Publications

Drosophila oogenesis is a powerful and tractable model for studies of cell and developmental biology due to the multitude of well-characterized events in both germline and somatic cells, the ease of genetic manipulation in fruit flies, and the large number of egg chambers produced by each fly. Recent improvements in live imaging and ex vivo culturing protocols have enabled researchers to conduct more detailed, longer-term studies of egg chamber development, enabling insights into fundamental biological processes. Here, we present a protocol for dissection, culturing, and imaging of late-stage egg chambers to study intercellular and directional cytoplasmic flow during “nurse cell dumping.” This critical developmental process towards the latter stages of oogenesis (stages 10b/11) results in rapid growth of the oocyte and shrinkage of the nurse cells and is accompanied by dynamic changes in cell shape.

Fluorescent protein biomaterials have important applications such as bioimaging in pharmacological studies. Self-assembly of proteins, especially into fibrils, is known to produce fluorescence in the blue band. Capable of self-assembly into nanofibers, we have shown we can modulate its aggregation into mesofibers by encapsulation of a small hydrophobic molecule. Conversely, azobenzenes are hydrophobic small molecules that are virtually non-fluorescent in solution due to their highly efficient photoisomerization. However, they demonstrate fluorogenic properties upon confinement in nanoscale assemblies by reducing the non-radiative photoisomerization. Here, we report the fluorescence of a hybrid protein-small molecule system in which azobenzene is confined in our protein assembly leading to fiber thickening and increased fluorescence. We show our engineered protein Q encapsulates AzoCholine, bearing a photoswitchable azobenzene moiety, in the hydrophobic pore to produce fluorescent mesofibers. This study further investigates the photocontrol of protein conformation as well as fluorescence of an azobenze-containing biomaterial.

The fluid–structure interactions between flexible fibres and viscous flows play an essential role in various biological phenomena, medical problems and industrial processes. Of particular interest is the case of particles transported freely in time-dependent flows. This work elucidates the dynamics and morphologies of actin filaments under oscillatory shear flows by combining microfluidic experiments, numerical simulations and theoretical modelling. Our work reveals that, in contrast to steady shear flows, in which small orientational fluctuations from a flow-aligned state initiate tumbling and deformations, the periodic flow reversal allows the filament to explore many different configurations at the beginning of each cycle. Investigation of filament motion during half time periods of oscillation highlights the critical role of the initial filament orientation on the emergent dynamics. This strong coupling between orientation and deformation results in new deformation regimes and novel higher-order buckling modes absent in steady shear flows. The primary outcome of our analysis is the possibility of suppression of buckling instabilities for certain combinations of the oscillation frequency and initial filament orientation, even in very strong flows. We explain this unusual behaviour through a weakly nonlinear Landau theory of buckling, in which we treat the filaments as inextensible Brownian Euler–Bernoulli rods whose hydrodynamics is described by local slender-body theory.

The nucleation of actin filament branches by the Arp2/3 complex involves activation through nucleation promotion factors (NPFs), recruitment of actin monomers, and binding of the complex to the side of actin filaments. Because of the large system size and processes that involve flexible regions and diffuse components, simulations of branch formation using all-atom molecular dynamics are challenging. We applied a coarse-grained model that retains amino-acid level information and allows molecular dynamics simulations in implicit solvent, with globular domains represented as rigid bodies and flexible regions allowed to fluctuate. We used recent electron microscopy structures of the inactive Arp2/3 complex bound to NPF domains and to mother actin filament for the activated Arp2/3 complex. We studied interactions of Arp2/3 complex with the activating VCA domain of the NPF Wiskott-Aldrich syndrome protein, actin monomers, and actin filament. We found stable configurations with one or two actin monomers bound along the branch filament direction and with CA domain of VCA associated to the strong and weak binding sites of the Arp2/3 complex, supporting prior structural studies and validating our approach. We reproduced delivery of actin monomers and CA to the Arp2/3 complex under different conditions, providing insight into mechanisms proposed in previous studies. Simulations of active Arp2/3 complex bound to a mother actin filament indicate the contribution of each subunit to the binding. Addition of the C-terminal tail of Arp2/3 complex subunit ArpC2, which is missing in the cryo-EM structure, increased binding affinity, indicating a possible stabilizing role of this tail.

Recent experiments have shown that the nematode {\it T. aceti} can assemble into collectively undulating groups at the edge of fluid drops. This coordinated state consists of metachronal waves and drives fluid circulation inside the drop. We find that the circulation velocity is about 2 mm/s and nearly half the speed of the metachronal wave. We develop a quasi two-dimensional hydrodynamics model using the Stokes flow approximation. The periodic motion of the nematodes constitute our moving boundary condition that drives the flow. Our model suggests that large amplitude excursions of the nematodes tails produce the fluid circulation. We discuss the constraints on containers that would enhance fluid motion, which could be used in the future design of on demand flow generating systems.

The rupture potential of an atherosclerotic plaque is dependent on both the plaque's composition and the shear stresses it encounters from blood flow. Because plaques move and deform throughout the cardiac cycle, resulting in changes to plaque position and shape as well as to the encountered shear stresses, concurrent imaging of both risk factors over time is required to accurately predict plaque vulnerability. To evaluate the potential to achieve as much, multi-angle plane wave (PW) ARFI and least-squares vector Doppler data were acquired in a calibrated flow phantom with channels of 4–8 mm diameters and flow rates of 100–600 ml/min. The wall shear stress (WSS) was measured to within 15% of the ground-truth analytical solutions. The same methods were then implemented in an excised human cadaveric carotid with a x% stenotic plaque. ARFI VoA detected plaque regions of calcium and intraplaque hemorrhage that were validated by spatially-matched histology. Concurrent vector Doppler yielded a peak WSS of 5.2 Pa on the plaque shoulder, which was consistent with the 6.4 Pa WSS predicted by an immersed interface fluid-solid interation (FSI) model developed using the specific geometry of the examined cadaveric carotid. Overall our results demonstrate the feasibility of concurrent imaging of carotid plaque composition by ARFI VoA, vector flow, and WSS to better assess stroke risk.

Marine recruits training at Parris Island experienced an unexpectedly high rate of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, despite preventive measures including a supervised, 2-week, pre-entry quarantine. We characterize SARS-CoV-2 transmission in this cohort.

Astrocytes have been shown to play a central role in Alzheimer’s Disease (AD). However, the genes and biological pathways underlying disease manifestation are unknown, and it is unclear whether regional molecular differences among astrocytes contribute to regional specificity of disease. Here, we began to address these challenges with integrated experimental and computational approaches. We constructed a human astrocyte-specific functional gene network using Bayesian integration of a large compendium of human functional genomics data, as well as regional astrocyte gene expression profiles we generated in the mouse. This network identifies likely region-specific astrocyte pathways that operate in healthy brains. We leveraged our findings to compile genome-wide astrocyte-associated disease-gene predictions, employing a novel network-guided differential expression analysis (NetDIFF). We also used this data to predict a list of astrocyte-expressed genes mediating region-specific human disease, using a network-guided shortest path method (NetPATH). Both the network and our results are publicly available using an interactive web interface at http://astrocyte.princeton.edu. Our experimental and computational studies propose a strategy for disease gene and pathway prediction that may be applied to a host of human neurological disorders.

Novel design of proteins to target receptors for treatment or tissue augmentation has come to the fore owing to advancements in computing power, modeling frameworks, and translational successes. Shorter proteins, or peptides, can offer combinatorial synergies with dendrimer, polymer, or other peptide carriers for enhanced local signaling, which larger proteins may sterically hinder. Here, we present a generalized method for designing a novel peptide. We first show how to create a script protocol that can be used to iteratively optimize and screen novel peptide sequences for binding a target protein. We present a step-by-step introduction to utilizing file repositories, data bases, and the Rosetta software suite. RosettaScripts, an .xml interface that allows for sequential functions to be performed, is used to order the functions for repeatable performance. These strategies may lead to more groups venturing into computational design, which may result in synergies from artificial intelligence/machine learning (AI/ML) to phage display and screening. Importantly, the beginner is expected to be able to design their first peptide ligand and begin their journey in peptide drug discovery. Generally, these peptides potentially could be used to interact with any enzyme or receptor, for example, in the study of chemokines and their interactions with glycosoaminoglycans and their receptors.

Inspired by the recent realization of a two-dimensional (2-D) chiral fluid as an active monolayer droplet moving atop a 3-D Stokesian fluid, we formulate mathematically its free-boundary dynamics. The surface droplet is described as a general 2-D linear, incompressible and isotropic fluid, having a viscous shear stress, an active chiral driving stress and a Hall stress allowed by the lack of time-reversal symmetry. The droplet interacts with itself through its driven internal mechanics and by driving flows in the underlying 3-D Stokes phase. We pose the dynamics as the solution to a singular integral–differential equation, over the droplet surface, using the mapping from surface stress to surface velocity for the 3-D Stokes equations. Specializing to the case of axisymmetric droplets, exact representations for the chiral surface flow are given in terms of solutions to a singular integral equation, solved using both analytical and numerical techniques. For a disc-shaped monolayer, we additionally employ a semi-analytical solution that hinges on an orthogonal basis of Bessel functions and allows for efficient computation of the monolayer velocity field, which ranges from a nearly solid-body rotation to a unidirectional edge current, depending on the subphase depth and the Saffman–Delbrück length. Except in the near-wall limit, these solutions have divergent surface shear stresses at droplet boundaries, a signature of systems with codimension-one domains embedded in a 3-D medium. We further investigate the effect of a Hall viscosity, which couples radial and transverse surface velocity components, on the dynamics of a closing cavity. Hall stresses are seen to drive inward radial motion, even in the absence of edge tension.