Publications

Phosphotriesterases (PTEs) represent a class of enzymes capable of efficient neutralization of organophosphates (OPs), a dangerous class of neurotoxic chemicals. PTEs suffer from low catalytic activity, particularly at higher temperatures, due to low thermostability and low solubility. Supercharging, a protein engineering approach via selective mutation of surface residues to charged residues, has been successfully employed to generate proteins with increased solubility and thermostability by promoting charge–charge repulsion between proteins. We set out to overcome the challenges in improving PTE activity against OPs by employing a computational protein supercharging algorithm in Rosetta. Here, we discover two supercharged PTE variants, one negatively supercharged (with −14 net charge) and one positively supercharged (with +12 net charge) and characterize them for their thermodynamic stability and catalytic activity. We find that positively supercharged PTE possesses slight but significant losses in thermostability, which correlates to losses in catalytic efficiency at all temperatures, whereas negatively supercharged PTE possesses increased catalytic activity across 25°C–55°C while offering similar thermostability characteristic to the parent PTE. The impact of supercharging on catalytic efficiency will inform the design of shelf-stable PTE and criteria for enzyme engineering.

This work probes the role of cell geometry in orienting self-organized fluid flows in the late stage Drosophila oocyte. Recent theoretical work has shown that a model, which relies only on hydrodynamic interactions of flexible, cortically anchored microtubules (MTs) and the mechanical loads from molecular motors moving upon them, is sufficient to generate observed flows. While the emergence of flows has been studied in spheres, oocytes change shape during streaming and it was unclear how robust these flows are to the geometry of the cell. Here we use biophysical theory and computational analysis to investigate the role of geometry and find that the axis of rotation is set by the shape of the domain and that the flow is robust to biologically relevant perturbations of the domain shape. Using live imaging and 3D flow reconstruction, we test the predictions of the theory/simulation, finding consistency between the model and live experiments, further demonstrating a geometric dependence on flow direction in late-stage Drosophila oocytes.

Signaling pathways induce stereotyped transcriptional changes as stem cells progress into mature cell types during embryogenesis. Signaling perturbations are necessary to discover which genes are responsive or insensitive to pathway activity. However, gene regulation is additionally dependent on cell state-specific factors like chromatin modifications or transcription factor binding. Thus, transcriptional profiles need to be assayed in single cells to identify potentially multiple, distinct perturbation responses among heterogeneous cell states in an embryo. In perturbation studies, comparing heterogeneous transcriptional states among experimental conditions often requires samples to be collected over multiple independent experiments. Datasets produced in such complex experimental designs can be confounded by batch effects. We present Design-Aware Integration of Single Cell ExpEriments (DAISEE), a new algorithm that models perturbation responses in single-cell datasets with a complex experimental design. We demonstrate that DAISEE improves upon a previously available integrative non-negative matrix factorization framework, more efficiently separating perturbation responses from confounding variation. We use DAISEE to integrate newly collected single-cell RNA-sequencing datasets from 5-hour old zebrafish embryos expressing optimized photoswitchable MEK (psMEK), which globally activates the extracellular signal-regulated kinase (ERK), a signaling molecule involved in many cell specification events. psMEK drives some cells that are normally not exposed to ERK signals towards other wild type states and induces novel states expressing a mixture of transcriptional programs, including precociously activated endothelial genes. ERK signaling is therefore capable of introducing profoundly new gene expression states in developing embryos.

We introduce OpenRAND, a C++17 library aimed at facilitating reproducible scientific research by generating statistically robust yet replicable random numbers in as little as two lines of code, overcoming some of the unnecessary complexities of existing RNG libraries. OpenRAND accommodates single and multi-threaded applications on CPUs and GPUs and offers a simplified, user-friendly API that complies with the C++ standard’s random number engine interface. It is lightweight; provided as a portable, header-only library. It is statistically robust: a suite of built-in tests ensures no pattern exists within single or multiple streams. Despite its simplicity and portability, it remains performant—matching and sometimes outperforming native libraries. Our tests, including a Brownian walk simulation, affirm its reproducibility and ease-of-use while highlight its computational efficiency, outperforming CUDA’s cuRAND by up to 1.8 times.

The motion of rigid particles in complex fluids is ubiquitous in natural and industrial processes. The most popular toy model for understanding the physics of such systems is the settling of a solid sphere in a viscoelastic fluid. There is general agreement that an elastic wake develops downstream of the sphere, causing the breakage of fore-and-aft symmetry, while the flow remains axisymmetric, independent of fluid viscoelasticity and flow conditions. Using a continuum mechanics model, we reveal that axisymmetry holds only for weak viscoelastic flows. Beyond a critical value of the settling velocity, steady, non-axisymmetric disturbances develop peripherally of the rear pole of the sphere, giving rise to a four-lobed fingering instability. The transition from axisymmetric to non-axisymmetric flow fields is characterized by a regular bifurcation and depends solely on the interplay between shear and extensional properties of the viscoelastic fluid under different flow regimes. At higher settling velocities, each lobe tip is split into two new lobes, resembling fractal fingering in interfacial flows. For the first time, we capture an elastic fingering instability under steady-state conditions, and provide the missing information for understanding and predicting such instabilities in the response of viscoelastic fluids and soft media.

We present a comprehensive investigation combining numerical simulations with experimental validation, focusing on the creeping flow behavior of a shear-banding, viscoelastic wormlike micellar (WLM) solution over concavities with various depths (D) and lengths (L). The fluid is modeled using the diffusive Giesekus model, with model parameters set to quantitatively describe the shear rheology of a 100 : 60 mM cetylpyridinium chloride:sodium salicylate aqueous WLM solution used for the experimental validation. We observe a transition from “cavity flow” to “expansion–contraction flow” as the length L exceeds the sum of depth D and channel width W. This transition is manifested by a change of vortical structures within the concavity. For L ≤ D + W, “cavity flow” is characterized by large scale recirculations spanning the concavity length. For L > D + W, the recirculations observed in “expansion–contraction flow” are confined to the salient corners downstream of the expansion plane and upstream of the contraction plane. Using the numerical dataset, we construct phase diagrams in L–D at various fixed Weissenberg numbers Wi, characterizing the transitions and describing the evolution of vortical structures influenced by viscoelastic effects.

Unraveling the phenotypic and genetic complexity of autism is extremely challenging yet critical for understanding the biology, inheritance, trajectory, and clinical manifestations of the many forms of the condition. Here, we leveraged broad phenotypic data from a large cohort with matched genetics to characterize classes of autism and their patterns of core, associated, and co-occurring traits, ultimately demonstrating that phenotypic patterns are associated with distinct genetic and molecular programs. We used a generative mixture modeling approach to identify robust, clinically-relevant classes of autism which we validate and replicate in a large independent cohort. We link the phenotypic findings to distinct patterns of de novo and inherited variation which emerge from the deconvolution of these genetic signals, and demonstrate that class-specific common variant scores strongly align with clinical outcomes. We further provide insights into the distinct biological pathways and processes disrupted by the sets of mutations in each class. Remarkably, we discover class-specific differences in the developmental timing of genes that are dysregulated, and these temporal patterns correspond to clinical milestone and outcome differences between the classes. These analyses embrace the phenotypic complexity of children with autism, unraveling genetic and molecular programs underlying their heterogeneity and suggesting specific biological dysregulation patterns and mechanistic hypotheses.

Cryo-electron microscopy (cryo-EM) is a powerful technique for determining high-resolution 3D biomolecular structures from imaging data. As this technique can capture dynamic biomolecular complexes, 3D reconstruction methods are increasingly being developed to resolve this intrinsic structural heterogeneity. However, the absence of standardized benchmarks with ground truth structures and validation metrics limits the advancement of the field. Here, we propose CryoBench, a suite of datasets, metrics, and performance benchmarks for heterogeneous reconstruction in cryo-EM. We propose five datasets representing different sources of heterogeneity and degrees of difficulty. These include conformational heterogeneity generated from simple motions and random configurations of antibody complexes and from tens of thousands of structures sampled from a molecular dynamics simulation. We also design datasets containing compositional heterogeneity from mixtures of ribosome assembly states and 100 common complexes present in cells. We then perform a comprehensive analysis of state-of-the-art heterogeneous reconstruction tools including neural and non-neural methods and their sensitivity to noise, and propose new metrics for quantitative comparison of methods. We hope that this benchmark will be a foundational resource for analyzing existing methods and new algorithmic development in both the cryo-EM and machine learning communities.

Huntington’s disease (HD) is a fatal neurodegenerative disorder resulting from an abnormal expansion of polyglutamine (polyQ) repeats in the N-terminus of the huntingtin protein. When the polyQ tract surpasses 35 repeats, the mutated protein undergoes misfolding, culminating in the formation of intracellular aggregates. Research in mouse models suggests that HD pathogenesis involves the aggregation of N-terminal fragments of the huntingtin protein (htt). These early oligomeric assemblies of htt, exhibiting diverse characteristics during aggregation, are implicated as potential toxic entities in HD. However, a consensus on their specific structures remains elusive. Understanding the heterogeneous nature of htt oligomers provides crucial insights into disease mechanisms, emphasizing the need to identify various oligomeric conformations as potential therapeutic targets. Employing coarse-grained molecular dynamics, our study aims to elucidate the mechanisms governing the aggregation process and resultant aggregate architectures of htt. The polyQ tract within htt is flanked by two regions: an N-terminal domain (N17) and a short C-terminal proline-rich segment. We conducted self-assembly simulations involving five distinct N17 + polyQ systems with polyQ lengths ranging from 7 to 45, utilizing the ProMPT force field. Prolongation of the polyQ domain correlates with an increase in β-sheet-rich structures. Longer polyQ lengths favor intramolecular β-sheets over intermolecular interactions due to the folding of the elongated polyQ domain into hairpin-rich conformations. Importantly, variations in polyQ length significantly influence resulting oligomeric structures. Shorter polyQ domains lead to N17 domain aggregation, forming a hydrophobic core, while longer polyQ lengths introduce a competition between N17 hydrophobic interactions and polyQ polar interactions, resulting in densely packed polyQ cores with outwardly distributed N17 domains. Additionally, at extended polyQ lengths, we observe distinct oligomeric conformations with varying degrees of N17 bundling. These findings can help explain the toxic gain-of-function that htt with expanded polyQ acquires.