Harnessing Water Competition to Drive Enzyme Crosstalk.

In nature, enzymatic pathways often involve compartmentalization effects that can modify the intrinsic activity and specificity of the different enzymes involved. Consequently, extensive research has focused on replicating and studying the compartmentalization effects on individual enzymes and on multistep enzyme "cascade" reactions. This study explores the influence of compartmentalization achieved using molecular crowding on the glucose oxidase/horseradish peroxidase (GOx/HRP) cascade reaction.

DNA origami characterized a solid-state nanopore: insights into nanostructure dimensions, rigidity and yield.

Due to their programmability specific base pairing, self-assembled DNA origami structures have proven to be useful for a wide variety of applications, including diagnostics, molecular computation, drug delivery, and therapeutics. Measuring and characterizing these structures is therefore of great interest and an important part of quality control. Here, we show the extent to which DNA nanostructures can be characterized by a solid-state nanopore; a non-destructive, label-free, single-molecule sensor capable of electrically detecting and characterizing charged biomolecules.

Deconvolution volumetric additive manufacturing.

Volumetric additive manufacturing techniques are a promising pathway to ultra-rapid light-based 3D fabrication. Their widespread adoption, however, demands significant improvement in print fidelity. Currently, volumetric additive manufacturing prints suffer from systematic undercuring of fine features, making it impossible to print objects containing a wide range of feature sizes, precluding effective adoption in many applications.

Parallel computing for mobilities in periodic geometries.

We examine methods for calculating the effective mobilities of molecules driven through periodic geometries in the context of particle-based simulation. The standard formulation of the mobility, based on the long-time limit of the mean drift velocity, is compared to a formulation based on the mean first-passage time of molecules crossing a single period of the system geometry. The equivalence of the two definitions is derived under weaker assumptions than similar conclusions obtained previously, requiring only that the state of the system at subsequent period crossings satisfy the Markov property.

Overcoming PEG─Protein Mutual Repulsion to Improve the Efficiency of PEGylation.

Bioconjugation reactions, such as protein PEGylation, generally require excess reagents because of their inefficiency. Intriguingly, few reports have investigated the fundamental causes of this inefficiency. This study demonstrates that the excluded volume effect (EVE)─caused by the mutual repulsion of methoxy poly(ethylene glycol) (mPEG) and proteins under typical PEGylation conditions─causes proteins and protein-reactive mPEG (5 kDa) to self-associate into separate "protein-rich" and "mPEG-rich" nano-domains (i.

Studying first passage problems using neural networks: A case study in the slit-well microfluidic device.

This study presents deep neural network solutions to a time-integrated Smoluchowski equation modeling the mean first passage time of nanoparticles traversing the slit-well microfluidic device. This physical scenario is representative of a broader class of parametrized first passage problems in which key output metrics are dictated by a complicated interplay of problem parameters and system geometry. Specifically, whereas these types of problems are commonly studied using particle simulations of stochastic differential equation models, here the corresponding partial differential equation model is solved using a method based on deep neural networks.

Characterization of adjacent charged residues near the agonist binding site of the nematode UNC-49 GABA receptor.

The UNC-49 receptor is a Cys-loop GABA receptor that is unique to the nematode phylum. The receptor differs from mammalian GABA receptors both in amino acid sequence and pharmacology which highlights its potential as a novel anthelmintic target. Sequence differences within and near the various ligand-binding loops of the nematode receptor suggest that there could be structural differences compared to mammalian receptors that result in different pharmacological and functional features.

Using geometric criteria to study helix-like structures produced in molecular dynamics simulations of single amylose chains in water.

Amylose is a linear polymer chain of α-d-glucose units connected through α(1 → 4) glycosidic bonds. Experimental studies show that in non-polar solvents, single amylose chains form helical structures containing precise H-bond patterns. However, both experimental and computational studies indicate that these perfectly H-bonded helices are not stable in pure water.

Direct printing of functional 3D objects using polymerization-induced phase separation.

3D printing has enabled materials, geometries and functional properties to be combined in unique ways otherwise unattainable via traditional manufacturing techniques, yet its adoption as a mainstream manufacturing platform for functional objects is hindered by the physical challenges in printing multiple materials. Vat polymerization offers a polymer chemistry-based approach to generating smart objects, in which phase separation is used to control the spatial positioning of materials and thus at once, achieve desirable morphological and functional properties of final 3D printed objects. This study demonstrates how the spatial distribution of different material phases can be modulated by controlling the kinetics of gelation, cross-linking density and material diffusivity through the judicious selection of photoresin components.

Collective Dynamics of Model Pili-Based Twitcher-Mode Bacilliforms.

Pseudomonas aeruginosa, like many bacilliforms, are not limited only to swimming motility but rather possess many motility strategies. In particular, twitching-mode motility employs hair-like pili to transverse moist surfaces with a jittery irregular crawl. Twitching motility plays a critical role in redistributing cells on surfaces prior to and during colony formation.

Entropic Trapping of DNA with a Nanofiltered Nanopore.

Elucidating the kinetics of DNA passage through a solid-state nanopore is a fertile field of research, and mechanisms for controlling capture, passage, and trapping of biopolymers are likely to find numerous technological applications. Here we present a nanofiltered nanopore device, which forms an entropic cage for DNA following first passage through the nanopore, trapping the translocated DNA and permitting recapture for subsequent reanalysis and investigation of kinetics of passage under confinement. We characterize the trapping properties of this nanodevice by driving individual DNA polymers into the nanoscale gap separating the nanofilter and the pore, forming an entropic cage similar to a "two pores in series" device, leaving polymers to diffuse in the cage for various time lengths, and attempting to recapture the same molecule.

Electrophoretic ratcheting of spherical particles in well/channel microfluidic devices: Making particles move against the net field.

We examine the electrophoresis of spherical particles in microfluidic devices made of alternating wells and narrow channels, including a system previously used to separate DNA molecules. Our computer simulations predict that such systems can be used to separate spherical particles of different sizes that share the same free-solution mobility. Interestingly, the electrophoretic velocity shows an inversion as the field intensity is increased: while small particles have higher velocities at low field, the situation is reversed at high fields with the larger particles then moving faster.

Ultra-sub-stoichiometric "Dynamic" Bioconjugation Reduces Viscosity by Disrupting Immunoglobulin Oligomerization.

Monoclonal antibodies (mAb) are a major focus of the pharmaceutical industry, and polyclonal immunoglobulin G (IgG) therapy is used to treat a wide variety of health conditions. As some individuals require mAb/IgG therapy their entire life, there is currently a great desire to formulate antibodies for bolus injection rather than infusion. However, to achieve the required doses, very concentrated antibody solutions may be required.

Determination of the degree of PEGylation of protein bioconjugates using data from proton nuclear magnetic resonance spectroscopy.

The average number of methoxy poly(ethylene glycol) (mPEG) chains grafted to a protein - also known as the degree of PEGylation - is a fundamental parameter for characterizing a bioconjugate. The degree of PEGylation is typically determined by chromatographic or electrophoretic methods, which are subject to certain biases. This contribution describes an analytical approach alongside technical precautions for quantitatively determining the degree of PEGylation of protein bioconjugates by H NMR spectroscopy.

Evidence, Manipulation, and Termination of pH 'Nanobuffering' for Quantitative Homogenous Scavenging of Monoclonal Antibodies.

This study demonstrates that pH-responsive polymers have a very high buffering capacity in their immediate vicinity, a phenomenon termed "nanobuffering". This can be exploited to dissociate local nanoscale pH from bulk solution pH. Herein, a series of pH-responsive polymers were conjugated to Protein-A to rationally manipulate the latter's binding affinity toward antibodies via nanobuffering ( i.

A sequential nanopore-channel device for polymer separation.

In this work, we investigated whether a series of nanopores connected by channels can be used to separate polymer mixtures by molecular size. We conducted multiscale coarse-grained simulations of semiflexible polymers driven through such a device. Polymers were modelled as chains of beads near the nanopores and as single particles in the bulk of the channels.

A mutational and molecular dynamics study of the cys-loop GABA receptor Hco-UNC-49 from Haemonchus contortus: Agonist recognition in the nematode GABA receptor family.

The UNC-49 receptor is a unique nematode γ-aminobutyric acid (GABA)-gated chloride channel that may prove to be a novel target for the development of nematocides. Here we have characterized various charged amino acid residues in and near the agonist binding site of the UNC-49 receptor from the parasitic nematode Haemonchus contorts. Utilizing the Caenorhabditis elegans GluCl crystal structure as a template, a model was generated and various charged residues [D83 (loop D), E131 (loop A), H137 (pre-loop E), R159 (Loop E), E185 (Loop B) and R241 (Loop C)] were investigated based on their location and conservation.

Morphology of depletant-induced erythrocyte aggregates.

Red blood cells suspended in quiescent plasma tend to aggregate into multicellular assemblages, including linearly stacked columnar rouleaux, which can reversibly form more complex clusters or branching networks. While these aggregates play an essential role in establishing hemorheological and pathological properties, the biophysics behind their self-assembly into dynamic mesoscopic structures remains under-explored. We employ coarse-grained molecular simulations to model low-hematocrit erythrocytes subject to short-range implicit depletion forces, and demonstrate not only that depletion interactions are sufficient to account for a sudden dispersion-aggregate transition, but also that the volume fraction of depletant macromolecules controls small aggregate morphology.

Reducing the variance in the translocation times by prestretching the polymer.

Langevin dynamics simulations of polymer translocation are performed where the polymer is stretched via two opposing forces applied on the first and last monomer before and during translocation. In this setup, polymer translocation is achieved by imposing a bias between the two pulling forces such that there is net displacement towards the trans side. Under the influence of stretching forces, the elongated polymer ensemble contains less variations in conformations compared to an unstretched ensemble.

Molecular Characterization of Binding Loop E in the Nematode Cys-Loop GABA Receptor.

Nematodes exhibit a vast array of cys-loop ligand-gated ion channels with unique pharmacologic characteristics. However, many of the structural components that govern the binding of various ligands are unknown. The nematode cys-loop GABA receptor uncoordinated 49 (UNC-49) is an important receptor found at neuromuscular junctions that plays an important role in the sinusoidal movement of worms.

Hemoglobin-driven iron-directed assembly of gold nanoparticles.

The ability to form complex 3D architectures using nanoparticles (NPs) as the building blocks and complex macromolecules that direct these assemblies remains a challenging objective for nanotechnology. Here we report results in which the partial substitution of classical Turkevich citrate-capped gold NPs by a novel, heteroaromatic ligand (L) results in NPs able to form coordination-driven assemblies mediated by free or protein-bound iron ions. The morphology of these assemblies can be tuned depending on the source of iron.

Buckling Causes Nonlinear Dynamics of Filamentous Viruses Driven through Nanopores.

Measurements and Langevin dynamics simulations of filamentous viruses driven through solid-state nanopores reveal a superlinear rise in the translocation velocity with driving force. The mobility also scales with the length of the virus in a nontrivial way that depends on the force. These dynamics are consequences of the buckling of the leading portion of a virus as it emerges from the nanopore and is put under compressive stress by the viscous forces it encounters.

DNA Translocations through Nanopores under Nanoscale Preconfinement.

To reduce unwanted variation in the passage speed of DNA through solid-state nanopores, we demonstrate nanoscale preconfinement of translocating molecules using an ultrathin nanoporous silicon nitride membrane separated from a single sensing nanopore by a nanoscale cavity. We present comprehensive experimental and simulation results demonstrating that the presence of an integrated nanofilter within nanoscale distances of the sensing pore eliminates the dependence of molecular passage time distributions on pore size, revealing a global minimum in the coefficient of variation of the passage time. These results provide experimental verification that the inter- and intramolecular passage time variation depends on the conformational entropy of each molecule prior to translocation.

Sorting polymers by size via an array of viscous posts.

DNA fragments can be sorted according to size by forcing them through an array of nanoposts. Whereas previous studies have explored solid nanoposts, this work examines nanoposts constructed out of viscous inclusions. Langevin dynamics simulations are used to study the dynamics of polymers driven through arrays of these viscous nanoposts for a range of post viscosities.

Translocation Time through a Nanopore with an Internal Cavity Is Minimal for Polymers of Intermediate Length.

The translocation of polymers through nanopores with large internal cavities bounded by two narrow pores is studied via Langevin dynamics simulations. The total translocation time is found to be a nonmonotonic function of polymer length, reaching a minimum at intermediate length, with both shorter and longer polymers taking longer to translocate. The location of the minimum is shown to shift with the magnitude of the applied force, indicating that the pore can be dynamically tuned to favor different polymer lengths.