In situ analysis of osmolyte mechanisms of proteome thermal stabilization.

Organisms use organic molecules called osmolytes to adapt to environmental conditions. In vitro studies indicate that osmolytes thermally stabilize proteins, but mechanisms are controversial, and systematic studies within the cellular milieu are lacking. We analyzed Escherichia coli and human protein thermal stabilization by osmolytes in situ and across the proteome. Using structural proteomics, we probed osmolyte effects on protein thermal stability, structure and aggregation, revealing common mechanisms but also osmolyte- and protein-specific effects. All tested osmolytes (trimethylamine N-oxide, betaine, glycerol, proline, trehalose and glucose) stabilized many proteins, predominantly via a preferential exclusion mechanism, and caused an upward shift in temperatures at which most proteins aggregated. Thermal profiling of the human proteome provided evidence for intrinsic disorder in situ but also identified potential structure in predicted disordered regions. Our analysis provides mechanistic insight into osmolyte function within a complex biological matrix and sheds light on the in situ prevalence of intrinsically disordered regions.

Fast deep learning reconstruction techniques for preclinical magnetic resonance fingerprinting.

We propose a deep learning (DL) model and a hyperparameter optimization strategy to reconstruct T1 and T2 maps acquired with the magnetic resonance fingerprinting (MRF) methodology. We applied two different MRF sequence routines to acquire images of ex vivo rat brain phantoms using a 7-T preclinical scanner. Subsequently, the DL model was trained using experimental data, completely excluding the use of any theoretical MRI signal simulator. The best combination of the DL parameters was implemented by an automatic hyperparameter optimization strategy, whose key aspect is to include all the parameters to the fit, allowing the simultaneous optimization of the neural network architecture, the structure of the DL model, and the supervised learning algorithm. By comparing the reconstruction performances of the DL technique with those achieved from the traditional dictionary-based method on an independent dataset, the DL approach was shown to reduce the mean percentage relative error by a factor of 3 for T1 and by a factor of 2 for T2 , and to improve the computational time by at least a factor of 37. Furthermore, the proposed DL method enables maintaining comparable reconstruction performance, even with a lower number of MRF images and a reduced k-space sampling percentage, with respect to the dictionary-based method. Our results suggest that the proposed DL methodology may offer an improvement in reconstruction accuracy, as well as speeding up MRF for preclinical, and in prospective clinical, investigations.

The S/T-Rich Motif in the DNAJB6 Chaperone Delays Polyglutamine Aggregation and the Onset of Disease in a Mouse Model.

Expanded CAG repeats lead to debilitating neurodegenerative disorders characterized by aggregation of proteins with expanded polyglutamine (polyQ) tracts. The mechanism of aggregation involves primary and secondary nucleation steps. We show how a noncanonical member of the DNAJ-chaperone family, DNAJB6, inhibits the conversion of soluble polyQ peptides into amyloid fibrils, in particular by suppressing primary nucleation. This inhibition is mediated by a serine/threonine-rich region that provides an array of surface-exposed hydroxyl groups that bind to polyQ peptides and may disrupt the formation of the H bonds essential for the stability of amyloid fibrils. Early prevention of polyQ aggregation by DNAJB6 occurs also in cells and leads to delayed neurite retraction even before aggregates are visible. In a mouse model, brain-specific coexpression of DNAJB6 delays polyQ aggregation, relieves symptoms, and prolongs lifespan, pointing to DNAJB6 as a potential target for disease therapy and tool for unraveling early events in the onset of polyQ diseases.

The binding of the small heat-shock protein αB-crystallin to fibrils of α-synuclein is driven by entropic forces.

Molecular chaperones are key components of the cellular proteostasis network whose role includes the suppression of the formation and proliferation of pathogenic aggregates associated with neurodegenerative diseases. The molecular principles that allow chaperones to recognize misfolded and aggregated proteins remain, however, incompletely understood. To address this challenge, here we probe the thermodynamics and kinetics of the interactions between chaperones and protein aggregates under native solution conditions using a microfluidic platform. We focus on the binding between amyloid fibrils of α-synuclein, associated with Parkinson's disease, to the small heat-shock protein αB-crystallin, a chaperone widely involved in the cellular stress response. We find that αB-crystallin binds to α-synuclein fibrils with high nanomolar affinity and that the binding is driven by entropy rather than enthalpy. Measurements of the change in heat capacity indicate significant entropic gain originates from the disassembly of the oligomeric chaperones that function as an entropic buffer system. These results shed light on the functional roles of chaperone oligomerization and show that chaperones are stored as inactive complexes which are capable of releasing active subunits to target aberrant misfolded species.

α-Synuclein Aggregation Is Triggered by Oligomeric Amyloid-ß 42 via Heterogeneous Primary Nucleation.

An increasing number of cases where amyloids of different proteins are found in the same patient are being reported. This observation complicates diagnosis and clinical intervention. Amyloids of the amyloid-ß peptide or the protein α-synuclein are traditionally considered hallmarks of Alzheimer's and Parkinson's diseases, respectively. However, the co-occurrence of amyloids of these proteins has also been reported in patients diagnosed with either disease. Here, we show that soluble species containing amyloid-ß can induce the aggregation of α-synuclein. Fibrils formed under these conditions are solely composed of α-synuclein to which amyloid-ß can be found associated but not as part of the core of the fibrils. Importantly, by global kinetic analysis, we found that the aggregation of α-synuclein under these conditions occurs via heterogeneous primary nucleation, triggered by soluble aggregates containing amyloid-ß.

Multiparametric Orthogonal Characterization of Extracellular Vesicles by Liquid Chromatography Combined with In-Line Light Scattering and Fluorescence Detection.

Extracellular vesicles (EVs) are membrane-enclosed biological nanoparticles with potential as diagnostic markers and carriers for therapeutics. Characterization of EVs poses severe challenges due to their complex structure and composition, requiring the combination of orthogonal analytical techniques. Here, we demonstrate how liquid chromatography combined with multi-angle light scattering (MALS) and fluorescence detection in one single apparatus can provide multiparametric characterization of EV samples, including concentration of particles, average diameter of the particles, protein amount to particle number ratio, presence of EV surface markers and lipids, EV shape, and sample purity. The method requires a small amount of sample of approximately 107 EVs, limited handling of the sample and data analysis time in the order of minutes; it is fully automatable and can be applied to both crude and purified samples.

High-Yield Separation of Extracellular Vesicles Using Programmable Zwitterionic Coacervates.

Programmable coacervates based on zwitterionic polymers are designed as dynamic materials for ion exchange bioseparation. These coacervates are proposed as promising materials for the purification of soft nanoparticles such as liposomes and extracellular vesicles (EVs). It is shown that the stimulus-responsiveness of the coacervates and the recruitment of desired molecules can be independently programmed by polymer design. Moreover, the polymeric coacervates can recruit and release intact liposomes, human EVs, and nanoalgosomes in high yields and separate vesicles from different types of impurities, including proteins and nucleic acids. This approach combines the speed and simplicity of precipitation methods and the programmability of chromatography with the gentleness of aqueous two-phase separation, thereby guaranteeing product stability. This material represents a promising alternative for providing a low-shear, gentle, and selective purification method for EVs.

AF4 and PEG Precipitation as Predictive Assays for Antibody Self-Association.

Monoclonal antibodies (mAbs) are often formulated as high-protein-concentration solutions, which in some cases can exhibit physical stability issues such as high viscosity and opalescence. To ensure that mAb-based drugs can meet their manufacturing, stability, and delivery requirements, it is advantageous to screen for and select mAbs during discovery that are not prone to such behaviors. It has been recently shown that both these macroscopic properties can be predicted to a certain extent from the diffusion interaction parameter (kD), which is a measure of self-association under dilute conditions.1 However, kD can be challenging to measure at the early stage of discovery, where a relatively large amount of a high-purity material, which is required by traditional methods, is often not available. In this study, we demonstrate asymmetric field-flow fractionation (AF4) as a tool to measure self-association and therefore identify antibodies with problematic issues at high concentrations. The principle lies on the ability to concentrate the sample close to the membrane during the injection mode, which can reach formulation-relevant concentrations (>100 mg/mL).2 By analyzing a well-characterized library of commercial antibodies, we show that the measured retention time of the antibodies allows us to pinpoint molecules that exhibit issues at high concentrations. Remarkably, our AF4 assay requires very little (30 µg) sample under dilute conditions and does not need extensive sample purification. Furthermore, we show that a polyethylene glycol (PEG) precipitation assay provides results consistent with AF4 and moreover can further differentiate molecules with issues of opalescence or high viscosity. Overall, our results delineate a two-step strategy for the identification of problematic variants at high concentrations, with AF4 for early developability screening, followed by a PEG assay to validate the problematic molecules and further discriminate between opalescence or high-viscosity issues. This two-step antibody selection strategy enables us to select antibodies early in the discovery process, which are compatible with high-concentration formulations.

Surface Passivation Method for the Super-repellence of Aqueous Macromolecular Condensates.

Solutions of macromolecules can undergo liquid-liquid phase separation to form droplets with ultralow surface tension. Droplets with such low surface tension wet and spread over common surfaces such as test tubes and microscope slides, complicating in vitro experiments. The development of a universal super-repellent surface for macromolecular droplets has remained elusive because their ultralow surface tension requires low surface energies. Furthermore, the nonwetting of droplets containing proteins poses additional challenges because the surface must remain inert to a wide range of chemistries presented by the various amino acid side chains at the droplet surface. Here, we present a method to coat microscope slides with a thin transparent hydrogel that exhibits complete dewetting (contact angles θ ≈ 180°) and minimal pinning of phase-separated droplets in aqueous solution. The hydrogel is based on a swollen matrix of chemically cross-linked polyethylene glycol diacrylate of molecular weight 12 kDa (PEGDA), and can be prepared with basic chemistry laboratory equipment. The PEGDA hydrogel is a powerful tool for in vitro studies of weak interactions, dynamics, and the internal organization of phase-separated droplets in aqueous solutions.

Thermodynamic and kinetic design principles for amyloid-aggregation inhibitors.

Understanding the mechanism of action of compounds capable of inhibiting amyloid-fibril formation is critical to the development of potential therapeutics against protein-misfolding diseases. A fundamental challenge for progress is the range of possible target species and the disparate timescales involved, since the aggregating proteins are simultaneously the reactants, products, intermediates, and catalysts of the reaction. It is a complex problem, therefore, to choose the states of the aggregating proteins that should be bound by the compounds to achieve the most potent inhibition. We present here a comprehensive kinetic theory of amyloid-aggregation inhibition that reveals the fundamental thermodynamic and kinetic signatures characterizing effective inhibitors by identifying quantitative relationships between the aggregation and binding rate constants. These results provide general physical laws to guide the design and optimization of inhibitors of amyloid-fibril formation, revealing in particular the important role of on-rates in the binding of the inhibitors.

Effect of Phosphate and Ferritin Subunit Composition on the Kinetics, Structure, and Reactivity of the Iron Core in Human Homo- and Heteropolymer Ferritins.

Ferritins are highly conserved supramolecular protein nanostructures that play a key role in iron homeostasis. Thousands of iron atoms can be stored inside their hollow cavity as a hydrated ferric oxyhydroxide mineral. Although phosphate associates with the ferritin iron nanoparticles, the effect of physiological concentrations on the kinetics, structure, and reactivity of ferritin iron cores has not yet been explored. Here, the iron loading and mobilization kinetics were studied in the presence of 1-10 mM phosphate using homopolymer and heteropolymer ferritins having different H to L subunit ratios. In the absence of ferritin, phosphate enhances the rate of ferrous ion oxidation and forms large and soluble polymeric Fe(III)-phosphate species. In the presence of phosphate, Fe(II) oxidation and core formation in ferritin is significantly accelerated with oxidation rates several-fold higher than with phosphate alone. High-angle annular dark-field scanning transmission electron microscopy measurements revealed a strong phosphate effect on both the size and morphology of the iron mineral in H-rich (but not L-rich) ferritins. While iron nanoparticles in L-rich ferritins have spherical shape in the absence and presence of phosphate, iron nanoparticles in H-rich ferritins change from irregular shapes in the absence of phosphate to spherical particles in the presence of phosphate with larger size distribution and smaller particle size. In the presence of phosphate, the kinetics of iron-reductive mobilization from ferritin releases twice as much iron than in its absence. Altogether, our results demonstrate an important role for phosphate, and the ferritin H and L subunit composition toward the kinetics of iron oxidation and removal from ferritin, as well as the structure and reactivity of the iron mineral, and may have an important implication on ferritin iron management in vivo.

Droplet-Based Microfluidic Temperature-Jump Platform for the Rapid Assessment of Biomolecular Kinetics.

Protein folding, unfolding, and aggregation are important in a variety of biological processes and intimately linked to "protein misfolding diseases". The ability to perform experiments at different temperatures allows the extraction of important information regarding the kinetics and thermodynamics of such processes. Unfortunately, conventional stopped-flow methods are difficult to implement, generate limited information, and involve complex sample handling. To address this issue, we present a temperature-controlled droplet-based microfluidic platform that allows measurement of reaction kinetics on millisecond to second timescales and at temperatures between ambient and 90 °C. The utility of the microfluidic platform for measuring fast biomolecular kinetics at high temperatures is showcased through the investigation of the unfolding kinetics of haloalkane dehalogenases and the elongation of fibrils composed of the amyloid ß peptide associated with Alzheimer's disease. In addition, a deep-ultraviolet (UV) fluorescence microscope was developed for the on-chip recording of protein intrinsic fluorescence spectrum originating from aromatic amino acid residues. We envision that the developed optofluidic platform will find wide applicability in the analysis of biological processes, such as protein refolding and phase separation.

Droplet Microfluidics for the Label-Free Extraction of Complete Phase Diagrams and Kinetics of Liquid-Liquid Phase Separation in Finite Volumes.

Liquid-liquid phase separation of polymer and protein solutions is central in many areas of biology and material sciences. Here, an experimental and theoretical framework is provided to investigate the thermodynamics and kinetics of liquid-liquid phase separation in volumes comparable to cells. The strategy leverages droplet microfluidics to accurately measure the volume of the dense phase generated by liquid-liquid phase separation of solutions confined in micro-sized compartments. It is shown that the measurement of the volume fraction of the dense phase at different temperatures allows the evaluation of the binodal lines that determine the coexistence region of the two phases in the temperature-concentration phase diagram. By applying a thermodynamic model of phase separation in finite volumes, it is further shown that the platform can predict and validate kinetic barriers associated with the formation of a dense droplet in a parent dilute phase, therefore connecting thermodynamics and kinetics of liquid-liquid phase separation.

New Advances in Iron Metabolism, Ferritin and Hepcidin Research.

The interest in the regulation of iron metabolism has increased in recent years with the clarification of the mechanism by which hepcidin regulates systemic iron homeostasis and the discovery of ferritinophagy, the major mechanism of ferritin degradation, which plays a major role in intracellular iron homeostasis and ferroptosis [...].

Iron Mobilization from Ferritin in Yeast Cell Lysate and Physiological Implications.

Most in vitro iron mobilization studies from ferritin have been performed in aqueous buffered solutions using a variety of reducing substances. The kinetics of iron mobilization from ferritin in a medium that resembles the complex milieu of cells could dramatically differ from those in aqueous solutions, and to our knowledge, no such studies have been performed. Here, we have studied the kinetics of iron release from ferritin in fresh yeast cell lysates and examined the effect of cellular metabolites on this process. Our results show that iron release from ferritin in buffer is extremely slow compared to cell lysate under identical experimental conditions, suggesting that certain cellular metabolites present in yeast cell lysate facilitate the reductive release of ferric iron from the ferritin core. Using filtration membranes with different molecular weight cut-offs (3, 10, 30, 50, and 100 kDa), we demonstrate that a cellular component >50 kDa is implicated in the reductive release of iron. When the cell lysate was washed three times with buffer, or when NADPH was omitted from the solution, a dramatic decrease in iron mobilization rates was observed. The addition of physiological concentrations of free flavins, such as FMN, FAD, and riboflavin showed about a two-fold increase in the amount of released iron. Notably, all iron release kinetics occurred while the solution oxygen level was still high. Altogether, our results indicate that in addition to ferritin proteolysis, there exists an auxiliary iron reductive mechanism that involves long-range electron transfer reactions facilitated by the ferritin shell. The physiological implications of such iron reductive mechanisms are discussed.

Biochemical, Biophysical and Functional Characterization of an Insoluble Iron Containing Hepcidin-Ferritin Chimeric Monomer Assembled Together with Human Ferritin H/L Chains at Different Molar Ratios.

Hepcidin and ferritin are key proteins of iron homeostasis in mammals. In this study, we characterize a chimera by fusing camel hepcidin to a human ferritin H-chain to verify if it retained the properties of the two proteins. The construct (HepcH) is expressed in E. coli in an insoluble and iron-containing form. To characterize it, the product was incubated with ascorbic acid and TCEP to reduce and solubilize the iron, which was quantified with ferrozine. HepcH bound approximately five times more iron than the wild type human ferritin, due to the presence of the hepcidin moiety. To obtain a soluble and stable product, the chimera was denatured and renatured together with different amounts of L-ferritin of the H-chain in order to produce 24-shell heteropolymers with different subunit proportions. They were analyzed by denaturing and non-denaturing PAGE and by mass spectroscopy. At the 1:5 ratio of HepcH to H- or L-ferritin, a stable and soluble molecule was obtained. Its biological activity was verified by its ability to both bind specifically cell lines that express ferroportin and to promote ferroportin degradation. This chimeric molecule showed the ability to bind both mouse J774 macrophage cells, as well as human HepG2 cells, via the hepcidin-ferroportin axis. We conclude that the chimera retains the properties of both hepcidin and ferritin and might be exploited for drug delivery.

Broad-Band Spectrum, High-Sensitivity Absorbance Spectroscopy in Picoliter Volumes.

Picoliter-volume droplets within segmented flows can be probed in a rapid and efficient manner using optical detection methods. To date, however, most detection schemes for droplet content analysis have relied on the use of time-integrated fluorescence measurements. Despite its undoubted utility, the implementation of absorbance-based detectors is particularly challenging due to the reduced optical path lengths that are characteristic of microfluidic systems and deleterious scattering at droplet-oil interfaces. Unsurprisingly, efforts to develop sensitive absorbance-based detection schemes for the interrogation of rapidly moving droplets have primarily focused on ensuring adequate analytical sensitivity and, to date, have been exclusively limited to single-wavelength measurements. To address this limitation, and expand the information content associated with absorbance measurements on-chip, we herein describe a detection scheme for the extraction of broad-band absorbance spectra from pL-volume droplets with high sensitivity. The combination of a confocal optical system (that confines incident light to a reduced detection volume) and a postprocessing algorithm (that effectively removes the contribution of the carrier oil from the extracted spectra) engenders significant improvements in signal-to-noise ratios. Our system is initially calibrated by acquiring absorbance spectra from aqueous solutions of fluorescein isothiocyanate. These measurements confirm both excellent linearity over the studied range (from 0 to 100 µM) and a concentration limit of detection of 800 nM. The methodology is then used to monitor the salt-induced aggregation of gold nanoparticles with millisecond time resolution. This approach for small-volume absorbance spectroscopy allows for both high-throughput and high-information content measurements in subnanoliter volumes and will be highly desirable in a wide variety of bioanalytical applications where sensitivity and throughput are priorities.

Design of Biopharmaceutical Formulations Accelerated by Machine Learning.

In addition to activity, successful biological drugs must exhibit a series of suitable developability properties, which depend on both protein sequence and buffer composition. In the context of this high-dimensional optimization problem, advanced algorithms from the domain of machine learning are highly beneficial in complementing analytical screening and rational design. Here, we propose a Bayesian optimization algorithm to accelerate the design of biopharmaceutical formulations. We demonstrate the power of this approach by identifying the formulation that optimizes the thermal stability of three tandem single-chain Fv variants within 25 experiments, a number which is less than one-third of the experiments that would be required by a classical DoE method and several orders of magnitude smaller compared to detailed experimental analysis of full combinatorial space. We further show the advantage of this method over conventional approaches to efficiently transfer historical information as prior knowledge for the development of new biologics or when new buffer agents are available. Moreover, we highlight the benefit of our technique in engineering multiple biophysical properties by simultaneously optimizing both thermal and interface stabilities. This optimization minimizes the amount of surfactant in the formulation, which is important to decrease the risks associated with corresponding degradation processes. Overall, this method can provide high speed of converging to optimal conditions, the ability to transfer prior knowledge, and the identification of new nonlinear combinations of excipients. We envision that these features can lead to a considerable acceleration in formulation design and to parallelization of operations during drug development.

Iron distribution in different tissues of homozygous Mask (msk/msk) mice and the effects of oral iron treatments.

Iron-refractory iron deficiency anemia (IRIDA) is an autosomal recessive disorder caused by genetic mutations on TMPRSS6 gene which encodes Matriptase2 (MT2). An altered MT2 cannot appropriately suppress hepatic BMP6/SMAD signaling in case of low iron, hence hepcidin excess blocks dietary iron absorption, leading to a form of anemia resistant to oral iron supplementation. In this study, using the IRIDA mouse model Mask, we characterized homozygous (msk/msk) compared to asymptomatic heterozygous (msk/wt) mice, assessing the major parameters of iron status in different organs, at different ages in both sexes. The effect of carbonyl iron diet was analyzed as control iron supplementation being used for many studies in mice. It resulted effective in both anemic control and msk/msk mice, as expected, even if there is no information about its mechanism of absorption. Then, we mainly compared two forms of oral iron supplement, largely used for humans: ferrous sulfate and Sucrosomial iron. In anemic control mice, the two oral formulations corrected hemoglobin levels from 11.40 ± 0.60 to 15.38 ± 1.71 g/dl in 2-4 weeks. Interestingly, in msk/msk mice, ferrous sulfate did not increase hemoglobin likely due to ferroportin/hepcidin-dependent absorption, whereas Sucrosomial iron increased it from 11.50 ± 0.60 to 13.53 ± 0.64 g/dl mainly in the first week followed by a minor increase at 4 weeks with a stable level of 13.30 ± 0.80 g/dl, probably because of alternative absorption. Thus, Sucrosomial iron, already used in other conditions of iron deficiency, may represent a promising option for oral iron supplementation in IRIDA patients.

Longitudinal and transverse NMR relaxivities of Ln(III)-DOTA complexes: A comprehensive investigation.

Longitudinal and transverse 1H nuclear magnetic resonance relaxivities of Ln(III)-DOTA complexes (with Ln = Gd, Tb, Dy, Er; DOTA = 1,4,7,10-tetraazacyclododecane-N,N',N″,N‴-tetraacetic acid) and Mn(II) aqueous solutions were measured in a wide range of frequencies, 10 kHz to 700 MHz. The experimental data were interpreted by means of models derived from the Solomon-Bloembergen-Morgan theory. The data analysis was performed assuming the orbital angular momentum L = 0 for Gd-DOTA and the aqua ion [Mn(H2O)6]2+ and L ≠ 0 for Dy-, Tb-, and Er-DOTA. A refined estimation of the zero-field-splitting barrier Δ and of the modulation correlation time τv was obtained for [Mn(H2O)6]2+ by extending the fitting of nuclear magnetic relaxation dispersion profiles to the low-field regime. The Gd-DOTA fitting parameters resulted in good agreement with the literature, and the fit of transverse relaxivity data confirmed the negligibility of the scalar interaction in the nuclear relaxation mechanism. Larger transverse relaxivities of Dy-DOTA and Tb-DOTA (∼10 mM-1 s-1) with respect to Er-DOTA (∼1 mM-1 s-1) were observed at 16 T. Such higher values are suggested to be due to a shorter residence time τm that is possibly linked to the fluctuations of the hyperfine interaction and the different shape of the magnetic anisotropy. The possible employment of Dy-DOTA, Tb-DOTA, and Er-DOTA as negative magnetic resonance imaging contrast agents for high-field applications was envisaged by collecting spin-echo images at 7 T. Particularly in Dy- and Tb-derivatives, the transverse relaxivity at 16 T is of the order of the Gd-one at 1.5 T.