European Biophysics Journal

The author has withdrawn his manuscript because: The withdrawn preprint was about methodological aspects in Isothermal Titration Calorimetry (ITC) used to obtain thermodynamic information about reactions like A + B {rightleftarrows} C where A is initially in the cell and B injected from a syringe. The preprint considered the two possible methods in ITC: 1/ the Multiple Injection Method (MIM) making use of short-time injections separated by sufficient time to allow the reaction to reach equilibrium before a new injection. 2/ the Single Injection Method (SIM) making use of a slow continuous injection. The first result mentioned is about a new equation linking the rate of heat evolution with the injected volume (equations 9 and 10). With this equation and with the hypothesis that there is always perfect mixing of the cell content it was concluded that an ideal titration curve (i.e. not affected by any external influence) for a simple reaction like A + B {rightleftarrows} C cannot change sign (section 3.2). This conclusion turns out to be incorrect when taking in consideration real conditions with imperfect mixing, particularly with MIM using injections often of very short duration, which prevents from reaching perfect mixing. The major problem is that this erroneous conclusion was accompanied with comparisons of the results from well-established programs, which led to the conclusion that these were in error on this point (section 3.6). I therefore felt necessary to withdraw this preprint to avoid casting doubts unduly on these programs used extensively. Note that many other aspects in this preprint remain correct (section 3.8). A new version of this work, limited to SIM and considering imperfect mixing, will be submitted for publication under the title: "Isothermal titration calorimetry in the single-injection mode with imperfect mixing". If you have any questions, please contact me at dumasp@igbmc.fr or at p.dumas@unistra.fr Sorry for the inconvenience. Philippe Dumas November 6, 2021

From cellular mechanotransduction to the formation of embryonic tissues and organs, mechanics has been shown to play an important role in the control of cell behavior and embryonic development. Most of our existing knowledge of how mechanics affects cell behavior comes from in vitro studies, mainly because measuring cell and tissue mechanics in 3D multicellular systems, and especially in vivo, remains challenging. Oil microdroplet sensors, and more recently gel microbeads, use surface deformations to directly quantify mechanical stresses within developing tissues, in vivo and in situ, as well as in 3D in vitro systems like organoids or multicellular spheroids. However, an automated analysis software able to quantify the spatiotemporal evolution of stresses and their characteristics from particle deformations is lacking. Here we develop STRESS (Surface Topography Reconstruction for Evaluation of Spatiotemporal Stresses), an analysis software to quantify the geometry of deformable particles of spherical topology, such as microdroplets or gel microbeads, that enables the automatic quantification of the temporal evolution of stresses in the system and the spatiotemporal features of stress inhomogeneities in the tissue. As a test case, we apply these new code to measure the temporal evolution of mechanical stresses using oil microdroplets in developing zebrafish tissues. Starting from a 3D timelapse of a droplet, the software automatically calculates the statistics of local anisotropic stresses, decouples the deformation modes associated with tissue- and cell-scale stresses, obtains their spatial features on the droplet surface and analyzes their spatiotemporal variations using spatial and temporal stress autocorrelations. The automated nature of the analysis will help users obtain quantitative information about mechanical stresses in a wide range of 3D multicellular systems, from developing embryos or tissue explants to organoids. Author summaryThe measurement of mechanical stresses in 3D multicellular systems, such as living tissues, has been very challenging because of a lack in technologies for this purpose. Novel microdroplet techniques enable direct, quantitative in situ measurements of mechanical stresses in these systems. However, computational tools to obtain mechanical stresses from 3D images of microdroplets in an automated and accurate manner are lacking. Here we develop STRESS, an automated analysis software to analyze the spatiotemporal characteristics of mechanical stresses from microdroplet deformations in a wide range of systems, from living embryonic tissues and tissue explants to organoids and multicellular spheroids.

Colloid suspensions in the form of mammalian or bacterial cell mixtures in orbital shakers are commonly encountered in biomedical research. An understanding of particle motion in these conditions would provide insight into bulk colloid behavior and distribution under short and long timeframes. Such data can be used to supplement and clarify existing concepts of biological phenomena encountered in the laboratory setting. It can also aid biomedical researchers in experiment design and data interpretation. We present a MATLAB based simulation of colloid motion under rotary agitation. Our simulation setup is modular and therefore designed to act as a scaffold that can be customized to simulate different mammalian, bacterial, or particle properties in liquid suspension.

Sedimentation velocity analytical ultracentrifugation (SV-AUC) is an indispensable tool for the study of particle size distributions in biopharmaceutical industry, for example, to characterize protein therapeutics and vaccine products. In particular, the diffusion-deconvoluted sedimentation coefficient distribution analysis, in the software SEDFIT, has found widespread applications due to its relatively high resolution and sensitivity. However, a lack of available software compatible with Good Manufacturing Practices (GMP) has hampered the use of SV-AUC in this regulatory environment. To address this, we have created an interface for SEDFIT so that it can serve as an automatically spawned module with controlled data input through command line parameters and output of key results in files. The interface can be integrated in custom GMP compatible software, and in scripts that provide documentation and meta-analyses for replicate or related samples, for example, to streamline analysis of large families of experimental data, such as binding isotherm analyses in the study of protein interactions. To test and demonstrate this approach we provide a MATLAB script mlSEDFIT.

Rheology and the study of viscoelastic materials is an integral part of engineering and the study of biophysical systems however the cost of a rheometer is only feasible for colleges, universities and research laboratories. Even if a rheometer can be purchased it is bulky and delicately calibrated limiting its usefulness to the laboratory itself. The design presented here is less than a tenth of the cost of a professional rheometer and portable making it the ideal solution for high school students as a way to introduce viscoelasticity at a younger age as well as for use in the field for obtaining preliminary rheological data.

When optimized, sedimentation velocity analytical ultracentrifugation (SV-AUC) provides the most-accurate, broadest-range, and highest-resolution size distribution analysis of any method. Generating simulated data for an adeno-associated virus (AAV) sample consisting of four species differing only in their DNA content and having closely spaced sedimentation coefficients, allows manipulation of the SV-AUC experimental protocol to optimize the size distribution resolution. In developing this high speed SV-AUC (hs-SV-AUC) protocol several experimental challenges must be overcome: 1) the need for rapid data acquisition, 2) avoiding optical artifacts from steep boundaries and 3) overcoming the increased potential for convection. A protocol, hs-SV-AUC, has been developed that uses high rotor speeds, interference detection and low temperatures to overcome these challenges. By confining data analysis to a limited radial-time window and using a very short run time (< 20 min after temperature equilibration), the need to match the sample and reference solvent composition and meniscus positions is relaxed, making interference detection is as simple to employ as absorbance detection. Experimental size distributions from the same AAV sample by hs-SV-AUC at 45K rpm and 10 {degrees}C versus low-speed SV-AUC at 15K rpm, and 10 {degrees}C illustrates the improved size distribution resolution offered by the hs-SV-AUC protocol.

Actin is a major intracellular protein with key functions in cellular motility, signalling and structural rearrangements. Its dynamic behavior with actin filaments (F-actin) polymerising and depolymerising in response to intracellular changes, is controlled by actin-binding proteins (ABPs). Gelsolin is one of the most potent filament severing ABPs. However, myosin motors that interact with actin in the presence of ATP also produce actin filament fragmentation through motor induced shearing forces. To test the idea that gelsolin and myosin cooperate in these processes we used the in vitro motility assay, where actin filaments are propelled by surface-adsorbed heavy meromyosin (HMM) motor fragments. This allows studies of both motility and filament dynamics using isolated proteins. Gelsolin (5 nM) at very low [Ca2+] (free [Ca2+] [~]6.8 nM) appreciably enhanced actin filament severing caused by HMM-induced forces at 1 mM [MgATP], an effect that was increased at increased HMM motor density. This finding is consistent with cooperativity between actin filament severing by myosin-induced forces and by gelsolin. As further support of myosin-gelsolin cooperativity we observed reduced sliding velocity of the HMM propelled filaments in the presence of gelsolin. Overall, the results corroborate ideas for cooperative effects between gelsolin-induced alterations in the actin filaments and changes due to myosin motor activity, leading among other effects to enhanced F-actin severing of possible physiological relevance.

Surfactants and film-forming polymers are common ingredients in consumer spray products such as cleaning products, hair care products, and anti-perspirants. Spraying eases application by creating aerosolised droplets of the product that can distribute evenly over the treated surface. However, these aerosols can potentially be inhaled during their normal application. Droplets that reach the alveoli can interact with the pulmonary surfactant; a complex mixture of phospholipids and proteins that regulates the surface tension at the air-liquid interface. This interaction could elevate the minimum surface tension at maximum compression and change the surface rheology of the pulmonary surfactant at the interface. We tested four surfactants and seven polymers for their ability to inhibit pulmonary surfactant function in vitro and investigated if the inhibition is dose-rate dependent i.e., the product of the concentration (mg/mL) and aerosolisation rate (mL/min). We found that independent of chemical class (surfactant or polymer) there was a clear dose-rate dependent inhibition of pulmonary surfactant function and that different chemicals inhibited function at different dose-rates. We compared the points of departure of inhibitory chemicals to a polymer with known dose-rate dependent lung toxicity. When assessing the risk of chemicals that might be inhaled, it is essential to ensure normal use would not inhibit pulmonary surfactant function leading to immediate effects on the lungs. Lay summarySpray products create a cloud of tiny droplets in the air when they are used. This cloud can be inhaled, and if it reaches the deepest parts of the lungs, it can interact with the thin layer of liquid, called pulmonary surfactant, that covers the cells. It protects the lung tissue during the constant movement of breathing. Droplets can sometimes disrupt the pulmonary surfactant function, making breathing difficult. Chemicals that are used in spray products must be tested to assess if they are harmful if inhaled. In this project we studied the effect of chemicals that are commonly found in spray products on the functioning of the pulmonary surfactant in vitro. The results can be combined with other in vitro methods to test if chemicals are harmful to inhale without testing on animals.

BackgroundMucus in the endocervix serves as fertility gatekeeper in the reproductive tract through hormonally regulated changes in biophysical properties. Cervical mucus can thicken to prevent ascension of sperm into the upper reproductive tract or thin to permit fertilization. Current reproductive studies of mucus viscoelastic properties rely on subjective visual appraisal of cervical mucus changes. Our goal was to use particle tracking microrheology (PTMR) to objectively assess cervical mucus viscoelastic properties and associate these measurements with in vitro measures of sperm velocity. MethodsUsing cervical mucus obtained from rhesus macaques (Macaca mulatta) at necropsy, we used to PTMR to measure viscoelasticity (*) under stepwise, serial dilutions. In parallel we measure sperm velocity using custom sperm tracking and analysis workflows. ResultsWe report that both mucus * and sperm velocity displayed a concentration-dependent behavior, where * increased as mucus concentration increased, and sperm velocity correspondingly decreased. Viscoelasticity and sperm velocity were strongly negatively correlated (p<0.001). ConclusionsPTMR and sperm tracking in mucus provide quantitative measure of viscoelastic mucus changes. PTMR is potentially a method for quantitatively assessing fertility potential in the cervix that could be applied to both infertility and contraceptives studies.

Spheroids are in vitro spherical structures of cell aggregates, eventually cultured within a hydrogel matrix, that are used, among other applications, as a technological platform to investigate tumor formation and evolution. Several interesting features can be replicated using this methodology, such as cell communication mechanisms, the effect of gradients of nutrients, or the creation of realistic 3D biological structures. In this paper, we propose a continuum mechanobiological model which accounts for the most relevant phenomena that take place in tumor spheroids evolution under in vitro suspension, namely, nutrients diffusion in the spheroid, kinetics of cellular growth and death, and mechanical interactions among the cells. The model is qualitatively validated, after calibration of the model parameters, versus in vitro experiments of spheroids of different glioblastoma cell lines. This preliminary validation allowed us to conclude that glioblastoma tumor spheroids evolution is mainly driven by mechanical interactions of the cell aggregate and the dynamical evolution of the cell population. In particular, it is concluded that our model is able to explain quite different setups, such as spheroids growth (up to six times the initial configuration for U-87 MG cell line) or shrinking (almost half of the initial configuration for U-251 MG cell line); as the result of the mechanical interplay of cells driven by cellular evolution. Indeed, the main contribution of this work is to link the spheroid evolution with the mechanical activity of cells, coupled with nutrient consumption and the subsequent cell dynamics. All this information can be used to further investigate mechanistic effects in the evolution of tumors and their role in cancer disease. Author summarySpheroids structures of cell aggregates are an available experimental platform to analyze the evolution and drug response of solid tumors. In particular, the dynamics of different glioblastoma cell lines have been studied in this work using spheroids. Interestingly, very different behaviors were observed, from a half of the initial configuration shrinking for U-251 MG cell line to six times the initial configuration growth for U-87 MG cell line. These results were replicated by means of a coupled mathematical model which accounts for nutrients diffusion in the spheroid, kinetics of cellular growth and death, and mechanical interactions among the cells. Tumor growth or shrinkage can be explained from a continuum mechanics view driven by cell activity and nutrients availability. This modeling put the focus on mechanistic effects and is aligned with novel experimental techniques to quantify the mechanical microenvironment in tumors. These techniques may be combined with the approach presented in this work to further investigate the role of mechanics in cancer disease.

Liposomes are widely used as model lipid membrane platforms in many fields, ranging from basic biophysical studies to drug delivery and biotechnology applications. Various methods exist to prepare liposomes, but common procedures include thin-film hydration followed by extrusion, freeze-thaw, and/or sonication. These procedures have the potential to produce liposomes at specific concentrations and membrane compositions, and researchers often assume that the concentration and composition of their liposomes are similar to, if not identical, to what would be expected if no lipid loss occurred during preparation. However, lipid loss and concomitant biasing of lipid composition can in principle occur at any preparation step due to nonideal mixing, lipid-surface interactions, etc. Here, we report a straightforward method using HPLC-ELSD to quantify the lipid concentration and membrane composition of liposomes, and apply that method to study the preparation of simple POPC/cholesterol liposomes. We examine many common steps in liposome formation, including vortexing during re-suspension, hydration of the lipid film, extrusion, freeze-thaw, sonication, and the percentage of cholesterol in the starting mixture. We found that the resuspension step can play an outsized role in determining the overall lipid loss (up to [~]50% under seemingly rigorous procedures). The extrusion step yielded smaller lipid losses ([~]10-20%). Freeze-thaw and sonication could both be employed to improve lipid yields. Hydration times up to 60 minutes and increasing cholesterol concentrations up to 50 mole% had little influence on lipid recovery. Fortunately, even conditions with large lipid loss did not substantially influence the target membrane composition more than [~]5% under the conditions we tested. From our results, we identify best practices for producing maximum levels of lipid recovery and minimal changes to lipid composition during liposome preparation protocols. We expect our results can be leveraged for improved preparation of model membranes by researchers in many fields. Statement of SignificanceLiposomes are spherical lipid membranes that can be prepared by a variety of biophysical techniques. Researchers use liposomes in a variety of ways, including fundamental biophysical studies of lipid membranes, in drug delivery, drug formulation, and other biotechnology applications. In this report, we study the process to prepare liposomes by several common techniques and validate how reliable each technique is at producing consistent liposome concentrations and lipid compositions. We identify best practices for researchers to produce reliable liposome preparations.

Many biophysical systems and proteins undergo mesoscopic conformational changes in order to perform their biological function. However, these conformational changes often result from a cascade of atomistic interactions within a much larger overall object. For simulations of such systems, the computational cost of retaining high-resolution structural and dynamical information whilst at the same time observing large scale motions over long times is high. Furthermore, this cost is only going to increase as ever larger biological objects are observed experimentally at high resolution. We derive a generalised mechano-kinetic simulation framework which aims to compensate for these disparate time scales, capable of dynamically exploring a defined mechanical energy landscape whilst at the same time performing kinetic transitions between discretely defined states. With insight from the theory of Markov state models and transition state theory, the framework models continuous dynamical motion at coarse-grained length scales whilst simultaneously including fine-detail, chemical reactions or conformational changes implicitly using a kinetic model. The kinetic model is coupled to the dynamic model in a highly generalised manner, such that kinetic transition rates are continuously modulated by the dynamic simulation. Further, it can be applied to any defined continuous energy landscape, and hence, any existing dynamic simulation framework. We present a series of analytical examples to validate the framework, and showcase its capabilities for studying more complex systems by simulating protein unfolding via single-molecule force spectroscopy on an atomic force microscope. Author summaryOur intention with this work is to provide a generalised, highly coarse-grained model to allow kinetic processes (conformational changes, protein unfolding, chemical reactions etc) to occur within the context of a dynamic simulation. Performing computationally intensive dynamic simulations to obtain kinetic information can be unjustifiably costly and scientifically inefficient, and so we instead want to emphasise that experimentally available kinetic information can be used to infer the underlying dynamics they result from. We hope our work can begin a discussion on the topic of computationally efficient science, and continue the drive towards collaborative science between theory and experimentation.

The post-detachment drifting phase of macrophytes, during which they can be alive, dead, or senescent, plays a crucial ecological and biogeochemical role by influencing long-range dispersal, transporting rafting species, affecting carbon sequestration, promoting blooms, and leading to beaching events. In order to predict the dispersal of macrophytes and macroplastic particles and where they will affect the ecosystem, it is important to be able to model how their drift velocities are influenced by hydrodynamic and aerodynamic factors. In this study, we investigated the drift velocity of macrophytes with diverse morphologies and macroplastic particles in a racetrack flume under different current conditions, in combination with and without wind in the same direction as the water current. Our data show that the drift velocity of macrophytes is highly dependent on their buoyancy and affected by morphological characteristics. Wind increased the velocity of the surface water, which in turn increased the drift velocity of both macrophytes and macroplastic particles. However, wind-induced turbulences reduced the overall effect, especially for macrophytes, which protruded minimally above the water surface in comparison to macroplastic particles. For positively buoyant specimens, an existing particle model was experimentally confirmed to predict macrophyte and macroplastic particle drift velocities reliably, irrespective of shape. For negatively buoyant species, we propose a novel equation to predict drift velocity, incorporating the diverse shapes of macrophytes, as well as their interaction with the bottom. These results represent the first step toward the development of trait-based models that represent macrophytes more realistically in dispersal simulations. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/709487v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@1ab9f6aorg.highwire.dtl.DTLVardef@6ef75dorg.highwire.dtl.DTLVardef@132334forg.highwire.dtl.DTLVardef@c6a3d8_HPS_FORMAT_FIGEXP M_FIG C_FIG

Bicelles have been demonstrated to be a valuable tool for studying membrane protein interactions and structure in vitro. They are distinguished by a distinct lipid bilayer that mimics the plasma membrane of cells making it more native-like than its detergent micelle counter-part. Bicelles are typically comprised of a long-chain phospholipid such as dimyristoylphosphatidylcholine (DMPC) and a short-chain phospholipid such as dihexanoylphosphatidylcholine (DHPC). When mixed together in solution DMPC-DHPC bicelles assume a discoidal structure comprised of a heterogeneous arrangement where the short-chain lipids gather around the rim of the disk and the long-chain lipids form the flat, planar, bilayer region. In this study, the nonionic surfactant, C8E5, was used to prepare mixtures with DMPC to determine if it adopts properties similar to bicelles with a q [&ge;] 0.5. At q [&ge;] 0.5, DMPC-DHPC bicelles are bilayered and DMPC is sequestered from the detergent micelle-like DHPC. Mixtures of DMPC and C8E5 were prepared at various q values, a parameter used to describe the mole ratio of DMPC to DHPC in the preparation of bicelles. Employing biophysical methods like dynamic light scattering, 31P-NMR and analytical ultracentrifugation, properties of these lipid-detergent complexes are described. Interestingly they adopted a spherical-shaped micellar structure morphology and did not assume a discoidal shape typical of bicelles at q [&ge;] 0.5. However, they appear to retain bilayer-like properties that may prove beneficial for in vitro biophysical studies of membrane proteins.

The article presents the results of current measurements in membrane systems with bacterial cellulose membranes, located in horizontal plane, for various initial quotients of NaCl concentrations on the membrane. The obtained time-current characteristics indicate a stable formation of concentration boundary layers near the membrane for the configuration with a solution of lower concentration and lower density above the membrane (A). In turn, for the configuration with a higher concentration and higher density solution above the membrane (B) and a sufficiently large initial concentration quotient on the membrane, greater than 50, current pulsations are observed over time, resulting from hydrodynamic instabilities occurring in the vicinity of the membrane. The increase of initial concentration quotient on the membrane in configuration B causes an increase in the frequency of current pulsations and a change in their amplitudes over time. Furthermore, significant differences were observed between the types of the temporal changes in membrane currents in both configurations, and these differences persisted even 24 hours after turning off mechanical stirring of solutions. To analyze the hydrodynamic instabilities in configuration B of the membrane system, Fourier analysis was used both in the range of observed pulsations of the measured currents (from 50 to 250 min) and in the twenty-minute intervals with the intervals centered at 20, 100, 150, 200 and 290 min). As the analysis shows in all tested time ranges, the average signal power of currents in the frequency range from 0.05 to 1 min-1, depends non-linearly on the initial concentration quotient on the membrane, showing a maximum for the concentration quotient on the membrane equal to 2500.

Microbial-induced calcium carbonate precipitation (MICP) is a biological process inducing biomineralization of CaCO3. This can be used to form a solid, concrete-like material. To be able to use MICP successfully for producing solid materials, it is important to understand the formation process of the material in detail. It is well known, that crystallization surfaces can influence the precipitation process. Therefore, we present in this contribution a systematic study investigating the influence of calcite seeds on the MICP processes. We focus on the pH changes during the crystallization process measured with absorption spectroscopy and on the optical density (OD) signal to analyze the precipitation process. Furthermore, optical microscopy was used to visualize the precipitation processes in the sample and connect them to changes in pH and OD. We show that there is a significant difference in the pH evolution between samples with and without calcite seeds present and that the shape of the pH evolution and the changes in OD can give detailed information about the mineral precipitation and transformations. In the presented experiments we show that amorphous calcium carbonate (ACC) can also precipitate in the presence of initial calcite seeds, which can have consequences for consolidated MICP materials.

Impermeant molecules inside a cell would lead to an inward osmotic flow of water, causing swelling, were it not for the pumping of permeant sodium ions out of the cell as soon as they leak in. The energy barrier model for a semipermeable membrane, first introduced by Debye to provide a molecular-level explanation of the vant Hoff equation for osmotic pressure, can be used to advantage in this situation, since the pump can be conceptualized as increasing the energy barrier for the sodium ion. The Debye model has previously been extended to include osmosis induced by electrostatically neutral solutes. Discussion of the effect of ion pumping on water transport requires an understanding of osmosis in systems containing permeant ions, that is, Donnan systems. We have obtained an equation for Donnan osmosis across a Debye energy barrier that separates an aqueous solution of permeant sodium, potassium, and chloride ions from a solution containing these permeant ions and additionally an impermeant anion, the latter representing intra-cellular impermeant charged species. Donnan osmosis occurs even if osmolarities on the two sides of the membrane are equal. Numerical representation shows that the Donnan-Debye model provides a quantitative theoretical framework for the action of the sodium/potassium/ATPase ion pump as effectively rendering the extracellular sodium ions impermeant, thus balancing the impermeant molecules inside the cell. Another application of Donnan osmosis shows that ion charge effects, missing from lists of Starling forces, are nonetheless expected to be a major contributor to transport across capillary walls. SummaryOsmosis as driven by Starling forces is applicable only if the solute is electrostatically neutral. For ions, Donnan charge effects dominate. An equation for Donnan osmosis is presented and applied to ion pumps and to transport across capillary walls.

We here present a novel and sensitive methodology for determining the melting point (MP) of Bovine Serum Albumin (BSA) from micromolar to picomolar concentration levels under label free conditions. At 1 pM we could model the melting with a sharp gaussian. However, from the transient state observed during the melting process by using a simple exponential decay model we determined a time constant of 67 seconds. We applied this methodology by studying a 3.3 pM sample of a botulinum toxin A (BoNT-A) (stabilized with 2.8 nanomolar denatured Human Serum Albumin (HSA)). We were able to determine the Tm of BoNT-A in the presence of the approximately 1000-fold more concentrated HSA. Entry for the Table of Contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=153 SRC="FIGDIR/small/624557v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@12d93a7org.highwire.dtl.DTLVardef@138d01dorg.highwire.dtl.DTLVardef@e725acorg.highwire.dtl.DTLVardef@15a5f77_HPS_FORMAT_FIGEXP M_FIG C_FIG Protein label-free melting point (MP) determination at ultralow concentrations is a huge problem which concern to the biopharmaceutical industry. Here, we present a novel method to determine the MP of bovine serum albumin (BSA) from 1M to 1pM under label-free conditions. The benefits of this study match the purposes of stability studies in formulations, in which the protein active component is successful at very low concentrations, such as botulinum toxin A (BoNT-A). We used BOCOUTRE, a commercially available pharmaceutical product based on BoNT-A, and Human Serum Albumin (HSA) as a stabilizer. Our method can detect the MP of the stabilizer protein, even if its concentration is markedly different from that of the active component protein (1000-fold) in the case of BOCOUTRE.

Bioprinting of living cells can cause major shape deformations, which may severely affect cell survival and functionality. While the shear stresses occurring during cell flow through the printer nozzle have been quantified to some extent, the extensional stresses occurring as cells leave the nozzle into the free printing strand have been mostly ignored. Here we use Lattice-Boltzmann simulations together with a finite-element based cell model to study cell deformation at the nozzle exit. Our simulation results are in good qualitative agreement with experimental microscopy images. We show that for cells flowing in the center of the nozzle extensional stresses can be significant, while for cells flowing off-center their deformation is dominated by the shear flow inside the nozzle. From the results of these simulations, we develop two simple methods that only require the printing parameters (nozzle diameter, flow rate, bioink rheology) to (i) accurately predict the maximum cell stress occurring during the 3D bioprinting process and (ii) approximately predict the cell strains caused by the elongational flow at the nozzle exit.

The eastern oyster is a keystone species and ecosystem engineer. However, restoration efforts of wild oysters are often unsuccessful, in that they do not produce a robust population of oysters that are able to successfully reproduce. Furthermore, the dynamics of wild oyster fertilization is not yet well understood. Through conducting an experiment predicated on quantifying the influence of elementary aspects of fertilization kinetics--sperm concentration, gamete age, and success rate--we found that, as stochastic as the mating process may seem, there are correlations which fundamentally serve as the framework for assessing long-term sustainability, reef structure, and hydrodynamic parameters in relation to fertilization. We then focused on mathematically defining a procedure which simulated a concentration distribution of a single sperm and egg release where there existed conditions necessary for breeding to take place. We found a very significant impact of both gamete age and sperm concentration on fertilization rate (p < 0.0001). Our hydrodynamic model demonstrates that distance can also drastically influence broadcast spawning. This could be used as a foundation for developing a flexible model for wild oyster fertilization based on placement, initial seawater conditions, and size of the starting population. The results of this research could be implemented into a more user-friendly program which would accept multiple variables as inputs and output the probability of fertilization given arbitrary values. By accounting for environmental deviations, this generalization would increase its compatibility with the public and actualize the projects intended purpose: enhance the planning of oyster reef restoration projects.