Mechanical properties of clot made from human and bovine whole blood differ significantly.

Thromboembolism - that is, clot formation and the subsequent fragmentation of clot - is a leading cause of death worldwide. Clots' mechanical properties are critical determinants of both the embolization process and the pathophysiological consequences thereof. Thus, understanding and quantifying the mechanical properties of clots is important to our ability to treat and prevent thromboembolic disease. However, assessing these properties from in vivo clots is experimentally challenging. Therefore, we and others have turned to studying in vitro clot mimics instead. Unfortunately, there are significant discrepancies in the reported properties of these clot mimics, which have been hypothesized to arise from differences in experimental techniques and blood sources. The goal of our current work is therefore to compare the mechanical behavior of clots made from the two most common sources, human and bovine blood, using the same experimental techniques. To this end, we tested clots under pure shear with and without initial cracks, under cyclic loading, and under stress relaxation. Based on these data, we computed and compared stiffness, strength, work-to-rupture, fracture toughness, relaxation time constants, and prestrain. While clots from both sources behaved qualitatively similarly, they differed quantitatively in almost every metric. We also correlated each mechanical metric to measures of blood composition. Thereby, we traced this inter-species variability in clot mechanics back to significant differences in hematocrit, but not platelet count. Thus, our work suggests that the results of past studies that have used bovine blood to make in vitro mimics - without adjusting blood composition - should be interpreted carefully. Future studies about the mechanical properties of blood clots should focus on human blood alone.

Lipid droplets as substrates for protein phase separation.

Membrane-associated protein phase separation plays critical roles in cell biology, driving essential cellular phenomena from immune signaling to membrane traffic. Importantly, by reducing dimensionality from three to two dimensions, lipid bilayers can nucleate phase separation at far lower concentrations compared with those required for phase separation in solution. How might other intracellular lipid substrates, such as lipid droplets, contribute to nucleation of phase separation? Distinct from bilayer membranes, lipid droplets consist of a phospholipid monolayer surrounding a core of neutral lipids, and they are energy storage organelles that protect cells from lipotoxicity and oxidative stress. Here, we show that intrinsically disordered proteins can undergo phase separation on the surface of synthetic and cell-derived lipid droplets. Specifically, we find that the model disordered domains FUS LC and LAF-1 RGG separate into protein-rich and protein-depleted phases on the surfaces of lipid droplets. Owing to the hydrophobic nature of interactions between FUS LC proteins, increasing ionic strength drives an increase in its phase separation on droplet surfaces. The opposite is true for LAF-1 RGG, owing to the electrostatic nature of its interprotein interactions. In both cases, protein-rich phases on the surfaces of synthetic and cell-derived lipid droplets demonstrate molecular mobility indicative of a liquid-like state. Our results show that lipid droplets can nucleate protein condensates, suggesting that protein phase separation could be key in organizing biological processes involving lipid droplets.

A solid beta-sheet structure is formed at the surface of FUS droplets during aging.

Phase transitions are important to understand cell dynamics, and the maturation of liquid droplets is relevant to neurodegenerative disorders. We combined NMR and Raman spectroscopies with microscopy to follow, over a period of days to months, droplet maturation of the protein fused in sarcoma (FUS). Our study reveals that the surface of the droplets plays a critical role in this process, while RNA binding prevents it. The maturation kinetics are faster in an agarose-stabilized biphasic sample compared with a monophasic condensed sample, owing to the larger surface-to-volume ratio. In addition, Raman spectroscopy reports structural differences upon maturation between the inside and the surface of droplets, which is comprised of ß-sheet content, as revealed by solid-state NMR. In agreement with these observations, a solid crust-like shell is observed at the surface using microaspiration. Ultimately, matured droplets were converted into fibrils involving the prion-like domain as well as the first RGG motif.

Collagen organization and structure in FLBN5-/- mice using label-free microscopy: implications for pelvic organ prolapse.

Pelvic organ prolapse (POP) is a gynecological disorder described by the descent of superior pelvic organs into or out of the vagina as a consequence of disrupted muscles and tissue. A thorough understanding of the etiology of POP is limited by the availability of clinically relevant samples, restricting longitudinal POP studies on soft-tissue biomechanics and structure to POP-induced models such as fibulin-5 knockout (FBLN5-/-) mice. Despite being a principal constituent in the extracellular matrix, little is known about structural perturbations to collagen networks in the FBLN5-/- mouse cervix. We identify significantly different collagen network populations in normal and prolapsed cervical cross-sections using two label-free, nonlinear microscopy techniques. Collagen in the prolapsed mouse cervix tends to be more isotropic, and displays reduced alignment persistence via 2-D Fourier Transform analysis of images acquired using second harmonic generation microscopy. Furthermore, coherent Raman hyperspectral imaging revealed elevated disorder in the secondary structure of collagen in prolapsed tissues. Our results underscore the need for in situ multimodal monitoring of collagen organization to improve POP predictive capabilities.

Intrinsic Burst-Blinking Nanographenes for Super-Resolution Bioimaging.

Single-molecule localization microscopy (SMLM) is a powerful technique to achieve super-resolution imaging beyond the diffraction limit. Although various types of blinking fluorophores are currently considered for SMLM, intrinsic blinking fluorophores remain rare at the single-molecule level. Here, we report the synthesis of nanographene-based intrinsic burst-blinking fluorophores for highly versatile SMLM. We image amyloid fibrils in air and in various pH solutions without any additive and lysosome dynamics in live mammalian cells under physiological conditions. In addition, the single-molecule labeling of nascent proteins in primary sensory neurons was achieved with azide-functionalized nanographenes via click chemistry. SMLM imaging reveals higher local translation at axonal branching with unprecedented detail, while the size of translation foci remained similar throughout the entire network. These various results demonstrate the potential of nanographene-based fluorophores to drastically expand the applicability of super-resolution imaging.

Liquid-Liquid Phase Separation of the Intrinsically Disordered Domain of the Fused in Sarcoma Protein Results in Substantial Slowing of Hydration Dynamics.

Formation of liquid condensates plays a critical role in biology via localization of different components or via altered hydrodynamic transport, yet the hydrogen-bonding environment within condensates, pivotal for solvation, has remained elusive. We explore the hydrogen-bond dynamics within condensates formed by the low-complexity domain of the fused in sarcoma protein. Probing the hydrogen-bond dynamics sensed by condensate proteins using two-dimensional infrared spectroscopy of the protein amide I vibrations, we find that frequency-frequency correlations of the amide I vibration decay on a picosecond time scale. Interestingly, these dynamics are markedly slower for proteins in the condensate than in a homogeneous protein solution, indicative of different hydration dynamics. All-atom molecular dynamics simulations confirm that lifetimes of hydrogen-bonds between water and the protein are longer in the condensates than in the protein in solution. Altered hydrogen-bonding dynamics may contribute to unique solvation and reaction dynamics in such condensates.

Visualizing molecular deformation in fibrin networks under tensile loading via FLIM-FRET.

Mapping molecular deformation and forces in protein biomaterials is critical to understanding mechanochemistry. Here we use intramolecular Förster resonance energy transfer (FRET) of dual-labeled fibrin to distinguish molecular conformations of proteins in situ during mechanical loading. The FRET approach offers increased spatial resolution compared to our previous vibrational imaging. By using fluorescence lifetime microscopy (FLIM), we demonstrate that the combination of FRET and FLIM can probe the molecular changes in fibrin with high spatial (nanometer) and temporal (nanosecond) resolution. Our results map changes in fibrin monomer deformation during the macroscopic loading of the fibrin network, paving the way to directly visualizing the biomaterial mechanics and structure in cell-ECM scaffolds for the first time.

Fibril formation and ordering of disordered FUS LC driven by hydrophobic interactions.

Biomolecular condensates, protein-rich and dynamic membrane-less organelles, play critical roles in a range of subcellular processes, including membrane trafficking and transcriptional regulation. However, aberrant phase transitions of intrinsically disordered proteins in biomolecular condensates can lead to the formation of irreversible fibrils and aggregates that are linked to neurodegenerative diseases. Despite the implications, the interactions underlying such transitions remain obscure. Here we investigate the role of hydrophobic interactions by studying the low-complexity domain of the disordered 'fused in sarcoma' (FUS) protein at the air/water interface. Using surface-specific microscopic and spectroscopic techniques, we find that a hydrophobic interface drives fibril formation and molecular ordering of FUS, resulting in solid-like film formation. This phase transition occurs at 600-fold lower FUS concentration than required for the canonical FUS low-complexity liquid droplet formation in bulk. These observations highlight the importance of hydrophobic effects for protein phase separation and suggest that interfacial properties drive distinct protein phase-separated structures.

Erratum: Tissue harvest with a laser microbiopsy (Erratum).

[This corrects the article DOI: 10.1117/1.JBO.27.12.125001.].

Comparing lipid remodeling of brown adipose tissue, white adipose tissue, and liver after one-week high fat diet intervention with quantitative Raman microscopy.

Brown adipose tissue (BAT) consists of highly metabolically active adipocytes that catabolize nutrients to produce heat. Playing an active role in triacylglycerol (TAG) clearance, research has shown that dietary fatty acids can modulate the TAG chemistry deposition in BAT after weeks-long dietary intervention, similar to what has been shown in white adipose tissue (WAT). Our objective was to compare the influence of sustained, nonchronic dietary intervention (a 1-week interval) on WAT and interscapular BAT lipid metabolism and deposition in situ. We use quantitative, label-free chemical microscopy to show that 1 week of high fat diet (HFD) intervention results in dramatically larger lipid droplet (LD) growth in BAT (and liver) compared to LD growth in inguinal WAT (IWAT). Moreover, BAT showed lipid remodeling as increased unsaturated TAGs in LDs, resembling the dietary lipid composition, while WAT (and liver) did not show lipid remodeling on this time scale. Concurrently, expression of genes involved in lipid metabolism, particularly desaturases, was reduced in BAT and liver from HFD-fed mice after 1 week. Our data show that BAT lipid chemistry remodels exceptionally fast to dietary lipid intervention compared WAT, which further points towards a role in TAG clearance.

Tissue harvest with a laser microbiopsy.

Significance: Traditional pathology workflow suffers from limitations including biopsy invasiveness, small fraction of large tissue samples being analyzed, and complex and time-consuming processing. Aim: We address limitations of conventional pathology workflow through development of a laser microbiopsy device for minimally invasive harvest of sub-microliter tissue volumes. Laser microbiopsy combined with rapid diagnostic methods, such as virtual hematoxylin and eosin (H&E) imaging has potential to provide rapid minimally invasive tissue diagnosis. Approach: Laser microbiopsies were harvested using an annular shaped Ho:YAG laser beam focused onto the tissue surface. As the annulus was ablated, the tissue section in the center of the annulus was ejected and collected directly onto a glass slide for analysis. Cryogen spray cooling was used before and after laser harvest to limit thermal damage. Microbiopsies were collected from porcine skin and kidney. Harvested microbiopsies were imaged with confocal microscopy and digitally false colored to provide virtual H&E images. Results: Microbiopsies were successfully harvested from porcine skin and kidney. Computational and experimental results show the benefit of cryogen pre- and post-cooling to limit thermal damage. Virtual H&E images of microbiopsies retained observable cellular features including cell nuclei. Conclusions: Laser microbiopsy with virtual H&E imaging shows promise as a potential rapid and minimally invasive tool for biopsy and diagnosis.

A-type lamins involvement in transport and implications in cancer?

Nuclear lamins and transport are intrinsically linked, but their relationship is yet to be fully unraveled. A multitude of complex, coupled interactions between lamins and nucleoporins (Nups), which mediate active transport into and out of the nucleus, combined with well documented dysregulation of lamins in many cancers, suggests that lamins and nuclear transport may play a pivotal role in carcinogenesis and the preservation of cancer. Changes of function related to lamin/Nup activity can principally lead to DNA damage, further increasing the genetic diversity within a tumor, which could lead to the reduction the effectiveness of antineoplastic treatments. This review discusses and synthesizes different connections of lamins to nuclear transport and offers a number of outlook questions, the answers to which could reveal a new perspective on the connection of lamins to molecular transport of cancer therapeutics, in addition to their established role in nuclear mechanics.

Lipid-driven condensation and interfacial ordering of FUS.

Protein condensation into liquid-like structures is critical for cellular compartmentalization, RNA processing, and stress response. Research on protein condensation has primarily focused on membraneless organelles in the absence of lipids. However, the cellular cytoplasm is full of lipid interfaces, yet comparatively little is known about how lipids affect protein condensation. Here, we show that nonspecific interactions between lipids and the disordered fused in sarcoma low-complexity (FUS LC) domain strongly affect protein condensation. In the presence of anionic lipids, FUS LC formed lipid-protein clusters at concentrations more than 30-fold lower than required for pure FUS LC. Lipid-triggered FUS LC clusters showed less dynamic protein organization than canonical, lipid-free FUS LC condensates. Lastly, we found that phosphatidylserine membranes promoted FUS LC condensates having ß sheet structures, while phosphatidylglycerol membranes initiated unstructured condensates. Our results show that lipids strongly influence FUS LC condensation, suggesting that protein-lipid interactions modulate condensate formation in cells.

Structural control of fibrin bioactivity by mechanical deformation.

Fibrin is the fibrous protein network that comprises blood clots; it is uniquely capable of bearing very large tensile strains (up to 200%) due to multiscale force accommodation mechanisms. Fibrin is also a biochemical scaffold for numerous enzymes and blood factors. The biomechanics and biochemistry of fibrin have been independently studied. However, comparatively little is known about how fibrin biomechanics and biochemistry are coupled: how does fibrin deformation influence its biochemistry? In this study, we show that mechanically induced protein structural changes in fibrin affect fibrin biochemistry. We find that tensile deformation of fibrin leads to molecular structural transitions of α-helices to ß-sheets, which reduced binding of tissue plasminogen activator (tPA), an enzyme that initiates fibrin lysis. Moreover, binding of tPA and Thioflavin T, a commonly used ß-sheet marker, were mutually exclusive, further demonstrating the mechano-chemical control of fibrin biochemistry. Finally, we demonstrate that structural changes in fibrin suppressed the biological activity of platelets on mechanically strained fibrin due to reduced αIIbß3 integrin binding. Our work shows that mechanical strain regulates fibrin molecular structure and biological activity in an elegant mechano-chemical feedback loop, which possibly extends to other fibrous biopolymers.

Biomechanical Dependence of SARS-CoV-2 Infections.

Older people have been disproportionately vulnerable to the current SARS-CoV-2 pandemic, with an increased risk of severe complications and death compared to other age groups. A mix of underlying factors has been speculated to give rise to this differential infection outcome including changes in lung physiology, weakened immunity, and severe immune response. Our study focuses on the impact of biomechanical changes in lungs that occur as individuals age, that is, the stiffening of the lung parenchyma and increased matrix fiber density. We used hydrogels with an elastic modulus of 0.2 and 50 kPa and conventional tissue culture surfaces to investigate how infection rate changes with parenchymal tissue stiffness in lung epithelial cells challenged with SARS-CoV-2 Spike (S) protein pseudotyped lentiviruses. Further, we employed electrospun fiber matrices to isolate the effect of matrix density. Given the recent data highlighting the importance of alternative virulent strains, we included both the native strain identified in early 2020 and an early S protein variant (D614G) that was shown to increase the viral infectivity markedly. Our results show that cells on softer and sparser scaffolds, closer resembling younger lungs, exhibit higher infection rates by the WT and D614G variant. This suggests that natural changes in lung biomechanics do not increase the propensity for SARS-CoV-2 infection and that other factors, such as a weaker immune system, may contribute to increased disease burden in the elderly.

Role of Solvent Compatibility in the Phase Behavior of Binary Solutions of Weakly Associating Multivalent Polymers.

Condensate formation of biopolymer solutions, prominently those of various intrinsically disordered proteins (IDPs), is often driven by "sticky" interactions between associating residues, multivalently present along the polymer backbone. Using a ternary mean-field "stickers-and-spacers" model, we demonstrate that if sticker association is of the order of a few times the thermal energy, a delicate balance between specific binding and nonspecific polymer-solvent interactions gives rise to a particularly rich ternary phase behavior under physiological circumstances. For a generic system represented by a solution comprising multiassociative scaffold and client polymers, the difference in solvent compatibility between the polymers modulates the nature of isothermal liquid-liquid phase separation (LLPS) between associative and segregative. The calculations reveal regimes of dualistic phase behavior, where both types of LLPS occur within the same phase diagram, either associated with the presence of multiple miscibility gaps or a flip in the slope of the tie-lines belonging to a single coexistence region.

Reversible Kinetic Trapping of FUS Biomolecular Condensates.

Formation of membrane-less organelles by self-assembly of disordered proteins can be triggered by external stimuli such as pH, salt, or temperature. These organelles, called biomolecular condensates, have traditionally been classified as liquids, gels, or solids with limited subclasses. Here, the authors show that a thermal trigger can lead to formation of at least two distinct liquid condensed phases of the fused in sarcoma low complexity (FUS LC) domain. Forming FUS LC condensates directly at low temperature leads to formation of metastable, kinetically trapped condensates that show arrested coalescence, escape from which to untrapped condensates can be achieved via thermal annealing. Using experimental and computational approaches, the authors find that molecular structure of interfacial FUS LC in kinetically trapped condensates is distinct (more ß-sheet like) compared to untrapped FUS LC condensates. Moreover, molecular motion within kinetically trapped condensates is substantially slower compared to that in untrapped condensates thereby demonstrating two unique liquid FUS condensates. Controlling condensate thermodynamic state, stability, and structure with a simple thermal switch may contribute to pathological protein aggregate stability and provides a facile method to trigger condensate mixing for biotechnology applications.

Biophysical studies of phase separation integrating experimental and computational methods.

Biomolecular phase separation that contributes to the formation of membraneless organelles and biomolecular condensates has recently gained tremendous attention because of the importance of these assemblies in physiology, disease, and engineering applications. Understanding and directing biomolecular phase separation requires a multiscale view of the biophysical properties of these phases. Yet, many classic tools to characterize biomolecular properties do not apply in these condensed phases. Here, we discuss insights obtained from spectroscopic methods, in particular nuclear magnetic resonance and optical spectroscopy, in understanding the molecular and atomic interactions that underlie the formation of protein-rich condensates. We also review approaches closely coupling nuclear magnetic resonance data with computational methods especially coarse-grained and all-atom molecular simulations, which provide insight into molecular features of phase separation. Finally, we point to future methodolical developments, particularly visualizing biophysical properties of condensates in cells.

Effect of ambient temperature on respiratory tract cells exposed to SARS-CoV-2 viral mimicking nanospheres-An experimental study.

The novel coronavirus caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has reached more than 160 countries and has been declared a pandemic. SARS-CoV-2 infects host cells by binding to the angiotensin-converting enzyme 2 (ACE-2) surface receptor via the spike (S) receptor-binding protein (RBD) on the virus envelope. Global data on a similar infectious disease spread by SARS-CoV-1 in 2002 indicated improved stability of the virus at lower temperatures facilitating its high transmission in the community during colder months (December-February). Seasonal viral transmissions are strongly modulated by temperatures, which can impact viral trafficking into host cells; however, an experimental study of temperature-dependent activity of SARS-CoV-2 is still lacking. We mimicked SARS-CoV-2 with polymer beads coated with the SARS-CoV-2 S protein to study the effect of seasonal temperatures on the binding of virus-mimicking nanospheres to lung epithelia. The presence of the S protein RBD on nanosphere surfaces led to binding by Calu-3 airway epithelial cells via the ACE-2 receptor. Calu-3 and control fibroblast cells with S-RBD-coated nanospheres were incubated at 33 and 37 °C to mimic temperature fluctuations in the host respiratory tract, and we found no temperature dependence in contrast to nonspecific binding of bovine serum ablumin-coated nanospheres. Moreover, the ambient temperature changes from 4 to 40 °C had no effect on S-RBD-ACE-2 ligand-receptor binding and minimal effect on the S-RBD protein structure (up to 40 °C), though protein denaturing occurred at 51 °C. Our results suggest that ambient temperatures from 4 to 40 °C have little effect on the SARS-CoV-2-ACE-2 interaction in agreement with the infection data currently reported.

Molecular Control of Interfacial Fibronectin Structure on Graphene Oxide Steers Cell Fate.

The use of graphene-based materials (GBMs) for tissue-engineering applications has been growing exponentially because of the seemingly endless multifunctional and tunable physicochemical properties of graphene that can be exploited to influence cellular behavior. Despite many demonstrations wherein cell physiology has been modulated on different GBMs, a clear mechanism connecting the different physicochemical properties of GBMs to cell fate has remained elusive. In this work, we demonstrate how different GBMs can be used to bias cell fate in a multiscale study-starting from serum protein (fibronectin) adsorption and its molecular scale morphology, structure, and bioactivity and ending with stem cell response. Using heat to chemically reduce graphene oxide without changing physical properties, we show that graphene chemistry controls surface-adsorbed molecular conformation and morphology, epitope presentation, and stem cell attachment. Moreover, this subtle change in the protein structure was found to drive increased bone differentiation of stem cells, suggesting that the physicochemical properties of graphene biases cell fate by directly influencing the adsorbed protein structure and subsequent biochemical activity.