by micron-scale pillars, could be subjected to controlled environments aiming to assess the effect of different conditions on the assembly process. Other microfluidics setups can produce droplets at a rate of over 100 s-1, with volumes as small as several picoliters. Under these conditions, each droplet can serve as an individual pico/nano-reactor allowing nucleation and assembly. These processes can be monitored, analyzed and imaged, by various techniques including simple bright-field microscopy. Elucidating the mechanism of such molecular events may serve as a conceptual stepping-stone for the rational control of the resulting physicochemical properties.

The Inhibitory Effect of Hydroxylated Carbon Nanotubes on the Aggregation of Human Islet Amyloid Polypeptide Revealed by a Combined Computational and Experimental Study.

Fibrillar deposits formed by the aggregation of the human islet amyloid polypeptide (hIAPP) are the major pathological hallmark of type 2 diabetes mellitus (T2DM). Inhibiting the aggregation of hIAPP is considered the primary therapeutic strategy for the treatment of T2DM. Hydroxylated carbon nanoparticles have received great attention in impeding amyloid protein fibrillation owing to their reduced cytotoxicity compared to the pristine ones. In this study, we investigated the influence of hydroxylated single-walled carbon nanotubes (SWCNT-OHs) on the first step of hIAPP aggregation: dimerization by performing explicit solvent replica exchange molecular dynamics (REMD) simulations. Extensive REMD simulations demonstrate that SWCNT-OHs can dramatically inhibit interpeptide ß-sheet formation and completely suppress the previously reported ß-hairpin amyloidogenic precursor of hIAPP. On the basis of our simulation results, we proposed that SWCNT-OH can hinder hIAPP fibrillation. This was further confirmed by our systematic turbidity measurements, thioflavin T fluorescence, circular dichroism (CD), transmission electron microscope (TEM), and atomic force microscopy (AFM) experiments. Detailed analyses of hIAPP-SWCNT-OH interactions reveal that hydrogen bonding, van der Waals, and π-stacking interactions between hIAPP and SWCNT-OH significantly weaken the inter- and intrapeptide interactions that are crucial for ß-sheet formation. Our collective computational and experimental data reveal not only the inhibitory effect but also the inhibitory mechanism of SWCNT-OH against hIAPP aggregation, thus providing new clues for the development of future drug candidates against T2DM.

Mechanistic perspective and functional activity of insulin in amylin aggregation.

Insulin is a key regulatory polypeptide that is secreted from pancreatic ß-cells and has several important effects on the synthesis of lipids, regulation of enzymatic activities, blood glucose levels and the prevention of hyperglycemia. Insulin was demonstrated to self-assemble into ordered amyloid fibrils upon repeated injections, although the possible biological significance of the supramolecular structures is enigmatic. Amylin is also an amyloidogenic polypeptide that is secreted from pancreatic ß-cells and plays an important role in glycemic regulation preventing post-prandial spikes in blood glucose levels. These two amyloidogenic proteins are secreted together from the pancreas and have the ability to interact and produce insulin-amylin aggregates. So far, the molecular architecture of insulin-amylin complexes at the atomic resolution has been unknown. The current work identifies for the first time the specific π-π interactions between Y16 in insulin and F19 in amylin that contribute to the stability of the insulin-amylin complex, by using experimental and molecular modeling techniques. We performed additional experiments that verify the functional activity of insulin in amylin aggregation. Our findings illustrate for the first time the specific interactions between insulin and amylin aggregates at the atomic resolution and provide a new mechanistic perspective on the effect of insulin on amylin aggregation and may pave the way towards pharmacological intervention in this process.

Opal-like Multicolor Appearance of Self-Assembled Photonic Array.

Molecular self-assembly of short peptide building blocks leads to the formation of various material architectures that may possess unique physical properties. Recent studies had confirmed the key role of biaromaticity in peptide self-assembly, with the diphenylalanine (FF) structural family as an archetypal model. Another significant direction in the molecular engineering of peptide building blocks is the use of fluorenylmethoxycarbonyl (Fmoc) modification, which promotes the assembly process and may result in nanostructures with distinctive features and macroscopic hydrogel with supramolecular features and nanoscale order. Here, we explored the self-assembly of the protected, noncoded fluorenylmethoxycarbonyl-ß,ß-diphenyl-Ala-OH (Fmoc-Dip) amino acid. This process results in the formation of elongated needle-like crystals with notable aromatic continuity. By altering the assembly conditions, arrays of spherical particles were formed that exhibit strong light scattering. These arrays display vivid coloration, strongly resembling the appearance of opal gemstones. However, unlike the Rayleigh scattering effect produced by the arrangement of opal, the described optical phenomenon is attributed to Mie scattering. Moreover, by controlling the solution evaporation rate, i.e., the assembly kinetics, we were able to manipulate the resulting coloration. This work demonstrates a bottom-up approach, utilizing self-assembly of a protected amino acid minimal building block, to create arrays of organic, light-scattering colorful surfaces.

NPT088 reduces both amyloid-ß and tau pathologies in transgenic mice.

INTRODUCTION: Alzheimer's disease (AD) is characterized by appearance of both extracellular senile plaques and intracellular neurofibrillary tangles, comprised of aggregates of misfolded amyloid-ß (Aß) and hyper-phosphorylated tau, respectively. In a previous study, we demonstrated that g3p, a capsid protein from bacteriophage M13, binds to and remodels misfolded aggregates of proteins that assume an amyloid conformation. We engineered a fusion protein ("NPT088") consisting of the active fragment of g3p and human-IgG1-Fc. METHODS: Aged Tg2576 mice or rTg4510 mice received NPT088 weekly via IP injection. Cognitive and/or functional motor endpoints were monitored during dosing. Pathology was quantified biochemically and immunohistochemically. RESULTS: NPT088-lowered Aß plaque and improved cognitive performance of aged Tg2576 mice. Moreover, NPT088 reduced phospho-tau pathology, reduced brain atrophy, and improved cognition in rTg4510 mice. DISCUSSION: These observations establish NPT088 as a novel therapeutic approach and potential drug class that targets both Aß and tau, the hallmark pathologies of AD.

A bacteriophage capsid protein provides a general amyloid interaction motif (GAIM) that binds and remodels misfolded protein assemblies.

Misfolded protein aggregates, characterized by a canonical amyloid fold, play a central role in the pathobiology of neurodegenerative diseases. Agents that bind and sequester neurotoxic intermediates of amyloid assembly, inhibit the assembly or promote the destabilization of such protein aggregates are in clinical testing. Here, we show that the gene 3 protein (g3p) of filamentous bacteriophage mediates potent generic binding to the amyloid fold. We have characterized the amyloid binding and conformational remodeling activities using an array of techniques, including X-ray fiber diffraction and NMR. The mechanism for g3p binding with amyloid appears to reflect its physiological role during infection of Escherichia coli, which is dependent on temperature-sensitive interdomain unfolding and cis-trans prolyl isomerization of g3p. In addition, a natural receptor for g3p, TolA-C, competitively interferes with Aß binding to g3p. NMR studies show that g3p binding to Aß fibers is predominantly through middle and C-terminal residues of the Aß subunit, indicating ß strand-g3p interactions. A recombinant bivalent g3p molecule, an immunoglobulin Fc (Ig) fusion of the two N-terminal g3p domains, (1) potently binds Aß fibers (fAß) (KD=9.4nM); (2); blocks fAß assembly (IC50~50nM) and (3) dissociates fAß (EC50=40-100nM). The binding of g3p to misfolded protein assemblies is generic, and amyloid-targeted activities can be demonstrated using other misfolded protein systems. Taken together, our studies show that g3p(N1N2) acts as a general amyloid interaction motif.

Apoptosis induced by islet amyloid polypeptide soluble oligomers is neutralized by diabetes-associated specific antibodies.

Soluble oligomeric assemblies of amyloidal proteins appear to act as major pathological agents in several degenerative disorders. Isolation and characterization of these oligomers is a pivotal step towards determination of their pathological relevance. Here we describe the isolation of Type 2 diabetes-associated islet amyloid polypeptide soluble cytotoxic oligomers; these oligomers induced apoptosis in cultured pancreatic cells, permeated model lipid vesicles and interacted with cell membranes following complete internalization. Moreover, antibodies which specifically recognized these assemblies, but not monomers or amyloid fibrils, were exclusively identified in diabetic patients and were shown to neutralize the apoptotic effect induced by these oligomers. Our findings support the notion that human IAPP peptide can form highly toxic oligomers. The presence of antibodies identified in the serum of diabetic patients confirms the pathological relevance of the oligomers. In addition, the newly identified structural epitopes may also provide new mechanistic insights and a molecular target for future therapy.

Photoacoustic Doppler measurement of flow using tone burst excitation.

In this paper a novel technique for flow measurement which is based on the photoacoustic (PA) Doppler effect is described. A significant feature of the proposed approach is that it can be implemented using tone burst optical excitation thus enabling simultaneous measurement of both velocity and position. The technique, which is based on external modulation and heterodyne detection, was experimentally demonstrated by measurement of the flow of a suspension of carbon particles in a silicon tube and successfully determined the particles mean velocity up to values of 130 mm/sec, which is about 10 times higher than previously reported PA Doppler set-ups. In the theoretical part a rigorous derivation of the PA response of a flowing medium is described and some important simplifying approximations are highlighted.

Simultaneous spatial and spectral mapping of flow using photoacoustic Doppler measurement.

We demonstrate the use of tone-burst excitation and time-gated spectral analysis for photoacoustic Doppler mapping of flow in an unperturbed vessel phantom and in a vessel with a spatially varying lumen. The method, which mimics pulsed Doppler ultrasound, enables simultaneous measurement of axial position and flow as well as complete characterization of the Doppler spectrum over a wide range of mean velocities (3.5 to 200 mm∕s). To generate the required optical excitation, a continuous cw laser source followed by an external electro-optic modulator is used. Stenoses at various levels are emulated in a C-flex tube with a flowing suspension of micrometer-scale carbon particles. Two-dimensional maps of spectral content versus axial position at different points along the vessel and for various levels of perturbations demonstrate the potential use of the method for characterization of flow irregularities.

The use of pulse synthesis for optimization of photoacoustic measurements.

In this paper the use of pulse shaping in photoacoustic (PA) measurements is presented. The benefits of this approach are demonstrated by utilizing it for optimization of either the responsivity or the sensitivity of PA measurements. The optimization is based on the observation that the temporal properties of the PA effect can be represented as a linear system which can be fully characterized by its impulse response. Accordingly, the response of the PA system to an input optical pulse, whose instantaneous power is arbitrarily shaped, can be analytically predicted via a convolution between the pulse envelope and the PA impulse response. Additionally, the same formalism can be used to show that the response of the PA system to a pulse whose instantaneous power is a reversed version of the impulse response, i.e. a matched pulse, would exhibit optimal peak amplitude when compared with all other pulses with the same energy. Pulses can also be designed to optimize the sensitivity of the measurement to a variation in a specific system parameter. The use of the matched pulses can improve SNR and enable a reduction in the total optical energy required for obtaining a detectable signal. This may be important for applications where the optical power is restricted or for dynamical measurements where long integration times are prohibited. To implement this new approach, a novel PA optical setup which enabled synthesis of excitation waveforms with arbitrary temporal envelopes was constructed. The setup was based on a tunable laser source, operating in the near-IR range, and an external electro-optic modulator. Using this setup, our approach for system characterization and response prediction was tested and the superiority of the matched pulses over other common types of pulses of equal energy was demonstrated.

The role of the 14-20 domain of the islet amyloid polypeptide in amyloid formation.

The molecular mechanism of amyloid formation by the islet amyloid polypeptide (IAPP) has been intensively studied since its identification in the late 1980s. The IAPP(20-29) region is considered to be the central amyloidogenic module of the polypeptide. This assumption is mainly based on the amyloidogenic properties of the region and on the large sequence diversity within this region between the human and mouse IAPP, as the mouse IAPP does not form amyloids. A few years ago, another region within IAPP was identified that seems to be at least as important as IAPP(20-29) in facilitation of molecular recognition that leads to amyloid formation. Here, we reinforce our and others' previous findings by analyzing supporting evidence from the recent literature. Moreover, we provide new proofs to our hypothesis by comparing between the amyloidogenic properties of the two regions derived from the IAPP of cats, which is also known to form amyloid fibrils.