Nucleation dynamics of a model biomolecular liquid.

Liquid-liquid phase separation in biology has recently been shown to play a major role in the spatial control of biomolecular components within the cell. However, as they are phase transitions, these processes also display nontrivial dynamics. A model phase-separating system of DNA nanostars provides unique access to nucleation physics in a biomolecular context, as phase separation is driven near room temperature by highly thermo-responsive DNA hybridization and at modest DNA concentrations. By measuring the delay time for phase-separated droplets to appear, we demonstrate that the dynamics of DNA nanostar phase separation reflect that of a metastable binary mixture of patchy particles. For sufficiently deep temperature quenches, droplets undergo spinodal decomposition and grow spontaneously, driven by Brownian motion and coalescence of phase-separated droplets, as confirmed by comparing experimental measurements to particle-based simulations. Near the coexistence boundary, droplet growth slows substantially, indicative of a nucleation process. The temperature dependence of droplet appearance times can be predicted by a classical nucleation picture with mean field exponents and demonstrates that a theory previously used to predict equilibrium phase diagrams can also distinguish spinodal and nucleation dynamical regimes. These dynamical principles are relevant to behaviors associated with liquid-liquid phase separating systems, such as their spatial patterning, reaction coupling, and biological function.

Nucleic acid liquids.

The confluence of recent discoveries of the roles of biomolecular liquids in living systems and modern abilities to precisely synthesize and modify nucleic acids (NAs) has led to a surge of interest in liquid phases of NAs. These phases can be formed primarily from NAs, as driven by base-pairing interactions, or from the electrostatic combination (coacervation) of negatively charged NAs and positively charged molecules. Generally, the use of sequence-engineered NAs provides the means to tune microsopic particle properties, and thus imbue specific, customizable behaviors into the resulting liquids. In this way, researchers have used NA liquids to tackle fundamental problems in the physics of finite valence soft materials, and to create liquids with novel structured and/or multi-functional properties. Here, we review this growing field, discussing the theoretical background of NA liquid phase separation, quantitative understanding of liquid material properties, and the broad and growing array of functional demonstrations in these materials. We close with a few comments discussing remaining open questions and challenges in the field.

Magnetic tweezers characterization of the entropic elasticity of intrinsically disordered proteins and peptoids.

Understanding the conformational behavior of biopolymers is essential to unlocking knowledge of their biophysical mechanisms and functional roles. Single-molecule force spectroscopy can provide a unique perspective on this by exploiting entropic elasticity to uncover key biopolymer structural parameters. A particularly powerful approach involves the use of magnetic tweezers, which can easily generate lower stretching forces (0.1-20 pN). For forces at the low end of this range, the elastic response of biopolymers is sensitive to excluded volume effects, and they can be described by Pincus blob elasticity model that allow robust extraction of the Flory polymer scaling exponent. Here, we detail protocols for the use of magnetic tweezers for force-extension measurements of intrinsically disordered proteins and peptoids. We also discuss procedures for fitting low-force elastic curves to the predictions of polymer physics models to extract key conformational parameters.

From isolated polyelectrolytes to star-like assemblies: the role of sequence heterogeneity on the statistical structure of the intrinsically disordered neurofilament-low tail domain.

Intrinsically disordered proteins (IDPs) are a subset of proteins that lack stable secondary structure. Given their polymeric nature, previous mean-field approximations have been used to describe the statistical structure of IDPs. However, the amino-acid sequence heterogeneity and complex intermolecular interaction network have significantly impeded the ability to get proper approximations. One such case is the intrinsically disordered tail domain of neurofilament low (NFLt), which comprises a 50 residue-long uncharged domain followed by a 96 residue-long negatively charged domain. Here, we measure two NFLt variants to identify the impact of the NFLt two main subdomains on its complex interactions and statistical structure. Using synchrotron small-angle x-ray scattering, we find that the uncharged domain of the NFLt induces attractive interactions that cause it to self-assemble into star-like polymer brushes. On the other hand, when the uncharged domain is truncated, the remaining charged N-terminal domains remain isolated in solution with typical polyelectrolyte characteristics. We further discuss how competing long- and short-ranged interactions within the polymer brushes dominate their ensemble structure and, in turn, their implications on previously observed phenomena in NFL native and diseased states.

Controlling the size and adhesion of DNA droplets using surface- enriched DNA molecules.

Liquid droplets of biomolecules serve as organizers of the cellular interior and are of interest in biosensing and biomaterials applications. Here, we investigate means to tune the interfacial properties of a model biomolecular liquid consisting of multi-armed DNA 'nanostar' particles. We find that long DNA molecules that have binding affinity for the nanostars are preferentially enriched on the interface of nanostar droplets, thus acting as surfactants. Fluorescent measurements indicate that, in certain conditions, the interfacial density of the surfactant is around 20 per square micron, indicative of a sparse brush-like structure of the long, polymeric DNA. Increasing surfactant concentration leads to decreased droplet size, down to the sub-micron scale, consistent with droplet coalesence being impeded by the disjoining pressure created by the brush-like surfactant layer. Added DNA surfactant also keeps droplets from adhering to both hydrophobic and hydrophilic solid surfaces, apparently due to this same disjoining effect of the surfactant layer. We thus demonstrate control of the size and adhesive properties of droplets of a biomolecular liquid, with implications for basic biophysical understanding of such droplets, as well as for their applied use.

The Effect of Visual Impairment and Its Severity on Vision-Related and Health-Related Quality of Life in Jordan: A Comparative Cross-Sectional Study.

Purpose: To assess the effect of visual impairment (VI), its severity, and ocular diseases on vision-related and health-related quality of life (QoL) in Jordan. Patients and Methods: A comparative, cross-sectional, hospital-based study was conducted among a group of 278 patients with VI aged ≥ 18 years, and age and sex-matched control group of 278 individuals with no VI. An interviewer administered the National Eye Institute Visual Function Questionnaire (NEI VFQ-25) and the Medical Outcomes Study 12-Item Short Form Health Survey (SF-12) to all participants. Results: All the mean VFQ-25 subscales scores, physical component scale (PCS) and the mental component scale (MCS) of the SF-12 were significantly lower in patients with VI compared to controls with no VI. The VFQ-25 subscales (except general health and ocular pain), PCS, and MCS scores significantly decreased with more severity of VI. In the adjusted multivariate analysis, lower level of education (p=0.013), male sex (p=0.016), and the presence of cerebrovascular disease (p=0.019) were significantly associated with lower VFQ-25 composite scores in visually impaired patients compared to controls. Ocular disease duration of >5 years and progressive VI were significantly associated with lower VFQ-25 composite scores (p= 0.026 and p<0.001) respectively, in patients with VI. Glaucoma had a significantly larger reduction in mean scores of all the VFQ-25 subscales, and the PCS of the SF-12 compared to all other ocular diseases. Conclusion: Both VI and increasing severity of impairment were associated with reduced vision-related and health-related quality of life in adult Jordanians. Glaucoma patients and less educated people were particularly affected. Routine assessment of QoL in visually impaired patients and improving referral protocols to vision rehabilitation services is recommended to improve the QoL in those patients.

Pincus blob elasticity in an intrinsically disordered protein.

Understanding the dynamic structure of intrinsically disordered proteins (IDPs) is important to deciphering their biological functions. Here, we exploit precision entropic elasticity measurements to infer the conformational behavior of a model IDP construct formed from the disordered tail of the neurofilament low molecular weight protein. The IDP construct notably displays a low-force power-law elastic regime, consistent with the Pincus blob model, which allows direct extraction of the Flory exponent, [Formula: see text], from the force-extension relationship. We find [Formula: see text] increases with added denaturant, transitioning from a nearly ideal chain to a swollen chain in a manner quantitatively consistent with measurements of IDP dimensions from other experimental techniques. We suggest that measurements of entropic elasticity could be broadly useful in the study of IDP structure.

Controlling liquid-liquid phase behaviour with an active fluid.

Demixing binary liquids is a ubiquitous transition explained using a well-established thermodynamic formalism that requires the equality of intensive thermodynamics parameters across phase boundaries. Demixing transitions also occur when binary fluid mixtures are driven away from equilibrium, but predicting and designing such out-of-equilibrium transitions remains a challenge. Here we study the liquid-liquid phase separation of attractive DNA nanostars driven away from equilibrium using a microtubule-based active fluid. We find that activity lowers the critical temperature and narrows the range of coexistence concentrations, but only in the presence of mechanical bonds between the liquid droplets and reconfiguring active fluid. Similar behaviours are observed in numerical simulations, suggesting that the activity suppression of the critical point is a generic feature of active liquid-liquid phase separation. Our work describes a versatile platform for building soft active materials with feedback control and providing an insight into self-organization in cell biology.

Pattern of Uveitis in a Tertiary Hospital in North Jordan and the Impact of Behcet's Disease.

Aim: The purpose of this study is to assess the prevalence of autoimmune-mediated uveitis in relation to other diseases and to describe the clinical patterns of uveitis in a single tertiary hospital in north Jordan. Methods: A cross-sectional retrospective review was performed. We included all patients diagnosed with uveitis in King Abdullah University Hospital (KAUH) ophthalmology clinic and/or patients referred to KAUH rheumatology clinics for evaluation of suspected autoimmune mediated uveitis or for difficult to treat uveitis. This included patients from January 2015 to January 2019. Data collected about patients' age, sex, anatomical location of the disease, etiology, treatment, complications, and outcomes on vision loss were analyzed. Results: A total of 221 patients were included in our study. The mean (±SD) age was 36 (±16) years. A total of 111 patients were female and 110 were male with a ratio of 1 : 1. Noninfectious uveitis was found to be more common than infectious uveitis (95% vs. 5% respectively). Autoimmune-mediated uveitis accounted for 40% of the total cases. The most common causes of autoimmune-mediated uveitis included Behcet's disease (n = 41, 19%) and seronegative spondyloarthropathy (n = 29, 13%). The majority of patients (n = 207, 94%) were treated with ophthalmic eye drops, cDMARDs (n = 101, 46%), biologics (n = 33, 15%), and colchicine (n = 30, 14%). Conclusion: Autoimmune-mediated uveitis, most commonly associated with Behcet's disease and seronegative spondyloarthropathy, represents a high percentage of uveitis in our area. This implies the need for a high index of suspicion at the time of evaluation.

Intramolecular structural heterogeneity altered by long-range contacts in an intrinsically disordered protein.

Short-range interactions and long-range contacts drive the 3D folding of structured proteins. The proteins' structure has a direct impact on their biological function. However, nearly 40% of the eukaryotes proteome is composed of intrinsically disordered proteins (IDPs) and protein regions that fluctuate between ensembles of numerous conformations. Therefore, to understand their biological function, it is critical to depict how the structural ensemble statistics correlate to the IDPs' amino acid sequence. Here, using small-angle X-ray scattering and time-resolved Förster resonance energy transfer (trFRET), we study the intramolecular structural heterogeneity of the neurofilament low intrinsically disordered tail domain (NFLt). Using theoretical results of polymer physics, we find that the Flory scaling exponent of NFLt subsegments correlates linearly with their net charge, ranging from statistics of ideal to self-avoiding chains. Surprisingly, measuring the same segments in the context of the whole NFLt protein, we find that regardless of the peptide sequence, the segments' structural statistics are more expanded than when measured independently. Our findings show that while polymer physics can, to some level, relate the IDP's sequence to its ensemble conformations, long-range contacts between distant amino acids play a crucial role in determining intramolecular structures. This emphasizes the necessity of advanced polymer theories to fully describe IDPs ensembles with the hope that it will allow us to model their biological function.

Vacuole dynamics and popping-based motility in liquid droplets of DNA.

Liquid droplets of biomolecules play key roles in organizing cellular behavior, and are also technologically relevant, yet physical studies of dynamic processes of such droplets have generally been lacking. Here, we investigate and quantify the dynamics of formation of dilute internal inclusions, i.e., vacuoles, within a model system consisting of liquid droplets of DNA 'nanostar' particles. When acted upon by DNA-cleaving restriction enzymes, these DNA droplets exhibit cycles of appearance, growth, and bursting of internal vacuoles. Analysis of vacuole growth shows their radius increases linearly in time. Further, vacuoles pop upon reaching the droplet interface, leading to droplet motion driven by the osmotic pressure of restriction fragments captured in the vacuole. We develop a model that accounts for the linear nature of vacuole growth, and the pressures associated with motility, by describing the dynamics of diffusing restriction fragments. The results illustrate the complex non-equilibrium dynamics possible in biomolecular condensates.

Emulsion imaging of a DNA nanostar condensate phase diagram reveals valence and electrostatic effects.

Liquid-liquid phase separation (LLPS) in macromolecular solutions (e.g., coacervation) is relevant both to technology and to the process of mesoscale structure formation in cells. The LLPS process is characterized by a phase diagram, i.e., binodal lines in the temperature/concentration plane, which must be quantified to predict the system's behavior. Experimentally, this can be difficult due to complications in handling the dense macromolecular phase. Here, we develop a method for accurately quantifying the phase diagram without direct handling: We confine the sample within micron-scale, water-in-oil emulsion droplets and then use precision fluorescent imaging to measure the volume fraction of the condensate within the droplet. We find that this volume fraction grows linearly with macromolecule concentration; thus, by applying the lever rule, we can directly extract the dense and dilute binodal concentrations. We use this approach to study a model LLPS system of self-assembled, fixed-valence DNA particles termed nanostars (NSs). We find that temperature/concentration phase diagrams of NSs display, with certain exceptions, a larger co-existence regime upon increasing salt or valence, in line with expectations. Aspects of the measured phase behavior validate recent predictions that account for the role of valence in modulating the connectivity of the condensed phase. Generally, our results on NS phase diagrams give fundamental insight into limited-valence phase separation, while the method we have developed will likely be useful in the study of other LLPS systems.

Tweezepy: A Python package for calibrating forces in single-molecule video-tracking experiments.

Single-molecule force spectroscopy (SMFS) instruments (e.g., magnetic and optical tweezers) often use video tracking to measure the three-dimensional position of micron-scale beads under an applied force. The force in these experiments is calibrated by comparing the bead trajectory to a thermal motion-based model with the drag coefficient, γ, and trap spring constant, κ, as parameters. Estimating accurate parameters is complicated by systematic biases from spectral distortions, the camera exposure time, parasitic noise, and least-squares fitting methods. However, while robust calibration methods exist that correct for these biases, they are not always used because they can be complex to implement computationally. To address this barrier, we present Tweezepy: a Python package for calibrating forces in SMFS video-tracking experiments. Tweezepy uses maximum likelihood estimation (MLE) to estimate parameters and their uncertainties from a single bead trajectory via the power spectral density (PSD) and Allan variance (AV). It is well-documented, fast, easy to use, and accounts for most common sources of biases in SMFS video-tracking experiments. Here, we provide a comprehensive overview of Tweezepy's calibration scheme, including a review of the theory underlying thermal motion-based parameter estimates, a discussion of the PSD, AV, and MLE, and an explanation of their implementation.

Flexible, charged biopolymers in monovalent and mixed-valence salt: Regimes of anomalous electrostatic stiffening and of salt insensitivity.

The conformations of biological polyelectrolytes (PEs), such as polysaccharides, proteins, and nucleic acids, affect how they behave and interact with other biomolecules. Relative to neutral polymers, PEs in solution are more locally rigid due to intrachain electrostatic repulsion, the magnitude of which depends on the concentration of added salt. This is typically quantified using the Odijk-Skolnick-Fixman (OSF) electrostatic-stiffening model, in which salt-dependent Debye-Hückel (DH) screening modulates intrachain repulsion. However, the applicability of this approach to flexible PEs has long been questioned. To investigate this, we use high-precision single-molecule elasticity measurements to infer the scaling with salt of the local stiffness of three flexible biopolymers (hyaluronic acid, single-stranded RNA, and single-stranded DNA) in both monovalent and mixed-valence salt solutions. In monovalent salt, we collapse the data across all three polymers by accounting for charge spacing, and find a common power-law scaling of the electrostatic persistence length with ionic strength with an exponent of 0.66±0.02. This result rules out simple OSF pictures of electrostatic stiffening. It is roughly compatible with a modified OSF picture developed by Netz and Orland; alternatively, we posit the exponent can be explained if the relevant electrostatic screening length is the interion spacing rather than the DH length. In mixed salt solutions, we find a regime where adding monovalent salt, in the presence of multivalent salt, does not affect PE stiffness. Using coarse-grained simulations, and a three-state model of condensed, chain-proximate, and bulk ions, we attribute this regime to a "jacket" of ions surrounding the PE that regulates the chain's effective charge density as ionic strength varies. The size of this jacket in simulations is again consistent with a screening length controlled by interion spacing rather than the DH length. Taken together, our results describe a unified picture of the electrostatic stiffness of polyelectrolytes in the mixed-valence salt conditions of direct relevance to cellular and intercellular biological systems.

Single-Molecule Stretching Shows Glycosylation Sets Tension in the Hyaluronan-Aggrecan Bottlebrush.

Large bottlebrush complexes formed from the polysaccharide hyaluronan (HA) and the proteoglycan aggrecan contribute to cartilage compression resistance and are necessary for healthy joint function. A variety of mechanical forces act on these complexes in the cartilage extracellular matrix, motivating the need for a quantitative description that links their structure and mechanical response. Studies using electron microscopy have imaged the HA-aggrecan brush but require adsorption to a surface, dramatically altering the complex from its native conformation. We use magnetic tweezers force spectroscopy to measure changes in extension and mechanical response of an HA chain as aggrecan monomers bind and form a bottlebrush. This technique directly measures changes undergone by a single complex with time and under varying solution conditions. Upon addition of aggrecan, we find a large swelling effect manifests when the HA chain is under very low external tension (i.e., stretching forces less than ∼1 pN). We use models of force-extension behavior to show that repulsion between the aggrecans induces an internal tension in the HA chain. Through reference to theories of bottlebrush polymer behavior, we demonstrate that the experimental values of internal tension are consistent with a polydisperse aggrecan population, likely caused by varying degrees of glycosylation. By enzymatically deglycosylating the aggrecan, we show that aggrecan glycosylation is the structural feature that causes HA stiffening. We then construct a simple stochastic binding model to show that variable glycosylation leads to a wide distribution of internal tensions in HA, causing variations in the mechanics at much longer length scales. Our results provide a mechanistic picture of how flexibility and size of HA and aggrecan lead to the brush architecture and mechanical properties of this important component of cartilage.

Clinical Experience in the Administration of Intravitreal Injection Therapy at a Tertiary University Hospital in Jordan During the COVID-19 Lockdown.

PURPOSE: To describe the clinical experience with the delivery of intravitreal injection therapy to patients with various indications at a tertiary university hospital during the COVID-19 lockdown in Jordan. METHODS: This is a retrospective observational study of patients who received intravitreal injections between April 12th and May 9th, 2020, a period during the national COVID-19 lockdown (March 16th to June 6th, 2020). Special medical and logistic arrangements, priority and visual risk assessment and strict infection control precautions were implemented. Demographics, diagnosis, intravitreal injection history, medical history, ophthalmic examinations and optical coherence tomography data were collected and analyzed. RESULTS: Intravitreal injections were successfully administered to 132 patients with diabetic retinopathy, age-related macular degeneration and retinal vein occlusion. All logistic and transmission control measures were followed by the medical staff and patients with no incidents. No new exposures or COVID-19 positive cases were traced to our location or time of therapy. No complications related to the injections were recorded. The mean period of delay due to the lockdown from the original scheduled appointment was six weeks. Mean visual acuity significantly decreased from 20/55 before the lockdown to 20/70 after the lockdown, and mean central macular thickness significantly increased from 329 to 370 µ. CONCLUSION: The administration of intravitreal injection therapy during the COVID-19 lockdown under special safety precautions was feasible and successful. Resumption of the essential therapies and medical services during periods of pandemic restrictions while adhering to strict transmission control measures is encouraged.

Sequence-Controlled Adhesion and Microemulsification in a Two-Phase System of DNA Liquid Droplets.

Membrane-less organelles, the liquid droplets formed via liquid-liquid phase separation (LLPS) of biomolecules in cells, act to organize intracellular components into multiple compartments. As a model for this process, and as a potential vehicle for in vitro exploitation of its properties, we explore here a synthetic multiphase LLPS system consisting of a mixture of self-assembled DNA particles. The particles, termed "DNA nanostars" (NSs), consist of four double-stranded DNA arms that each terminate in a single-stranded overhang. NSs condense into droplets due to overhang hybridization. Using two types of NSs with orthogonal overhangs enables the creation of two types of immiscible DNA droplets. Adhesion between the droplets can be tuned by the addition of "cross-linker NSs" that have two overhang sequences of each type. We find that increasing the amount of the cross-linker NSs decreases the droplet/droplet surface tension until a microemulsion transition occurs. Controlled droplet adhesion can also be achieved, without cross-linkers, using overhangs that can weakly hybridize. Finally, we show that solutes can be specifically targeted to the DNA phases by labeling them with appropriate sticky-ends. Overall, our findings demonstrate the ability to create a multiphase LLPS system, and to control its mesoscale configuration, via sequence design of the component molecules.

Characteristics, fates and complications of long-term silicone oil tamponade after pars plana vitrectomy.

BACKGROUND: Silicone oil tamponade has become a mainstay in treatment of advanced retinal detachment due to multiple etiologies. The aim of this study is to assess the characteristics, fates and complications of long-term silicone oil tamponade after par plana vitrectomy (PPV), and to compare the outcomes of different silicone oil viscosities used in a cohort of consecutive patients. METHODS: This is a retrospective comparative case series of eyes undergoing vitrectomy with silicone oil tamponade for retinal detachment by a single surgeon using different oil viscosities that were followed for one year with the silicone oil in situ. Visual acuity (VA), intraocular pressure (IOP) and complications associated with the follow up period were analyzed and compared. RESULTS: Eighty-five eyes of 85 patients were included in this study. Forty three patients had 1000 centistoke (cs) oil injected and 42 patients had 5000cs oil utilized. Demographic, cause of retinal detachment and preoperative ocular characteristics were similar in both groups. Long term complications in both groups included ocular hypertension (67.4% vs 66.7%), keratopathy due to silicone oil emulsification and migration to the anterior chamber (7.0% vs 11.9%), recurrent retinal detachment (4.7% vs 19%) and epiretinal membrane formation (7% vs 19%). In the 1000cs oil group, there was no significant difference between baseline IOP and any subsequent visit. There was a significant difference between baseline IOP and visits at day 1 (with IOP difference of 2.61 mmHg (±6.5)) (p = 0.028), 1 month (with IOP difference of 3.52 mmHg (±8.1)) (p = 0.026), 4 months (with IOP difference of 6.38 mmHg (±9.3)) (p = 0.005), and one year (with IOP difference of 4.24 mmHg (±11.1)) (p = 0.048), all higher in the post-operative period in the 5000cs oil group. Excluding the first post-operative day, no significant difference was found for VA between baseline visits and subsequent visits for either silicone oil groups. CONCLUSION: In this cohort of patients with long-term silicone oil tamponade after PPV to treat retinal detachment, IOP increased significantly in patients who received 5000cs silicone oil. There was no significant difference between other complication rates in patients receiving either oil viscosities. Long term silicone oil tamponade remains a viable option in certain cases, and a vigilant follow up for complications is necessary to limit any adverse effects and improve visual and surgical outcomes.

Glassy Dynamics and Memory Effects in an Intrinsically Disordered Protein Construct.

Glassy, nonexponential relaxations in globular proteins are typically attributed to conformational behaviors that are missing from intrinsically disordered proteins. Yet, we show that single molecules of a disordered-protein construct display two signatures of glassy dynamics, logarithmic relaxations and a Kovacs memory effect, in response to changes in applied tension. We attribute this to the presence of multiple independent local structures in the chain, which we corroborate with a model that correctly predicts the force dependence of the relaxation. The mechanism established here likely applies to other disordered proteins.