CARPs regulate STUB1 and its pathogenic mutants aggregation kinetics by mono-ubiquitination.

The development of neurological pathologies is linked to the accumulation of protein aggregates like alpha-synuclein in Parkinson's disease and tau protein in Alzheimer's disease. Mono- or di-ubiquitination of these molecules has been reported to stabilize aggregates and contribute to the disorders. STIP1 Homologous and U-Box-containing protein 1 (STUB1) is a multifunctional protein that maintains proteostasis and insulin signalling. In spinocerebellar ataxia 16 (SCAR16), an autosomal recessive neurodegenerative disease, mutations in and aggregation of STUB1 are reported. Despite the well-accepted neuroprotective role of STUB1, very little is known of regulatory mechanisms that control the dynamics of STUB1 aggregate assembly. Here, we report that CARP2, a ubiquitin ligase, is a novel regulator of STUB1. CARP2 interacts and mono-ubiquitinates STUB1. Furthermore, we found that CARP2 regulates STUB1 through its TPR motif, a domain that is also associated with HSP70. Modification of STUB1 by CARP2 leads to detergent-insoluble aggregate formation. Importantly, pathogenic mutants of STUB1 are more prone than the wild-type to CARP2-mediated aggregate assembly. Hence our findings revealed CARPs (CARP1 & CARP2) as novel regulators of STUB1 and controlled its cytosolic versus aggregate dynamics.

Endosomal-associated RFFL facilitates mitochondrial clearance by enhancing PRKN/parkin recruitment to mitochondria.

Mutations in the ubiquitin ligase PRKN (parkin RBR E3 ubiquitin protein ligase) are associated with Parkinson disease and defective mitophagy. Conceptually, PRKN-dependent mitophagy is classified into two phases: 1. PRKN recruits to and ubiquitinates mitochondrial proteins; 2. formation of phagophore membrane, sequestering mitochondria for degradation. Recently, endosomal machineries are reported to contribute to the later stage for membrane assembly. We reported a role for endosomes in the events upstream of phase 1. We demonstrate that the endosomal ubiquitin ligase RFFL (ring finger and FYVE like domain containing E3 ubiquitin protein ligase) associated with damaged mitochondria, and this association preceded that of PRKN. RFFL interacted with PRKN, and stable recruitment of PRKN to damaged mitochondria was substantially reduced in RFFL KO cells. Our study unraveled a novel role of endosomes in modulating upstream pathways of PRKN-dependent mitophagy initiation.Abbreviations CCCP: carbonyl cyanide 3-chlorophenylhydrazone; DMSO: dimethyl sulfoxide; EGFP: enhanced green fluorescence protein; KO: knockout; PRKN: parkin RBR E3 ubiquitin protein ligase; RFFL: ring finger and FYVE like domain containing E3 ubiquitin protein ligase; UQCRC1: ubiquinol-cytochrome c reductase core protein 1; WT: wild-type.

Parameterized MDPs and Reinforcement Learning Problems-A Maximum Entropy Principle-Based Framework.

We present a framework to address a class of sequential decision-making problems. Our framework features learning the optimal control policy with robustness to noisy data, determining the unknown state and action parameters, and performing sensitivity analysis with respect to problem parameters. We consider two broad categories of sequential decision-making problems modeled as infinite horizon Markov decision processes (MDPs) with (and without) an absorbing state. The central idea underlying our framework is to quantify exploration in terms of the Shannon entropy of the trajectories under the MDP and determine the stochastic policy that maximizes it while guaranteeing a low value of the expected cost along a trajectory. This resulting policy enhances the quality of exploration early on in the learning process, and consequently allows faster convergence rates and robust solutions even in the presence of noisy data as demonstrated in our comparisons to popular algorithms, such as Q -learning, Double Q -learning, and entropy regularized Soft Q -learning. The framework extends to the class of parameterized MDP and RL problems, where states and actions are parameter dependent, and the objective is to determine the optimal parameters along with the corresponding optimal policy. Here, the associated cost function can possibly be nonconvex with multiple poor local minima. Simulation results applied to a 5G small cell network problem demonstrate the successful determination of communication routes and the small cell locations. We also obtain sensitivity measures to problem parameters and robustness to noisy environment data.

Mapping the Fibril Core of the Prion Subdomain of the Mammalian CPEB3 that is Involved in Long Term Memory Retention.

Long-term memory storage is modulated by the prion nature of CPEB3 forming the molecular basis for the maintenance of synaptic facilitation. Here we report that the first prion sub-domain PRD1 of mouse CPEB3 can autonomously form amyloid fibrils in vitro and punctate-like structures in vivo. A ninety-four amino acid sequence within the PRD1 domain, PRD1-core, displays high propensity towards aggregation and associated amyloid characteristics. PRD1-core is characterized using electron microscopy, X-ray diffraction, and solution-state NMR deuterium exchange experiments. Secondary structure elements deduced from solid-state NMR reveal a ß-rich core comprising of forty amino acids at the N-terminus of PRD1-core. The synthesized twenty-three amino acid long peptide containing the longest rigid segment (E124-H145) of the PRD1-core rapidly self-aggregates and forms fibrils, indicating a limited aggregation-prone region that could potentially activate the aggregation of the full-length protein. This study provides the first step in identifying the structural trigger for the CPEB3 aggregation process.

Conformational Preferences of an Intrinsically Disordered Protein Domain: A Case Study for Modern Force Fields.

Molecular simulations of intrinsically disordered proteins (IDPs) are challenging because they require sampling a very large number of relevant conformations, corresponding to a multitude of shallow minima in a flat free energy landscape. However, in the presence of a binding partner, the free energy landscape of an IDP can be dominated by few deep minima. This characteristic imposes high demands on the accuracy of the force field used to describe the molecular interactions. Here, as a model system for an IDP that is unstructured in solution but folds upon binding to a structured interaction partner, the transactivation domain of c-Myb was studied both in the unbound (free) form and when bound to the KIX domain. Six modern biomolecular force fields were systematically tested and compared in terms of their ability to describe the structural ensemble of the IDP. The protein force field/water model combinations included in this study are AMBER ff99SB-disp with its corresponding water model that was derived from TIP4P-D, CHARMM36m with TIP3P, ff15ipq with SPC/Eb, ff99SB*-ILDNP with TIP3P and TIP4P-D, and FB15 with TIP3P-FB water. Comparing the results from REST2-enhanced sampling simulations with experimental CD spectra and secondary chemical shifts reveals that the ff99SB-disp force field can realistically capture the broad and mildly helical structural ensemble of free c-Myb. The structural ensembles yielded by CHARMM36m, ff99SB*-ILDNP together with TIP4P-D water, and FB15 are also mildly helical; however, each of these force fields can be assigned a specific subset of c-Myb residues for which the simulations could not reproduce the experimental secondary chemical shifts. In addition, microsecond-timescale MD simulations of the KIX/c-Myb complex show that most force fields used preserve a stable helix fold of c-Myb in the complex. Still, all force fields predict a KIX/c-Myb complex interface that differs slightly from the structures provided by NMR because several NOE-derived distances between KIX and c-Myb were exceeded in the simulations. Taken together, the ff99SB-disp force field in the first place but also CHARMM36m, ff99SB*-ILDNP together with TIP4P-D water, and FB15 can be suitable choices for future simulation studies of the coupled folding and binding mechanism of the KIX/c-Myb complex and potentially also other IDPs.

Vanadium pentoxide nanoparticle mediated perturbations in cellular redox balance and the paradigm of autophagy to apoptosis.

The redox-active transition metals such as copper, iron, chromium, vanadium, and silica are known for its ROS generation via mechanisms such as Haber-Weiss and Fenton-type reactions. Nanoparticles of these metals induce oxidative stress due to acellular factors owing to their small size and more reactive surface area, leading to various cellular responses. The intrinsic enzyme-like activity of nano vanadium has fascinated the scientific community. However, information concerning their cellular uptake and time-dependent induced effects on their cellular organelles and biological activity is lacking. This comprehensive study focuses on understanding the precise molecular interactions of vanadium pentoxide nanoparticles (VnNp) and evaluate their specific "nano" induced effects on MDA-MB-231 cancer cells. Understanding the mechanism behind NP-induced ROS generation could help design a model for selective NP induced toxicity, useful for cancer management. The study demonstrated the intracellular persistence of VnNp and insights into its molecular interactions with various organelles and its overall effects at the cellular level. Where triple-negative breast cancer MDA-MB-231 cells resulted in 59.6% cell death towards 48 h of treatment and the normal fibroblast cells showed only 15.4% cell death, indicating an inherent anticancer property of VnNp. It acts as an initial reactive oxygen species quencher, by serving itself as an antioxidant, while; it was also found to alter the cellular antioxidant system with prolonged incubation. The VnNp accumulated explicitly in the lysosomes and mitochondria and modulated various cellular processes including impaired lysosomal function, mitochondrial damage, and autophagy. At more extended time points, VnNp influenced cell cycle arrest, inhibited cell migration, and potentiated the onset of apoptosis. Results are indicative of the fact that VnNp selectively induced breast cancer cell death and hence could be developed as a future drug molecule for breast cancer management. This could override the most crucial challenge of chemo-resistance that still remain as the main hurdle to cancer therapy.

Lipid-dependent conformational landscape of the ErbB2 growth factor receptor dimers.

Altered lipid metabolism has been linked to cancer development and progression. Several roles have been attributed to the increased saturation and length of lipid acyl tails observed in tumors, but its effect on signaling receptors is still emerging. In this work, we have analyzed the lipid dependence of the ErbB2 growth factor receptor dimerization that plays an important role in the pathogenesis of breast cancer. We have performed coarse-grain ensemble molecular dynamics simulations to comprehensively sample the ErbB2 monomer-dimer association. Our results indicate a dynamic dimer state with a complex conformational landscape that is modulated with increasing lipid tail length. We resolve the native N-terminal "active" and C-terminal "inactive" conformations in all membrane compositions. However, the relative population of the N-terminal and C-terminal conformers is dependent on length of the saturated lipid tails. In short-tail membranes, additional non-specific dimers are observed which are reduced or absent in long-tailed bilayers. Our results indicate that the relative population as well as the structure of the dimer state is modulated by membrane composition. We have correlated these differences to local perturbations of the membrane around the receptor. Our work is an important step in characterizing ErbB dimers in healthy and diseased states and emphasize the importance of sampling lipid dynamics in understanding receptor association.

Naturally occurring and tumor-associated variants of RNF167 promote lysosomal exocytosis and plasma membrane resealing.

Lysosomal exocytosis and resealing of damaged plasma membrane are essential for cellular homeostasis and tumor invasion. However, very little is known of the molecular machinery that regulates these physiological processes. Moreover, no mutations in any of the known regulators of lysosomal exocytosis in primary tumors of patients have been characterized. Here we demonstrate that RNF167-a, a lysosomal-associated ubiquitin ligase, negatively regulates lysosomal exocytosis by inducing perinuclear clustering of lysosomes. Importantly, we also characterized a set of novel natural mutations in RNF167-a, which are commonly found in diverse tumor types. We found that RNF167-a-K97N mutant, unlike the wild type, localizes in the cytoplasm and does not promote perinuclear lysosomal clustering. Furthermore, cells expressing RNF167-a-K97N exhibit dispersed lysosomes, increased exocytosis and enhanced plasma membrane repair. Interestingly, these functional features of RNF167-a-K97N were shared with a naturally occurring short version of RNF167 (isoform RNF167-b). In brief, the results presented here reveal a novel role of RNF167-a, as well as its natural variants RNF167-a-K97N and RNF167-b, as an upstream regulator of lysosomal exocytosis and plasma membrane resealing.

Efficacy and safety of evogliptin versus sitagliptin as an add-on therapy in Indian patients with type 2 diabetes mellitus inadequately controlled with metformin: A 24-week randomized, double-blind, non-inferiority, EVOLUTION INDIA study.

AIM: This study aimed to assess efficacy and safety of evogliptin versus sitagliptin, when added to background metformin therapy in Indian patients with uncontrolled type 2 diabetes. METHOD: Overall, 184 patients with uncontrolled type 2 diabetes (7% ≤ HbA1c < 10%) receiving ≥8 weeks of stable metformin monotherapy (≥1 g/day), were randomized to receive add-on treatment (evogliptin 5 mg or sitagliptin 100 mg) for 24 weeks. Primary endpoint was change in HbA1c from baseline to 12 weeks (non-inferiority margin: <0.35). RESULTS: Mean reductions in HbA1c at 12 weeks in evogliptin- and sitagliptin-treated patients were -0.37 (1.06) and -0.32 (1.14), respectively. The adjusted mean difference between treatment groups was -0.022 (95% CI: -0.374, 0.330; P = 0.901), that demonstrated non-inferiority. Reductions in FPG and PPG were similar between evogliptin and sitagliptin at 12 and 24 weeks. Changes in body weight were comparable between the treatment groups. Patients achieving target HbA1c < 7.0% (evogliptin, 26.7% vs. sitagliptin, 20%) was almost equal in both groups. Treatment-emergent adverse events occured in 52 patients (evogliptin, 25% and sitagliptin, 31.5%) and were generally mild. CONCLUSIONS: Evogliptin was non-inferior to sitagliptin in HbA1c reduction. It effectively improved glycemic control and was well tolerated in type 2 diabetes patients inadequately controlled by metformin alone.

Solvent effects on ligand binding to a serine protease.

Solvation plays an important role in virtually all biomolecular recognition and binding processes. However, the consequences of changes in solvation conditions often remain elusive. In this work, we combined isothermal titration calorimetry (ITC) and molecular dynamics (MD) simulations to investigate the effect of solvent composition on the thermodynamics of protein-ligand binding. We studied the binding of p-aminobenzamidine (PAB) to trypsin in various water/methanol mixtures as a model system for a biomolecular complex. Our ITC experiments show that the free energy of binding changes only very modestly with methanol concentration, and that this small change is due to strong enthalpy-entropy compensation. The MD and free energy simulations not only reproduce the experimental binding free energies, but also provide atomic-level insights into the mechanisms behind the thermodynamic observations. The more favorable binding enthalpy at increased methanol concentrations (when compared to pure water) is attributed to stronger protein-ligand and intramolecular protein-protein interactions. The stronger protein-ligand interaction is linked to a small-scale conformational rearrangement of the L2 binding pocket loop, which senses the solvent environment. Remarkably, the stronger interactions do not substantially reduce the configurational entropy of the protein. Instead, the more disfavorable entropy contribution to the binding free energy at increased methanol concentrations is due to the desolvation of the ligand from the bulk, which is more favorable in pure aqueous solution than in the presence of methanol. Our work thus underpins the importance of including conformational flexibility, even for an overall rather rigid complex, since even small-amplitude motions can significantly alter the binding energetics. Furthermore, the ability of our combined ITC/MD approach to assign different thermodynamic contributions to distinct conformational states might contribute to an enhanced understanding of biomolecular binding processes in general.

Robust atomic force microscopy using multiple sensors.

Atomic force microscopy typically relies on high-resolution high-bandwidth cantilever deflection measurements based control for imaging and estimating sample topography and properties. More precisely, in amplitude-modulation atomic force microscopy (AM-AFM), the control effort that regulates deflection amplitude is used as an estimate of sample topography; similarly, contact-mode AFM uses regulation of deflection signal to generate sample topography. In this article, a control design scheme based on an additional feedback mechanism that uses vertical z-piezo motion sensor, which augments the deflection based control scheme, is proposed and evaluated. The proposed scheme exploits the fact that the piezo motion sensor, though inferior to the cantilever deflection signal in terms of resolution and bandwidth, provides information on piezo actuator dynamics that is not easily retrievable from the deflection signal. The augmented design results in significant improvements in imaging bandwidth and robustness, especially in AM-AFM, where the complicated underlying nonlinear dynamics inhibits estimating piezo motions from deflection signals. In AM-AFM experiments, the two-sensor based design demonstrates a substantial improvement in robustness to modeling uncertainties by practically eliminating the peak in the sensitivity plot without affecting the closed-loop bandwidth when compared to a design that does not use the piezo-position sensor based feedback. The contact-mode imaging results, which use proportional-integral controllers for cantilever-deflection regulation, demonstrate improvements in bandwidth and robustness to modeling uncertainties, respectively, by over 30% and 20%. The piezo-sensor based feedback is developed using H∞ control framework.

On Using Atomistic Solvent Layers in Hybrid All-Atom/Coarse-Grained Molecular Dynamics Simulations.

Hybrid all-atom/coarse-grained (AA-CG) simulations in which AA solutes are embedded in a CG environment can provide a significant computational speed-up over conventional fully atomistic simulations and thus alleviate the current length and time scale limitations of molecular dynamics (MD) simulations of large biomolecular systems. On one hand, coarse graining the solvent is particularly appealing, since it typically constitutes the largest part of the simulation system and thus dominates computational cost. On the other hand, retaining atomic-level solvent layers around the solute is desirable for a realistic description of hydrogen bonds and other local solvation effects. Here, we devise and systematically validate fixed resolution AA-CG schemes, both with and without atomistic water layers. To quantify the accuracy and diagnose possible pitfalls, Gibbs free energies of solvation of amino acid side chain analogues were calculated, and the influence of the nature of the CG solvent surrounding (polarizable vs nonpolarizable CG water) and the size of the AA solvent region was investigated. We show that distance restraints to keep the AA solvent around the solute lead to too high of a density in the inner shell. Together with a long-ranged effect due to orientational ordering of water molecules at the AA-CG boundary, this affects solvation free energies. Shifting the onset of the distance restraints slightly away from the central solute significantly improves solvation free energies, down to mean unsigned errors with respect to experiment of 2.3 and 2.6 kJ/mol for the polarizable and nonpolarizable CG water surrounding, respectively. The speed-up of the nonpolarizable model renders it computationally more attractive. The present work thus highlights challenges, and outlines possible solutions, involved with modeling the boundary between different levels of resolution in hybrid AA-CG simulations.

Fast and robust control of nanopositioning systems: Performance limits enabled by field programmable analog arrays.

This paper aims at control design and its implementation for robust high-bandwidth precision (nanoscale) positioning systems. Even though modern model-based control theoretic designs for robust broadband high-resolution positioning have enabled orders of magnitude improvement in performance over existing model independent designs, their scope is severely limited by the inefficacies of digital implementation of the control designs. High-order control laws that result from model-based designs typically have to be approximated with reduced-order systems to facilitate digital implementation. Digital systems, even those that have very high sampling frequencies, provide low effective control bandwidth when implementing high-order systems. In this context, field programmable analog arrays (FPAAs) provide a good alternative to the use of digital-logic based processors since they enable very high implementation speeds, moreover with cheaper resources. The superior flexibility of digital systems in terms of the implementable mathematical and logical functions does not give significant edge over FPAAs when implementing linear dynamic control laws. In this paper, we pose the control design objectives for positioning systems in different configurations as optimal control problems and demonstrate significant improvements in performance when the resulting control laws are applied using FPAAs as opposed to their digital counterparts. An improvement of over 200% in positioning bandwidth is achieved over an earlier digital signal processor (DSP) based implementation for the same system and same control design, even when for the DSP-based system, the sampling frequency is about 100 times the desired positioning bandwidth.

Model based control of dynamic atomic force microscope.

A model-based robust control approach is proposed that significantly improves imaging bandwidth for the dynamic mode atomic force microscopy. A model for cantilever oscillation amplitude and phase dynamics is derived and used for the control design. In particular, the control design is based on a linearized model and robust H(∞) control theory. This design yields a significant improvement when compared to the conventional proportional-integral designs and verified by experiments.

Saturation oxygen pressure index for assessment of pulmonary disease in neonates on non-invasive ventilation.

This prospective observational study on 36 neonates aimed to estimate the correlation between the new Saturation Oxygen distending Pressure Index (SOPI) and Oxygenation index. SOPI had high correlation (r=0.94) with oxygenation index. SOPI of <2, 2, and 3.7 represented mild, moderate and severe pulmonary disease, respectively with high sensitivity and specificity.

Systematic evaluation of bundled SPC water for biomolecular simulations.

In bundled SPC water models, the relative motion of groups of four water molecules is restrained by distance-dependent potentials. Bundled SPC models have been used in hybrid all-atom/coarse-grained (AA/CG) multiscale simulations, since they enable to couple atomistic SPC water with supra-molecular CG water models that effectively represent more than a single water molecule. In the present work, we systematically validated and critically tested bundled SPC water models as solvent for biomolecular simulations. To that aim, we investigated both thermodynamic and structural properties of various biomolecular systems through molecular dynamics (MD) simulations. Potentials of mean force of dimerization of pairs of amino acid side chains as well as hydration free energies of single side chains obtained with bundled SPC and standard (unrestrained) SPC water agree closely with each other and with experimental data. Decomposition of the hydration free energies into enthalpic and entropic contributions reveals that in bundled SPC, this favorable agreement of the free energies is due to a larger degree of error compensation between hydration enthalpy and entropy. The Ramachandran maps of Ala3, Ala5, and Ala7 peptides are similar in bundled and unrestrained SPC, whereas for the (GS)2 peptide, bundled water leads to a slight overpopulation of extended conformations. Analysis of the end-to-end distance autocorrelation times of the Ala5 and (GS)2 peptides shows that sampling in more viscous bundled SPC water is about two times slower. Pronounced differences between the water models were found for the structure of a coiled-coil dimer, which is instable in bundled SPC but not in standard SPC. In addition, the hydration of the active site of the serine protease α-chymotrypsin depends on the water model. Bundled SPC leads to an increased hydration of the active site region, more hydrogen bonds between water and catalytic triad residues, and a significantly slower exchange of water molecules between the active site and the bulk. Our results form a basis for assessing the accuracy that can be expected from bundled SPC water models. At the same time, this study also highlights the importance of evaluating beforehand the effects of water bundling on the biomolecular system of interest for a particular multiscale simulation application.

Thermodynamic and kinetic characterization of transmembrane helix association.

The transient dimerization of transmembrane proteins is an important event in several cellular processes and computational methods are being increasingly used to quantify their underlying energetics. Here, we probe the thermodynamics and kinetics of a simple transmembrane dimer to understand membrane protein association. A multi-step framework has been developed in which the dimerization profiles are calculated from coarse-grain molecular dynamics simulations, followed by meso-scale simulations using parameters calculated from the coarse-grain model. The calculated value of ΔGassoc is approx. -20 kJ mol(-1) and is consistent between three methods. Interestingly, the meso-scale stochastic model reveals low dimer percentages at physiologically-relevant concentrations, despite a favorable ΔGassoc. We identify generic driving forces arising from the protein backbone and lipid bilayer and complementary factors, such as protein density, that govern self-interactions in membranes. Our results provide an important contribution in understanding membrane protein organization and linking molecular, nano-scale computational studies to meso-scale experimental data.

Formulation approaches to improving the delivery of an antiviral drug with activity against seasonal flu.

The main objective of the present study was to develop formulations of noscapine hydrochloride hydrate with enhanced solubility and bioavailability using co-solvent- and cyclodextrin-based approaches. Different combinations of co-solvents, which were selected on the basis of high-throughput solubility screening, were subjected to in vitro intestinal drug permeability studies conducted with Ussing chambers. Vitamin E tocopherol polyethylene glycol succinate and propylene glycol based co-solvent formulations provided the maximum permeability coefficient for the drug. Inclusion complexes of the drug were prepared using hydroxypropyl-ß-cyclodextrin and sulphobutylether cyclodextrins. Pharmacokinetic studies were carried out in male Sprague-Dawley rats for the selected formulations. The relative bioavailabilities of the drug with the co-solvent- and cyclodextrin-based formulations were found to be similar.

The power of coarse graining in biomolecular simulations.

Computational modeling of biological systems is challenging because of the multitude of spatial and temporal scales involved. Replacing atomistic detail with lower resolution, coarse grained (CG), beads has opened the way to simulate large-scale biomolecular processes on time scales inaccessible to all-atom models. We provide an overview of some of the more popular CG models used in biomolecular applications to date, focusing on models that retain chemical specificity. A few state-of-the-art examples of protein folding, membrane protein gating and self-assembly, DNA hybridization, and modeling of carbohydrate fibers are used to illustrate the power and diversity of current CG modeling.