Harnessing elastic instabilities for enhanced mixing and reaction kinetics in porous media.

Turbulent flows have been used for millennia to mix solutes; a familiar example is stirring cream into coffee. However, many energy, environmental, and industrial processes rely on the mixing of solutes in porous media where confinement suppresses inertial turbulence. As a result, mixing is drastically hindered, requiring fluid to permeate long distances for appreciable mixing and introducing additional steps to drive mixing that can be expensive and environmentally harmful. Here, we demonstrate that this limitation can be overcome just by adding dilute amounts of flexible polymers to the fluid. Flow-driven stretching of the polymers generates an elastic instability, driving turbulent-like chaotic flow fluctuations, despite the pore-scale confinement that prohibits typical inertial turbulence. Using in situ imaging, we show that these fluctuations stretch and fold the fluid within the pores along thin layers ("lamellae") characterized by sharp solute concentration gradients, driving mixing by diffusion in the pores. This process results in a [Formula: see text] reduction in the required mixing length, a [Formula: see text] increase in solute transverse dispersivity, and can be harnessed to increase the rate at which chemical compounds react by [Formula: see text]-enhancements that we rationalize using turbulence-inspired modeling of the underlying transport processes. Our work thereby establishes a simple, robust, versatile, and predictive way to mix solutes in porous media, with potential applications ranging from large-scale chemical production to environmental remediation.

Exploring student perceptions of their learning adaptions during the COVID-19 pandemic.

OBJECTIVE: With the shelter-in-place orders implemented during the COVID-19 pandemic, learning experiences abruptly changed from on campus to wholly online. This qualitative study explores the perceptions and attitudes of students as they adapted their study space, study time, and approach to learning. METHODS: One hundred five students enrolled in a doctor of chiropractic program were invited to participate in a survey to understand how shelter-in-place orders during the COVID-19 pandemic influenced their approach to learning. Fifty-two of 105 (49.5%) students completed the survey. The survey asked students to select their primary study strategy from a list of options and then prompted students to explain how the COVID-19 pandemic influenced their study space, use of technology, study time, and metacognitive cycle of planning, monitoring, and evaluating their approach to learning. A Thematic analysis of the participants' responses was performed. RESULTS: Nearly all study participants described a challenge in adapting their study space, study time, or approach to learning. Respondents reported that the use of technology did not change because assessments and resources were electronic before the pandemic. Respondents who selected high-impact study strategies such as self-quizzing or who demonstrated evidence of well-developed metacognition described a positive approach to learning more frequently than did respondents who selected low-impact study strategies such as repeated reading or who did not show evidence of metacognitive development. CONCLUSION: This study presents student perceptions related to promoting and developing self-regulated learning skills. Educators can use this information to understand the adaptations to changes in learning experiences that may promote successful learning.

Elastic turbulence generates anomalous flow resistance in porous media.

Many energy, environmental, industrial, and microfluidic processes rely on the flow of polymer solutions through porous media. Unexpectedly, the macroscopic flow resistance often increases above a threshold flow rate in a porous medium, but not in bulk solution. The reason why has been a puzzle for over half a century. Here, by directly visualizing flow in a transparent 3D porous medium, we demonstrate that this anomalous increase is due to the onset of an elastic instability in which the flow exhibits strong spatiotemporal fluctuations reminiscent of inertial turbulence, despite the small Reynolds number. Our measurements enable us to quantitatively establish that the energy dissipated by pore-scale fluctuations generates the anomalous increase in the overall flow resistance. Because the macroscopic resistance is one of the most fundamental descriptors of fluid flow, our results both help deepen understanding of complex fluid flows and provide guidelines to inform a broad range of applications.

Sulfopin is a covalent inhibitor of Pin1 that blocks Myc-driven tumors in vivo.

The peptidyl-prolyl isomerase, Pin1, is exploited in cancer to activate oncogenes and inactivate tumor suppressors. However, despite considerable efforts, Pin1 has remained an elusive drug target. Here, we screened an electrophilic fragment library to identify covalent inhibitors targeting Pin1's active site Cys113, leading to the development of Sulfopin, a nanomolar Pin1 inhibitor. Sulfopin is highly selective, as validated by two independent chemoproteomics methods, achieves potent cellular and in vivo target engagement and phenocopies Pin1 genetic knockout. Pin1 inhibition had only a modest effect on cancer cell line viability. Nevertheless, Sulfopin induced downregulation of c-Myc target genes, reduced tumor progression and conferred survival benefit in murine and zebrafish models of MYCN-driven neuroblastoma, and in a murine model of pancreatic cancer. Our results demonstrate that Sulfopin is a chemical probe suitable for assessment of Pin1-dependent pharmacology in cells and in vivo, and that Pin1 warrants further investigation as a potential cancer drug target.

A Time-Resolved Cryo-EM Study of Saccharomyces cerevisiae 80S Ribosome Protein Composition in Response to a Change in Carbon Source.

The role of the ribosome in the regulation of gene expression has come into increased focus. It is proposed that ribosomes are catalytic engines capable of changing their protein composition in response to environmental stimuli. Time-resolved cryo-electron microscopy (cryo-EM) techniques are employed to identify quantitative changes in the protein composition and structure of the Saccharomyces cerevisiae 80S ribosomes after shifting the carbon source from glucose to glycerol. Using cryo-EM combined with the computational classification approach, it is found that a fraction of the yeast cells' 80S ribosomes lack ribosomal proteins at the entrance and exit sites for tRNAs, including uL16(RPL10), eS1(RPS1), uS11(RPS14A/B), and eS26(RPS26A/B). This fraction increased after a change from glucose to glycerol medium. The quantitative structural analysis supports the hypothesis that ribosomes are dynamic complexes that alter their composition in response to changes in growth or environmental conditions.

Identification of a potent and selective covalent Pin1 inhibitor.

Peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (Pin1) is commonly overexpressed in human cancers, including pancreatic ductal adenocarcinoma (PDAC). While Pin1 is dispensable for viability in mice, it is required for activated Ras to induce tumorigenesis, suggesting a role for Pin1 inhibitors in Ras-driven tumors, such as PDAC. We report the development of rationally designed peptide inhibitors that covalently target Cys113, a highly conserved cysteine located in the Pin1 active site. The inhibitors were iteratively optimized for potency, selectivity and cell permeability to give BJP-06-005-3, a versatile tool compound with which to probe Pin1 biology and interrogate its role in cancer. In parallel to inhibitor development, we employed genetic and chemical-genetic strategies to assess the consequences of Pin1 loss in human PDAC cell lines. We demonstrate that Pin1 cooperates with mutant KRAS to promote transformation in PDAC, and that Pin1 inhibition impairs cell viability over time in PDAC cell lines.

Targeting the PI5P4K Lipid Kinase Family in Cancer Using Covalent Inhibitors.

The PI5P4Ks have been demonstrated to be important for cancer cell proliferation and other diseases. However, the therapeutic potential of targeting these kinases is understudied due to a lack of potent, specific small molecules available. Here, we present the discovery and characterization of a pan-PI5P4K inhibitor, THZ-P1-2, that covalently targets cysteines on a disordered loop in PI5P4Kα/ß/γ. THZ-P1-2 demonstrates cellular on-target engagement with limited off-targets across the kinome. AML/ALL cell lines were sensitive to THZ-P1-2, consistent with PI5P4K's reported role in leukemogenesis. THZ-P1-2 causes autophagosome clearance defects and upregulation in TFEB nuclear localization and target genes, disrupting autophagy in a covalent-dependent manner and phenocopying the effects of PI5P4K genetic deletion. Our studies demonstrate that PI5P4Ks are tractable targets, with THZ-P1-2 as a useful tool to further interrogate the therapeutic potential of PI5P4K inhibition and inform drug discovery campaigns for these lipid kinases in cancer metabolism and other autophagy-dependent disorders.

Targeted Identification of Protein Interactions in Eukaryotic mRNA Translation.

To identify protein-protein interactions and phosphorylated amino acid sites in eukaryotic mRNA translation, replicate TAP-MudPIT and control experiments are performed targeting Saccharomyces cerevisiae genes previously implicated in eukaryotic mRNA translation by their genetic and/or functional roles in translation initiation, elongation, termination, or interactions with ribosomal complexes. Replicate tandem affinity purifications of each targeted yeast TAP-tagged mRNA translation protein coupled with multidimensional liquid chromatography and tandem mass spectrometry analysis are used to identify and quantify copurifying proteins. To improve sensitivity and minimize spurious, nonspecific interactions, a novel cross-validation approach is employed to identify the most statistically significant protein-protein interactions. Using experimental and computational strategies discussed herein, the previously described protein composition of the canonical eukaryotic mRNA translation initiation, elongation, and termination complexes is calculated. In addition, statistically significant unpublished protein interactions and phosphorylation sites for S. cerevisiae's mRNA translation proteins and complexes are identified.

Pore-Scale Flow Characterization of Polymer Solutions in Microfluidic Porous Media.

Polymer solutions are frequently used in enhanced oil recovery and groundwater remediation to improve the recovery of trapped nonaqueous fluids. However, applications are limited by an incomplete understanding of the flow in porous media. The tortuous pore structure imposes both shear and extension, which elongates polymers; moreover, the flow is often at large Weissenberg numbers, Wi, at which polymer elasticity in turn strongly alters the flow. This dynamic elongation can even produce flow instabilities with strong spatial and temporal fluctuations despite the low Reynolds number, Re. Unfortunately, macroscopic approaches are limited in their ability to characterize the pore-scale flow. Thus, understanding how polymer conformations, flow dynamics, and pore geometry together determine these nontrivial flow patterns and impact macroscopic transport remains an outstanding challenge. This review describes how microfluidic tools can shed light on the physics underlying the flow of polymer solutions in porous media at high Wi and low Re. Specifically, microfluidic studies elucidate how steady and unsteady flow behavior depends on pore geometry and solution properties, and how polymer-induced effects impact nonaqueous fluid recovery. This work thus provides new insights for polymer dynamics, non-Newtonian fluid mechanics, and applications such as enhanced oil recovery and groundwater remediation.

Equine infectious disease surveillance: surveillance concepts and international outbreak reporting systems.

Fleur Whitlock of the Animal Health Trust takes a look at equine infectious disease surveillance and the sources of information available.

West Nile fever in Europe in 2018: an emerging problem or just an anomaly?

Last summer saw an unusually high number of cases of West Nile fever in horses and people in south and south-east Europe, but it is too early to tell if this was a one-off increase or a sign of things to come. Here, Christopher Browne, Eleanor Glendenning, Jolyon Medlock and Helen Roberts discuss the various West Nile fever surveillance and control mechanisms in place in the UK.

Cooperative size sorting of deformable particles in porous media.

Diverse applications-ranging from enhanced oil recovery, filtration, and lab on a chip sorting-rely on the flow-induced transport of deformable particles in porous media. However, how fluid flow can force such particles to squeeze through pore constrictions of complex geometries is poorly understood. Here, we study the transport of model deformable particles in millifluidic porous media with constrictions of tunable aspect ratio. We find that multiple particles can unexpectedly squeeze through large-aspect ratio constrictions, even when isolated particles cannot. This phenomenon arises from pairwise flow-mediated interactions between the particles: when one particle is trapped at a constriction, the increased fluid flow around it enables a second to squeeze past due to locally increased hydrodynamic stresses. This cooperative mechanism causes the particles to ultimately sort themselves by size through the pore space. By revealing a new mode of deformable particle transport in porous media, our work helps to inform real-world applications and provides a straightforward way to sort particles based on size.

Discovery of Covalent CDK14 Inhibitors with Pan-TAIRE Family Specificity.

Cyclin-dependent kinase 14 (CDK14) and other TAIRE family kinases (CDKs 15-18) are proteins that lack functional annotation but are frequent off-targets of clinical kinase inhibitors. In this study we develop and characterize FMF-04-159-2, a tool compound that specifically targets CDK14 covalently and possesses a TAIRE kinase-biased selectivity profile. This tool compound and its reversible analog were used to characterize the cellular consequences of covalent CDK14 inhibition, including an unbiased investigation using phospho-proteomics. To reduce confounding off-target activity, washout conditions were used to deconvolute CDK14-specific effects. This investigation suggested that CDK14 plays a supporting role in cell-cycle regulation, particularly mitotic progression, and identified putative CDK14 substrates. Together, these results represent an important step forward in understanding the cellular consequences of inhibiting CDK14 kinase activity.

Leveraging Compound Promiscuity to Identify Targetable Cysteines within the Kinome.

Covalent kinase inhibitors, which typically target cysteine residues, represent an important class of clinically relevant compounds. Approximately 215 kinases are known to have potentially targetable cysteines distributed across 18 spatially distinct locations proximal to the ATP-binding pocket. However, only 40 kinases have been covalently targeted, with certain cysteine sites being the primary focus. To address this disparity, we have developed a strategy that combines the use of a multi-targeted acrylamide-modified inhibitor, SM1-71, with a suite of complementary chemoproteomic and cellular approaches to identify additional targetable cysteines. Using this single multi-targeted compound, we successfully identified 23 kinases that are amenable to covalent inhibition including MKNK2, MAP2K1/2/3/4/6/7, GAK, AAK1, BMP2K, MAP3K7, MAPKAPK5, GSK3A/B, MAPK1/3, SRC, YES1, FGFR1, ZAK (MLTK), MAP3K1, LIMK1, and RSK2. The identification of nine of these kinases previously not targeted by a covalent inhibitor increases the number of targetable kinases and highlights opportunities for covalent kinase inhibitor development.

A Chemoproteomic Strategy for Direct and Proteome-Wide Covalent Inhibitor Target-Site Identification.

Despite recent clinical successes for irreversible drugs, potential toxicities mediated by unpredictable modification of off-target cysteines represents a major hurdle for expansion of covalent drug programs. Understanding the proteome-wide binding profile of covalent inhibitors can significantly accelerate their development; however, current mass spectrometry strategies typically do not provide a direct, amino acid level readout of covalent activity for complex, selective inhibitors. Here we report the development of CITe-Id, a novel chemoproteomic approach that employs covalent pharmacologic inhibitors as enrichment reagents in combination with an optimized proteomic platform to directly quantify dose-dependent binding at cysteine-thiols across the proteome. CITe-Id analysis of our irreversible CDK inhibitor THZ1 identified dose-dependent covalent modification of several unexpected kinases, including a previously unannotated cysteine (C840) on the understudied kinase PKN3. These data streamlined our development of JZ128 as a new selective covalent inhibitor of PKN3. Using JZ128 as a probe compound, we identified novel potential PKN3 substrates, thus offering an initial molecular view of PKN3 cellular activity. CITe-Id provides a powerful complement to current chemoproteomic platforms to characterize the selectivity of covalent inhibitors, identify new, pharmacologically addressable cysteine-thiols, and inform structure-based drug design programs.

Critical Role for Saccharomyces cerevisiae Asc1p in Translational Initiation at Elevated Temperatures.

The eukaryotic ribosomal protein RACK1/Asc1p is localized to the mRNA exit channel of the 40S subunit but lacks a defined role in mRNA translation. Saccharomyces cerevisiae deficient in ASC1 exhibit temperature-sensitive growth. Using this null mutant, potential roles for Asc1p in translation and ribosome biogenesis are evaluated. At the restrictive temperature the asc1Δ null mutant has reduced polyribosomes. To test the role of Asc1p in ribosome stability, cryo-EM is used to examine the structure of 80S ribosomes in an asc1Δ yeast deletion mutant at both the permissive and nonpermissive temperatures. CryoEM indicates that loss of Asc1p does not severely disrupt formation of this complex structure. No defect is found in rRNA processing in the asc1Δ null mutant. A proteomic approach is applied to survey the effect of Asc1p loss on the global translation of yeast proteins. At the nonpermissive temperature, the asc1Δ mutant has reduced levels of ribosomal proteins and other factors critical for translation. Collectively, these results are consistent with recent observations suggesting that Asc1p is important for ribosome occupancy of short mRNAs. The results show the Asc1 ribosomal protein is critical in translation during heat stress.

Identification of Changing Ribosome Protein Compositions using Mass Spectrometry.

The regulatory role of the ribosome in gene expression has come into sharper focus. It has been proposed that ribosomes are dynamic complexes capable of changing their protein composition in response to environmental stimuli. MS is applied to identify quantitative changes in the protein composition of S. cerevisiae 80S ribosomes in response to different environmental stimuli. Using quantitative MS, it is found that the paralog yeast ribosomal proteins RPL8A (eL8A) and RPL8B (eL8B) change their relative proportions in the 80S ribosome when yeast is switched from growth in glucose to glycerol. By using yeast genetics and polysome profiling, it is shown that yeast ribosomes containing either RPL8A or RPL8B are not functionally interchangeable. The quantitative proteomic data support the hypothesis that ribosomes are dynamic complexes that alter their composition and functional activity in response to changes in growth or environmental conditions.

Non-contact AFM measurement of the Hamaker constants of solids: Calibrating cantilever geometries.

HYPOTHESIS: Surface effects arising from roughness and deformation can negatively affect the results of AFM contact experiments. Using the non-contact portion of an AFM deflection curve is therefore desirable for estimating the Hamaker constant, A, of a solid material. A previously validated non-contact quasi-dynamic method for estimating A is revisited, in which the cantilever tip is now always represented by an "effective sphere". In addition to simplifying this previous method, accurate estimates of A can still be obtained even though precise knowledge of the nanoscale geometric features of the cantilever tip are no longer required. EXPERIMENTS: The tip's "effective" radius of curvature, Reff, is determined from a "calibration" step, in which the tip's deflection at first contact with the surface is measured for a substrate with a known Hamaker constant. After Reff is known for a given tip, estimates of A for other surfaces of interest are then determined. FINDINGS: An experimental study was conducted to validate the new method and the obtained results are in good agreement with predictions from the Lifshitz approximation, when available. Since Reff accounts for all geometric uncertainties of the tip through a single fitted parameter, no visual fitting of the tip shape was required.

A New "Quasi-Dynamic" Method for Determining the Hamaker Constant of Solids Using an Atomic Force Microscope.

In order to minimize the effects of surface roughness and deformation, a new method for estimating the Hamaker constant, A, of solids using the approach-to-contact regime of an atomic force microscope (AFM) is presented. First, a previous "jump-into-contact" quasi-static method for determining A from AFM measurements is analyzed and then extended to include various AFM tip-surface force models of interest. Then, to test the efficacy of the "jump-into-contact" method, a dynamic model of the AFM tip motion is developed. For finite AFM cantilever-surface approach speeds, a true "jump" point, or limit of stability, is found not to appear, and the quasi-static model fails to represent the dynamic tip behavior at close tip-surface separations. Hence, a new "quasi-dynamic" method for estimating A is proposed that uses the dynamically well-defined deflection at which the tip and surface first come into contact, dc, instead of the dynamically ill-defined "jump" point. With the new method, an apparent Hamaker constant, Aapp, is calculated from dc and a corresponding quasi-static-based equation. Since Aapp depends on the cantilever's approach speed, vc, and the AFM's sampling resolution, δ, a double extrapolation procedure is used to determine Aapp in the quasi-static (vc → 0) and continuous sampling (δ → 0) limits, thereby recovering the "true" value of A. The accuracy of the new method is validated using simulated AFM data. To enable the experimental implementation of this method, a new dimensionless parameter τ is introduced to guide cantilever selection and the AFM operating conditions. The value of τ quantifies how close a given cantilever is to its quasi-static limit for a chosen cantilever-surface approach speed. For sufficiently small values of τ (i.e., a cantilever that effectively behaves "quasi-statically"), simulated data indicate that Aapp will be within ∼3% or less of the inputted value of the Hamaker constant. This implies that Hamaker constants can be reliably estimated using a single measurement taken with an appropriately chosen cantilever and a slow, yet practical, approach speed (with no extrapolation required). This result is confirmed by the very good agreement found between the experimental AFM results obtained using this new method and previously reported predictions of A for amorphous silica, polystyrene, and α-Al2O3 substrates obtained using the Lifshitz method.