Compartmentalization in membrane-surrounded organelles has the potential to overcome obstacles associated with the engineering of metabolic pathways, such as unwanted side reactions, accumulation of toxic intermediates, drain of intermediates out of the cell, and long diffusion distances. Strategies utilizing natural organelles suffer from the presence of endogenous pathways. In our approach, we make use of endoplasmic reticulum-derived vesicles loaded with enzymes of a metabolic pathway (“metabolic vesicles”). They are generated by fusion of synthetic peptides containing the N-terminal proline-rich and self-assembling region of the maize storage protein gamma-Zein (“Zera”) to the pathway enzymes. We have applied a strategy to integrate three enzymes of a cis,cis-muconic acid production pathway into those vesicles in yeast. Using fluorescence microscopy and cell fractionation techniques, we have proven the formation of metabolic vesicles and the incorporation of enzymes. Activities of the enzymes and functionality of the compartmentalized pathway were demonstrated in fermentation experiments.Directed self-assembly (DSA) of block copolymers (BCPs) provides a powerful tool to fabricate various 2D nanostructures. However, it still remains a challenge to extend DSA to make uniform and complex 3D nanostructures through BCP self-assembly. In this paper, we introduce a method to fabricate various nanostructures in 3D and test it using simulations. In particular, we employ dissipative particle dynamics (DPD) simulation to demonstrate that uniform multilayer nanostructures can be achieved by alternating the stacking of two “orthogonal” BCPs films, AB copolymer film and AC copolymer film, without the need to cross-link or etch any of the components. The assembly of a new layer occurs on top of the previous bottom layer, and thus the structural information from the substrate is propagated upward in the film, a process we refer to as self-directed self-assembly (SDSA). If this process is repeated many times, one can have tailored multilayer nanostructures. Furthermore, the natural (bulk) phases of the block copolymers in each layer do not need to be the same, so one can achieve complex 3D assemblies that are not possible with a single-phase 3D system. This method in conjunction with grapho (or chemo) epitaxy is able to evolve a surface pattern into a 3D nanostructure. Here we show several examples of nanostructures fabricated by this process, which include aligned cylinders, spheres on top of cylinders, and orthogonal nanomeshes. Our work should be useful for creating complex 3D nanostructures using self-assembly.Here, we report on systematic investigation of the impact of coextraction of the aqueous electrolyte and anion interference on the response of cation-selective bulk optodes. It is evident that to deliberately manage the properties of chemical sensors and to apply them in routine analysis, one should have exhaustive insight into their operation mechanism. Despite the extensive research in the field of ionophore-based optodes and numerous attempts for their practical application, the understanding of how coextraction of an aqueous electrolyte influences its response characteristics has not been developed thus far. Meanwhile, the electrolyte coextraction determines the detection limits of analogous ion-selective electrodes. A theoretical model based on phase distribution equilibrium is proposed to quantitatively describe the effect of Donnan exclusion failure on the response of polymeric plasticized optodes. The theoretical conclusions are confirmed by the results obtained with Na+/pH-selective optodes based on a neutral chromoionophore as a model system in solutions containing anions of various lipophilicities (Cl-, NO3-, I-, SCN-, and ClO4-). For the first time, it is shown that coextraction leads to a significant shift of the response range of the optodes as well as to nonmonotonic response curves due to the transition from cationic to anionic response. An approach to estimate the coextraction constants of electrolytes from the optode response curves is proposed. The limitations in the applicability of optodes due to co-ion interference are explored. It is found that neglecting anion interference can cause dramatic errors in the results of analyses with optical sensors.Calcium binding to troponin C (TnC) activates striated muscle contraction by removing TnI (troponin I) from its inhibitory site on actin. Troponin T (TnT) links TnI with tropomyosin, causing tropomyosin to move from an inhibitory position on actin to an activating position. Positive charges within the C-terminal region of human cardiac TnT limit Ca2+ activation. We now show that the positively charged region of TnT has an even larger impact on skeletal muscle regulation. We prepared one variant of human skeletal TnT that had the C-terminal 16 residues truncated (Δ16) and another with an added C-terminal Cys residue and Ala substituted for the last 6 basic residues (251C-HAHA). see more Both mutants reduced (based on S1 binding kinetics) or eliminated (based on acrylodan-tropomyosin fluorescence) the first inactive state of actin at less then 10 nM free Ca2+. 251C-HAHA-TnT and Δ16-TnT mutants greatly increased ATPase activation at 0.2 mM Ca2+, even without high-affinity cross-bridge binding. They also shifted the force-pCa curve of muscle fibers to lower Ca2+ by 0.8-1.2 pCa units (the larger shift for 251C-HAHA-TnT). Shifts in force-pCa were maintained in the presence of para-aminoblebbistatin. The effects of modification of the C-terminal region of TnT on the kinetics of S1 binding to actin were somewhat different from those observed earlier with the cardiac analogue. In general, the C-terminal region of human skeletal TnT is critical to regulation, just as it is in the cardiac system, and is a potential target for modulating activity.Biofilms are one of the most challenging obstacles in bacterial infections. By providing protection against immune responses and antibiotic therapies, biofilms enable chronic colonization and the development of antibiotic resistance. As previous clinical observations and studies have shown, traditional antibiotic therapy alone cannot effectively treat and eliminate biofilm forming infections due to the protection conferred by the biofilm. A new strategy specifically targeting biofilms must be developed. Here, we specifically target and bind to the PAO1 biofilm and elucidate the molecular mechanism behind the interaction between a glycan targeted polymer and biofilm using a continuous flow biofilm model. The incubation of biofilms with fluorescent glycan targeted polymers demonstrated strong and persistent interactions with the mannose-containing polymer even after 24 h of continuous flow. To evaluate the role of major biofilm proteins LecB and CdrA, loss of function experiments with knockout variants established the dual involvement of both proteins in mannose targeted polymer retention.