Finding Druggable Sites in Proteins Using TACTICS.

Structure-based drug discovery efforts require knowledge of where drug-binding sites are located on target proteins. To address the challenge of finding druggable sites, we developed a machine-learning algorithm called TACTICS (trajectory-based analysis of conformations to identify cryptic sites), which uses an ensemble of molecular structures (such as molecular dynamics simulation data) as input. First, TACTICS uses k-means clustering to select a small number of conformations that represent the overall conformational heterogeneity of the data. Then, TACTICS uses a random forest model to identify potentially bindable residues in each selected conformation, based on protein motion and geometry. Lastly, residues in possible binding pockets are scored using fragment docking. As proof-of-principle, TACTICS was applied to the analysis of simulations of the SARS-CoV-2 main protease and methyltransferase and the Yersinia pestis aryl carrier protein. Our approach recapitulates known small-molecule binding sites and predicts the locations of sites not previously observed in experimentally determined structures. The TACTICS code is available at https://github.com/Albert-Lau-Lab/tactics_protein_analysis.

Parylene C topographic micropattern as a template for patterning PDMS and Polyacrylamide hydrogel.

Parylene C is a well-known polymer and it has been mainly employed as a protective layer for implantable electronics. In this paper, we propose a new approach to use Parylene C as a versatile template for patterning soft materials potentially applicable as scaffolds in cardiac tissue engineering (TE). Parylene C substrates were anisotropically patterned through standard lithographic process with hydrophilic channels separating raised hydrophobic strips. Ridges and grooves of the template are 10 µm width and depth ranging from 1 to 17 µm. Polydimethylsiloxane (PDMS) and Polyacrylamide (PAm) hydrogel have been chosen as soft polymers to be moulded. Thanks to their chemical and physical properties PDMS and PAm hydrogel mimic the extracellular matrix (ECM). PDMS was spin coated on micropatterned Parylene C obtaining composite substrates with 460 nm and 1.15 µm high grooves. The Young's modulus of the composite Parylene C/PDMS was evaluated and it was found to be almost half when compared to PDMS. PAm hydrogel was also printed using collagen coated micro-grooved Parylene C. Optical micrographs and fluorescence analysis show the successful topographic and protein pattern transfer on the hydrogel.

Photokinetic analysis of the forces and torques exerted by optical tweezers carrying angular momentum.

The theory of photokinetic effects expresses the forces and torques exerted by a beam of light in terms of experimentally accessible amplitude and phase profiles. We use this formalism to develop an intuitive explanation for the performance of optical tweezers operating in the Rayleigh regime, including effects arising from the influence of light's angular momentum. First-order dipole contributions reveal how a focused beam can trap small objects, and what features limit the trap's stability. The first-order force separates naturally into a conservative intensity-gradient term that forms a trap and a non-conservative solenoidal term that drives the system out of thermodynamic equilibrium. Neither term depends on the light's polarization; light's spin angular momentum plays no role at dipole order. Polarization-dependent effects, such as trap-strength anisotropy and spin-curl forces, are captured by the second-order dipole-interference contribution to the photokinetic force. The photokinetic expansion thus illuminates how light's angular momentum can be harnessed for optical micromanipulation, even in the most basic optical traps.This article is part of the themed issue 'Optical orbital angular momentum'.

Charge renormalization in nominally apolar colloidal dispersions.

We present high-resolution measurements of the pair interactions between dielectric spheres dispersed in a fluid medium with a low dielectric constant. Despite the absence of charge control agents or added organic salts, these measurements reveal strong and long-ranged repulsions consistent with substantial charges on the particles whose interactions are screened by trace concentrations of mobile ions in solution. The dependence of the estimated charge on the particles' radii is consistent with charge renormalization theory and, thus, offers insights into the charging mechanism in this interesting class of model systems. The measurement technique, based on optical-tweezer manipulation and artifact-free particle tracking, makes use of optimal statistical methods to reduce measurement errors to the femtonewton frontier while covering an extremely wide range of interaction energies.

Parameterized computational imaging: Data driven computational modeling for image extension.

Parameterized Computational Imaging (PCI) allows for a continuous, portable and remote imaging of physiology without the continuous need of complex imaging systems. The method trades complex imaging equipment for computing power and potentially wireless measured parameters. The PCI algorithm uses a baseline image along with computational models to calculate physically measurable parameters. As the physically measurable parameters change the computational model is iteratively run until computationally predicted parameters matches the measured values. Swarm optimization routines are implemented to accelerate the process of finding the new values. A gelatin model with circular object is presented to demonstrate the PCI algorithm's ability to locate the circular object from four voltage measurements.

High-speed tracking of rupture and clustering in freely falling granular streams.

Thin streams of liquid commonly break up into characteristic droplet patterns owing to the surface-tension-driven Plateau-Rayleigh instability. Very similar patterns are observed when initially uniform streams of dry granular material break up into clusters of grains, even though flows of macroscopic particles are considered to lack surface tension. Recent studies on freely falling granular streams tracked fluctuations in the stream profile, but the clustering mechanism remained unresolved because the full evolution of the instability could not be observed. Here we demonstrate that the cluster formation is driven by minute, nanoNewton cohesive forces that arise from a combination of van der Waals interactions and capillary bridges between nanometre-scale surface asperities. Our experiments involve high-speed video imaging of the granular stream in the co-moving frame, control over the properties of the grain surfaces and the use of atomic force microscopy to measure grain-grain interactions. The cohesive forces that we measure correspond to an equivalent surface tension five orders of magnitude below that of ordinary liquids. We find that the shapes of these weakly cohesive, non-thermal clusters of macroscopic particles closely resemble droplets resulting from thermally induced rupture of liquid nanojets.

Inverse computational feedback optimization imaging applied to time varying changes in a homogeneous structure.

The technique of inverse computational feedback optimization imaging allows for the imaging of varying tissue without the continuous need of a complex imaging systems such as an MRI or CT. Our method trades complex imaging equipment for computing power. The objective is to use a baseline scan from an imaging system along with finite element method computational software to calculate the physically measurable parameters (such as voltage or temperature). As the physically measurable parameters change the computational model is iteratively run until it matches the measured values. Optimization routines are implemented to accelerate the process of finding the new values. Presented is a computational model demonstrating how the inverse imaging technique would work with a simple homogeneous sample with a circular structure. It demonstrates the ability to locate an object with only a few point measurements. The presented computational model uses swarm optimization techniques to help find the object location from the measured data (which in this case is voltage).

Modeling the interaction of electric current and tissue: importance of accounting for time varying electric properties.

Time varying computer models of the interaction of electric current and tissue are very valuable in helping to understand the complexity of the human body and biological tissue. The electrical properties of tissue, permittivity and conductivity, are vital to accurately modeling the interaction of the human tissue with electric current. Past models have represented the electric properties of the tissue as constant or temperature dependent. This paper presents time dependent electric properties that change as a result of tissue damage, temperature, blood flow, blood vessels, and tissue property. Six models are compared to emphasize the importance of accounting for these different tissue properties in the computer model. In particular, incorporating the time varying nature of the electric properties of human tissue into the model leads to a significant increase in tissue damage. An important feature of the model is the feedback loop created between the electric properties, tissue damage, and temperature.

Decreased shoot stature and grain alpha-amylase activity following ectopic expression of a gibberellin 2-oxidase gene in transgenic wheat.

Ectopic expression of a gibberellin 2-oxidase gene (PcGA2ox1) decreased the content of bioactive gibberellins (GAs) in transgenic wheat, producing a range of dwarf plants with different degrees of severity. In at least one case, a single transformation event gave rise to T(1) plants with different degrees of dwarfism, the phenotypes being stably inherited over at least four generations. The dwarf phenotype, which included dark-green leaves, increased tillering and, in severe cases, a prostrate growth habit, was replicated by the application of a GA biosynthesis inhibitor to the wild type. Ear rachis length, grain set, and grain size were also decreased in the wheat transformants, compared with an azygous (null) line. The extent of post-germination alpha-amylase production in grains reflected the severity of the shoot phenotype of the transformants and both developmental processes were restored to normal by the application of gibberellic acid (GA(3)). Expression of two GA biosynthesis genes (TaGA20ox1 and TaGA3ox2) was up-regulated, and that of two alpha-amylase gene families (alpha-Amy1 and alpha-Amy2) down regulated, in scutella of semi-dwarf lines, compared with controls. The marked decline in transcript abundance of both alpha-amylase gene families in aleurone was associated with a decreased content of bioactive GAs in grains of the semi-dwarf lines.

Function and transcript analysis of gibberellin-biosynthetic enzymes in wheat.

The enzymes gibberellin (GA) 20-oxidase and 3-oxidase are major sites of regulation in GA biosynthesis. We have characterised one member of each of the gene families encoding these enzymes that are highly expressed in elongating stems and in developing and germinating grains of wheat and are therefore likely to have prominent developmental roles in these tissues. We mapped the three homoeologues of the GA 20-oxidase gene TaGA20ox1 to chromosomes 5BL, 5DL and 4AL. TaGA20ox1 is expressed mainly in the nodes and ears of the elongating stem, and also in developing and germinating embryos. Expression in the nodes, ears and germinating embryos is predominantly from the A and D genomes. Each homoeologous cDNA encodes a functional enzyme that catalyses the multi-step conversions of GA12-GA9, and GA53-GA20. Time course and enzyme kinetic studies indicate that the initial oxidation steps from GA12 and GA53 to the free alcohol forms of GA15 and GA44, respectively, occur rapidly but that subsequent steps occur more slowly. The intermediate GA19 has an especially low affinity for the enzyme, consistent with its accumulation in wheat tissues. The three homoeologous cDNAs for the 3-oxidase gene TaGA3ox2 encode functional enzymes, one of which was shown to possess low levels of 2beta-hydroxylase, 2,3-desaturase, 2,3-epoxidase and even 13-hydroxylase activities in addition to 3beta-hydroxylase activity. In contrast to TaGA20ox1, TaGA3ox2 is expressed in internodes, as well as nodes and the ear of the elongating stem. It is also highly expressed in developing and germinated embryos.