Scanning defocusing particle tracking for the experimental characterization of flows in demanding microfluidic systems.

A novel scanning particle image velocimetry technique, to the best of our knowledge, is proposed to characterize flows in microfluidic applications. Three-dimensional information is acquired by oscillating the target sample over a fixed focal plane, allowing the reconstruction of particle trajectories with micrometer accuracy over an extended depth. This technology is suited for investigating acoustic flows with unprecedented precision in microfluidic applications. In this contribution, we describe the experimental setup and the data processing pipeline in detail; we study the technique's performance by reconstructing pressure-driven flow; and we report the three-dimensional trajectory of a 2 µm particle in an acoustic flow in a 525µm×375µm microchannel with micrometric accuracy.

Rail configuration for lateral particle transport across intact co-flows: effect of wall and rail geometry on liquid and particle transport.

Layer-by-Layer coating technology is of great importance for many applications of microparticles whereby exposure of the particles to various reagents is needed. Mutual contamination of the reagents during this process is a key challenge, and this undesired effect should be avoided. Here we introduce a device that provides subsequent exposure of particles to various liquids and minimizes mixing of the liquids at the same time. The key element of the device is a rail (groove) at the bottom of a microfluidic channel. The rail forms an angle (between 0 and 90 degrees) and thus enables passive transport of particles through the intact co-flows of the different fluids. To avoid the undesirable effect of reagent stream mixing, internal walls are introduced to separate the different flows. Various designs of the proposed device are considered, and their performance is experimentally analyzed.

Competition in drug binding and the race to equilibrium.

Binding kinetics has become a popular topic in pharmacology due to its potential contribution to the selectivity and duration of drug action. Yet, the overall kinetic aspects of complex binding mechanisms are still merely described in terms of elaborate algebraic equations. Interestingly, it has been recommended some 10 years ago to examine such mechanisms in terms of binding fluxes instead of the conventional rate constants. Alike the velocity of product formation in enzymology, those fluxes refer to the velocity by which one target species converts into another one. Novel binding flux-based approaches are utilized to get a better visual insight into the "competition" between two drugs/ligands for a single target as well as between induced fit- and conformational selection pathways for a single ligand within a thermodynamic cycle. The present data were obtained by differential equation-based simulations. Early on, the ligand-binding steps "race" to equilibrium (i.e., when their forward and reverse fluxes are equal) at their individual pace. The overall/global equilibrium is only reached later on. For the competition association assays, this parting might produce a transient "overshoot" of one of the bound target species. A similar overshoot may also show up within a thermodynamic cycle and, at first glance, suggest that the induced fit pathway dominates. Yet, present findings show that under certain circumstances, it could rather be the other way round. Novel binding flux-based approaches offer visually attractive insights into crucial aspects of "complex" binding mechanisms under non-equilibrium conditions.

Influence of Centrifugation and Shaking on the Self-Assembly of Lysozyme Fibrils.

Protein self-assembly into fibrils and oligomers plays a key role in the etiology of degenerative diseases. Several pathways for this self-assembly process have been described and shown to result in different types and ratios of final assemblies, therewith defining the effective physiological response. Known factors that influence assembly pathways are chemical conditions and the presence or lack of agitation. However, in natural and industrial systems, proteins are exposed to a sequence of different and often complex mass transfers. In this paper, we compare the effect of two fundamentally different mass transfer processes on the fibrilization process. Aggregation-prone solutions of hen egg white lysozyme were subjected to predominantly non-advective mass transfer by employing centrifugation and to advective mass transport represented by orbital shaking. In both cases, fibrilization was triggered, while in quiescent only oligomers were formed. The fibrils obtained by shaking compared to fibrils obtained through centrifugation were shorter, thicker, and more rigid. They had rod-like protofibrils as building blocks and a significantly higher ß-sheet content was observed. In contrast, fibrils from centrifugation were more flexible and braided. They consisted of intertwined filaments and had low ß-sheet content at the expense of random coil. To the best of our knowledge, this is the first evidence of a fibrilization pathway selectivity, with the fibrilization route determined by the mass transfer and mixing configuration (shaking versus centrifugation). This selectivity can be potentially employed for directed protein fibrilization.

Rail induced lateral migration of particles across intact co-flowing liquids.

This paper presents a rail guided method to apply a Layer-by-Layer (LbL) coating on particles in a microfluidic device. The passive microfluidic approach allows handling suspensions of particles to be coated in the system. The trajectory of the particles is controlled using engraved rails, inducing lateral movement of particles while keeping the axially oriented liquid flow (and the interface of different liquids) undisturbed. The depth and angle of the rails together with the liquid velocity were studied to determine a workable geometry of the device. A discontinuous LbL coating procedure was converted into one continuous process, demonstrating that the chip can perform seven consecutive steps normally conducted in batch operation, further easily extendable to larger cycle numbers. Coating of the particles with two bilayers was confirmed by fluorescence microscopy.

Biological colloids: Unique properties of membraneless organelles in the cell.

Biomolecular condensates are membraneless, intracellular organelles that form via liquid-liquid phase separation (LLPS) and have the ability to concentrate a wide range of molecules in the cellular milieu. These organelles are highly dynamic and play pivotal roles in cellular organization and physiology. Many studies also link the formation and misregulation of condensates to diseases such as neurodegenerative disorders and cancer. Biomolecular condensates represent a special type of colloids that actively interact with their environment to sustain physiological functions, due to which their misregulation may upset cell signaling, resulting in pathological states. In this review, we discuss the mechanisms underlying the formation, dynamics, and evolution of these biological colloids, with a special focus on their surface properties that are critical in their interaction with other components of the cell. We also summarize experimental approaches that enable the detailed characterization of the formation, interactions, and functions of these cellular colloidal organelles.

F/YGG-motif is an intrinsically disordered nucleic-acid binding motif.

Heterogeneous nuclear ribonucleoproteins (hnRNP) function in RNA processing, have RNA-recognition motifs (RRMs) and intrinsically disordered, low-complexity domains (LCDs). While RRMs are drivers of RNA binding, there is only limited knowledge about the RNA interaction by the LCD of some hnRNPs. Here, we show that the LCD of hnRNPA2 interacts with RNA via an embedded Tyr/Gly-rich region which is a disordered RNA-binding motif. RNA binding is maintained upon mutating tyrosine residues to phenylalanines, but abrogated by mutating to alanines, thus we term the RNA-binding region 'F/YGG motif'. The F/YGG motif can bind a broad range of structured (e.g. tRNA) and disordered (e.g. polyA) RNAs, but not rRNA. As the F/YGG otif can also interact with DNA, we consider it a general nucleic acid-binding motif. hnRNPA2 LCD can form dense droplets, by liquid-liquid phase separation (LLPS). Their formation is inhibited by RNA binding, which is mitigated by salt and 1,6-hexanediol, suggesting that both electrostatic and hydrophobic interactions feature in the F/YGG motif. The D290V mutant also binds RNA, which interferes with both LLPS and aggregation thereof. We found homologous regions in a broad range of RNA- and DNA-binding proteins in the human proteome, suggesting that the F/YGG motif is a general nucleic acid-interaction motif.

Induced fit versus conformational selection: From rate constants to fluxes and back to rate constants.

Induced fit- (IF) and conformational selection (CS) binding mechanisms have long been regarded to be mutually exclusive. Yet, they are now increasingly considered to produce the final ligand-target complex alongside within a thermodynamic cycle. This viewpoint benefited from the introduction of binding fluxes as a tool for analyzing the overall behavior of such cycle. This study aims to provide more vivid and applicable insights into this emerging field. In this respect, combining differential equation- based simulations and hitherto little explored alternative modes of calculation provide concordant information about the intricate workings of such cycle. In line with previous reports, we observe that the relative contribution of IF increases with the ligand concentration at equilibrium. Yet the baseline contribution may vary from one case to another and simulations as well as calculations show that this parameter is essentially regulated by the dissociation rate of both pathways. Closer attention should be paid to how the contributions of IF and CS compare at physiologically relevant drug/ligand concentrations. To this end, a simple equation discloses how changing a limited set of "microscopic" rate constants can extend the concentration range at which CS contributes most effectively. Finally, it could also be beneficial to extend the utilization of flux- based approaches to more physiologically relevant time scales and alternative binding models.

High-throughput amplicon sequencing to assess the impact of processing factors on the development of microbial communities during spontaneous meat fermentation.

During spontaneous meat fermentation, diverse microbial communities develop over time. These communities consist mainly of lactic acid bacteria (LAB) and coagulase-negative staphylococci (CNS), of which the species composition is influenced by the fermentation temperature and the level of acidification. Recent development and application of amplicon-based high-throughput sequencing (HTS) methods have allowed to gain deeper insights into the microbial communities of fermented meats. The aim of the present study was to investigate the effect of different fermentation temperatures and acidification profiles on the CNS communities during spontaneous fermentation, using a previously developed amplicon-based HTS method targeting both the 16S rRNA and tuf genes. Spontaneous fermentations were performed with five different lots of meat to assess inter-lot variability. The process influence was investigated by fermenting the meat batters for seven days at different fermentation temperatures (23 °C, 30 °C, and 37 °C) and in the absence or presence of added glucose to simulate different acidification levels. Additionally, the results were compared with a starter culture-initiated fermentation process. The data revealed that the fermentation temperature was the most influential processing condition in shaping the microbial communities during spontaneous meat fermentation processes, whereas differences in pH were only responsible for minor shifts in the microbial profiles. Furthermore, the CNS communities showed a great level of variability, which depended on the initial microbial communities present and their competitiveness.

A generic approach to study the kinetics of liquid-liquid phase separation under near-native conditions.

Understanding the kinetics, thermodynamics, and molecular mechanisms of liquid-liquid phase separation (LLPS) is of paramount importance in cell biology, requiring reproducible methods for studying often severely aggregation-prone proteins. Frequently applied approaches for inducing LLPS, such as dilution of the protein from an urea-containing solution or cleavage of its fused solubility tag, often lead to very different kinetic behaviors. Here we demonstrate that at carefully selected pH values proteins such as the low-complexity domain of hnRNPA2, TDP-43, and NUP98, or the stress protein ERD14, can be kept in solution and their LLPS can then be induced by a jump to native pH. This approach represents a generic method for studying the full kinetic trajectory of LLPS under near native conditions that can be easily controlled, providing a platform for the characterization of physiologically relevant phase-separation behavior of diverse proteins.

Fluxes for Unraveling Complex Binding Mechanisms.

A decade ago, many high-affinity drugs were thought to bind to their target via an induced-fit pathway instead of conformational selection. Yet, both pathways make up part of a thermodynamic cycle, and, owing to binding flux-based approaches, it is now rather considered that they act in parallel and also that their relative contribution to the final ligand-target complex depends on the ligand concentration. Those approaches are of increasing interest, but published data still merely refer to the peculiar situation of equilibrium binding. This article draws attention to the benefit of extending those approaches to address more physiological nonequilibrium binding conditions and in vivo situations. For the presented example, they help to apprehend transient experimental manifestations of a 'conventional' thermodynamic cycle.

YtrASa, a GntR-Family Transcription Factor, Represses Two Genetic Loci Encoding Membrane Proteins in Sulfolobus acidocaldarius.

In bacteria, the GntR family is a widespread family of transcription factors responsible for the regulation of a myriad of biological processes. In contrast, despite their occurrence in archaea only a little information is available on the function of GntR-like transcription factors in this domain of life. The thermoacidophilic crenarchaeon Sulfolobus acidocaldarius harbors a GntR-like regulator belonging to the YtrA subfamily, encoded as the first gene in an operon with a second gene encoding a putative membrane protein. Here, we present a detailed characterization of this regulator, named YtrASa, with a focus on regulon determination and mechanistic analysis with regards to DNA binding. Genome-wide chromatin immunoprecipitation and transcriptome experiments, the latter employing a ytrA Sa overexpression strain, demonstrate that the regulator acts as a repressor on a very restricted regulon, consisting of only two targets including the operon encoding its own gene and a distinct genetic locus encoding another putative membrane protein. For both targets, a conserved 14-bp semi-palindromic binding motif was delineated that covers the transcriptional start site and that is surrounded by additional half-site motifs. The crystallographic structure of YtrASa was determined, revealing a compact dimeric structure in which the DNA-binding motifs are oriented ideally to enable a specific high-affinity interaction with the core binding motif. This study provides new insights into the functioning of a YtrA-like regulator in the archaeal domain of life.

Structure of the Prx6-subfamily 1-Cys peroxiredoxin from Sulfolobus islandicus.

Aerobic thermoacidophilic archaea belonging to the genus Sulfolobus harbor peroxiredoxins, thiol-dependent peroxidases that assist in protecting the cells from oxidative damage. Here, the crystal structure of the 1-Cys peroxiredoxin from Sulfolobus islandicus, named 1-Cys SiPrx, is presented. A 2.75 Šresolution data set was collected from a crystal belonging to space group P212121, with unit-cell parameters a = 86.8, b = 159.1, c = 189.3 Å, α = ß = γ = 90°. The structure was solved by molecular replacement using the homologous Aeropyrum pernix peroxiredoxin (ApPrx) structure as a search model. In the crystal structure, 1-Cys SiPrx assembles into a ring-shaped decamer composed of five homodimers. This quaternary structure corresponds to the oligomeric state of the protein in solution, as observed by size-exclusion chromatography. 1-Cys SiPrx harbors only a single cysteine, which is the peroxidatic cysteine, and lacks both of the cysteines that are highly conserved in the C-terminal arm domain in other archaeal Prx6-subfamily proteins such as ApPrx and that are involved in the association of dimers into higher-molecular-weight decamers and dodecamers. It is thus concluded that the Sulfolobus Prx6-subfamily protein undergoes decamerization independently of arm-domain cysteines.

Quantification of Intrinsically Disordered Proteins: A Problem Not Fully Appreciated.

Protein quantification is essential in a great variety of biochemical assays, yet the inherent systematic errors associated with the concentration determination of intrinsically disordered proteins (IDPs) using classical methods are hardly appreciated. Routinely used assays for protein quantification, such as the Bradford assay or ultraviolet absorbance at 280 nm, usually seriously misestimate the concentrations of IDPs due to their distinct and variable amino acid composition. Therefore, dependable method(s) have to be worked out/adopted for this task. By comparison to elemental analysis as the gold standard, we show through the example of four globular proteins and nine IDPs that the ninhydrin assay and the commercial QubitTM Protein Assay provide reliable data on IDP quantity. However, as IDPs can show extreme variation in amino acid composition and physical features not necessarily covered by our examples, even these techniques should only be used for IDPs following standardization. The far-reaching implications of these simple observations are demonstrated through two examples: (i) circular dichroism spectrum deconvolution, and (ii) receptor-ligand affinity determination. These actual comparative examples illustrate the potential errors that can be incorporated into the biophysical parameters of IDPs, due to systematic misestimation of their concentration. This leads to inaccurate description of IDP functions.

Characterization of the diffusive dynamics of particles with time-dependent asymmetric microscopy intensity profiles.

We put forth an algorithm to track isolated micron-size solid and liquid particles that produce time-dependent asymmetric intensity patterns. This method quantifies the displacement of a particle in the image plane from the peak of a spatial cross-correlation function with a reference image. The peak sharpness results in subpixel resolution. We demonstrate the utility of the method for tracking liquid droplets with changing shapes and micron-size particles producing images with exaggerated asymmetry. We compare the accuracy of diffusivity determination with particles of known size by this method to that by common tracking techniques and demonstrate that our algorithm is superior. We address several open questions on the characterization of diffusive behaviors. We show that for particles, diffusing with a root-mean-square displacement of 0.6 pixel widths in the time between two successive recorded frames, more accurate diffusivity determinations result from mean squared displacement (MSD) for lag times up to 5 time intervals and that MSDs determined from non-overlapping displacements do not yield more accurate diffusivities. We discuss the optimal length of image sequences and demonstrate that lower frame rates do not affect the accuracy of the estimated diffusivity.

The unexpected structure of the designed protein Octarellin V.1 forms a challenge for protein structure prediction tools.

Despite impressive successes in protein design, designing a well-folded protein of more 100 amino acids de novo remains a formidable challenge. Exploiting the promising biophysical features of the artificial protein Octarellin V, we improved this protein by directed evolution, thus creating a more stable and soluble protein: Octarellin V.1. Next, we obtained crystals of Octarellin V.1 in complex with crystallization chaperons and determined the tertiary structure. The experimental structure of Octarellin V.1 differs from its in silico design: the (αßα) sandwich architecture bears some resemblance to a Rossman-like fold instead of the intended TIM-barrel fold. This surprising result gave us a unique and attractive opportunity to test the state of the art in protein structure prediction, using this artificial protein free of any natural selection. We tested 13 automated webservers for protein structure prediction and found none of them to predict the actual structure. More than 50% of them predicted a TIM-barrel fold, i.e. the structure we set out to design more than 10years ago. In addition, local software runs that are human operated can sample a structure similar to the experimental one but fail in selecting it, suggesting that the scoring and ranking functions should be improved. We propose that artificial proteins could be used as tools to test the accuracy of protein structure prediction algorithms, because their lack of evolutionary pressure and unique sequences features.

Step Crowding Effects Dampen the Stochasticity of Crystal Growth Kinetics.

Crystals grow by laying down new layers of material which can either correspond in size to the height of one unit cell (elementary steps) or multiple unit cells (macrosteps). Surprisingly, experiments have shown that macrosteps can grow under conditions of low supersaturation and high impurity density such that elementary step growth is completely arrested. We use atomistic simulations to show that this is due to two effects: the fact that the additional layers bias fluctuations in the position of the bottom layer towards growth and by a transition, as step height increases, from a 2D to a 3D nucleation mechanism.

Do protein crystals nucleate within dense liquid clusters?

Protein-dense liquid clusters are regions of high protein concentration that have been observed in solutions of several proteins. The typical cluster size varies from several tens to several hundreds of nanometres and their volume fraction remains below 10(-3) of the solution. According to the two-step mechanism of nucleation, the protein-rich clusters serve as locations for and precursors to the nucleation of protein crystals. While the two-step mechanism explained several unusual features of protein crystal nucleation kinetics, a direct observation of its validity for protein crystals has been lacking. Here, two independent observations of crystal nucleation with the proteins lysozyme and glucose isomerase are discussed. Firstly, the evolutions of the protein-rich clusters and nucleating crystals were characterized simultaneously by dynamic light scattering (DLS) and confocal depolarized dynamic light scattering (cDDLS), respectively. It is demonstrated that protein crystals appear following a significant delay after cluster formation. The cDDLS correlation functions follow a Gaussian decay, indicative of nondiffusive motion. A possible explanation is that the crystals are contained inside large clusters and are driven by the elasticity of the cluster surface. Secondly, depolarized oblique illumination dark-field microscopy reveals the evolution from liquid clusters without crystals to newly nucleated crystals contained in the clusters to grown crystals freely diffusing in the solution. Collectively, the observations indicate that the protein-rich clusters in lysozyme and glucose isomerase solutions are locations for crystal nucleation.

Mesoscopic Impurities Expose a Nucleation-Limited Regime of Crystal Growth.

Nanoscale self-assembly is naturally subject to impediments at the nanoscale. The recently developed ability to follow processes at the molecular level forces us to resolve older, coarse-grained concepts in terms of their molecular mechanisms. In this Letter, we highlight one such example. We present evidence based on experimental and simulation data that one of the cornerstones of crystal growth theory, the Cabrera-Vermilyea model of step advancement in the presence of impurities, is based on incomplete physics. We demonstrate that the piercing of an impurity fence by elementary steps is not solely determined by the Gibbs-Thomson effect, as assumed by Cabrera-Vermilyea. Our data show that for conditions leading up to growth cessation, step retardation is dominated by the formation of critically sized fluctuations. The growth recovery of steps is counter to what is typically assumed, not instantaneous. Our observations on mesoscopic impurities for lysozyme expose a nucleation-dominated regime of growth that has not been hitherto considered, where the system alternates between zero and near-pure velocity. The time spent by the system in arrest is the nucleation induction time required for the step to amass a supercritical fluctuation that pierces the impurity fence.