Autopopulus: A Novel Framework for Autoencoder Imputation on Large Clinical Datasets.

The adoption of electronic health records (EHRs) has made patient data increasingly accessible, precipitating the development of various clinical decision support systems and data-driven models to help physicians. However, missing data are common in EHR-derived datasets, which can introduce significant uncertainty, if not invalidating the use of a predictive model. Machine learning (ML)-based imputation methods have shown promise in various domains for the task of estimating values and reducing uncertainty to the point that a predictive model can be employed. We introduce Autopopulus, a novel framework that enables the design and evaluation of various autoencoder architectures for efficient imputation on large datasets. Autopopulus implements existing autoencoder methods as well as a new technique that outputs a range of estimated values (rather than point estimates), and demonstrates a workflow that helps users make an informed decision on an appropriate imputation method. To further illustrate Autopopulus' utility, we use it to identify not only which imputation methods can most accurately impute on a large clinical dataset, but to also identify the imputation methods that enable downstream predictive models to achieve the best performance for prediction of chronic kidney disease (CKD) progression.

Efficient synthesis of antiviral agent uprifosbuvir enabled by new synthetic methods.

An efficient route to the HCV antiviral agent uprifosbuvir was developed in 5 steps from readily available uridine in 50% overall yield. This concise synthesis was achieved by development of several synthetic methods: (1) complexation-driven selective acyl migration/oxidation; (2) BSA-mediated cyclization to anhydrouridine; (3) hydrochlorination using FeCl3/TMDSO; (4) dynamic stereoselective phosphoramidation using a chiral nucleophilic catalyst. The new route improves the yield of uprifosbuvir 50-fold over the previous manufacturing process and expands the tool set available for synthesis of antiviral nucleotides.

Quantitative Performance Characterization of Radiation Dose for the Carestream CS9600 Cone-Beam Computed Tomography Machine.

INTRODUCTION: Cone-beam computed tomography (CBCT) machines produce relatively low levels of harmful ionizing radiation, as compared with the computed tomography devices used in medical practices. The Carestream CS9600 CBCT imaging device has been recently introduced into the marketplace, and the manufacturer reports the use of an increased x-ray tube voltage (120 kVp) for the device, along with a reduced patient dose that is achieved using added filtration. Independent dosimetry studies are performed to ensure appropriate radiation exposure dose levels are within recommended safety guidelines.The purpose of this study is to independently evaluate and measure the radiation exposure dose performance parameters of the CS9600 CBCT, including its multiple field of view, exposure settings, and filtration options. METHODS: A thimble ionization chamber and PMMA phantom were used to characterize dose index using the established SEDENTEXTCT evaluation method. RESULTS: The phantom-obtained radiation dose index measures ranged from 0.128782-13.848 milligrays (mGy) for the various scanning options evaluated. The field of view, type of filter used, and phantom size all had a direct impact on the relationship between the experimentally obtained dose index measures and the dose area product values reported by the manufacturer. CONCLUSIONS: A strong linear correlation was observed between the experimentally obtained dose index measures and the manufacturer-reported dose area product values. The 0.7 mm Cu filter that has been added to the CS9600 reduced the exposure dose index measures even with the x-ray tube kilovoltage peak (kVp) being increased to 120 kVp, as compared with the 0.15 mm Cu filter at 90 kVp.

Capillary electrophoresis analysis of affinity to assess carboxylation of multi-walled carbon nanotubes.

Surface oxidation improves the dispersion of carbon nanotubes in aqueous solutions and plays a key role in the development of biosensors, electrochemical detectors and polymer composites. Accurate characterization of the carbon nanotube surface is important because the development of these nano-based applications depends on the degree of functionalization, in particular the amount of carboxylation. Affinity capillary electrophoresis is used to characterize the oxidation of multi-walled carbon nanotubes. A polytryptophan peptide that contains a single arginine residue (WRWWWW) serves as a receptor in affinity capillary electrophoresis to assess the degree of carboxylation. The formation of peptide-nanotube receptor-ligand complex was detected with a UV absorbance detector. Apparent dissociation constants (KD) are obtained by observing the migration shift of the WRWWWW peptide through background electrolyte at increasing concentrations of multi-walled carbon nanotubes. A 20% relative standard deviation in method reproducibility and repeatability is determined with triplicate analysis within a single sample preparation and across multiple sample preparations for a commercially available carbon nanotube. Affinity capillary electrophoresis is applied to assess differences in degree of carboxylation across two manufacturers and to analyze acid treated carbon nanotubes. The results of these studies are compared to X-ray photoelectron spectroscopy and zeta potential. Affinity capillary electrophoresis comparisons of carbon nanotube samples prepared by varying acid treatment time from 30 min to 3 h yielded significant differences in degree of carboxylation. X-ray photoelectron spectroscopy analysis was inconclusive due to potential acid contamination, while zeta potential showed no change based on surface charge. This work is significant to research involving carbon nanotube-based applications because it provides a new metric to rapidly characterize carbon nanotubes obtained from different vendors, or synthesized in laboratories using different procedures.

Peptide Probe for Multiwalled Carbon Nanotubes: Electrophoretic Assessment of the Binding Interface and Evaluation of Surface Functionalization.

Noncovalent interactions of peptides and proteins with carbon nanotubes play a key role in sensing, dispersion, and biocompatibility. Advances in these areas require that the forces which contribute to physical adsorption are understood in order that the carbon nanotubes present a degree of functionalization appropriate to the desired application. Affinity analyses of peptides are employed to evaluate the role of tryptophan and arginine residues in physical adsorption to carboxylated multiwalled carbon nanotubes. Peptides containing arginine and tryptophan, WR(W) n, are used with affinity capillary electrophoresis to identify factors that lead to the formation of peptide-carbon nanotube complexes. The effects of changing the amino acid composition and residue length are evaluated by measuring dissociation constants. Electrostatic interactions contribute significantly to complexation, with the strongest interaction observed using the peptide WRWWWW and carboxylated carbon nanotube. Stronger interaction is observed when the tryptophan content is successively increased as follows: WR(W)4 > WR(W)3 > WR(W)2 > WRW > WR. However, as observed with polytryptophan (W5, W4, W3, and W2), removing the arginine residue significantly reduces the interaction with carbon nanotubes. Increasing the arginine content to WRWWRW does not improve binding, whereas replacing the arginine residue in WRWWWW with lysine (WKWWWW) reveals that lysine also contributes to surface adsorption, but not as effectively as arginine. These observations are used to guide a search of the primary sequence of lysozyme to identify short regions in the peptide that contain a single cationic residue and two aromatic residues. One candidate peptide sequence (WMCLAKW) from this search is analyzed by capillary electrophoresis. The dissociation constant of carboxylated multiwalled carbon nanotubes is measured for the peptide, WMCLAKW, to demonstrate the utility of affinity capillary electrophoresis analysis.

Rhodium(III)-Catalyzed C-H Activation: An Oxidative Intramolecular Heck-Type Reaction Directed by a Carboxylic Acid.

Carboxylic acids effectively direct C-H activation for Rhodium(III)-catalyzed intramolecular Heck-type reactions. A catalytic amount of Cu(OAc)2 is used as the external oxidant with oxygen likely acting as the terminal oxidant. Additionally, a novel electron-deficient RhIII catalyst was found to be more effective that [RhCp*Cl2]2 with some substrates. A wide variety of complex dihydrobenzofurans, dihydrobenzopyrans, and other bicycles that can be easily further functionalized are now accessible through relatively mild reaction conditions.

Mdm2 and aurora kinase a inhibitors synergize to block melanoma growth by driving apoptosis and immune clearance of tumor cells.

Therapeutics that induce cancer cell senescence can block cell proliferation and promote immune rejection. However, the risk of tumor relapse due to senescence escape may remain high due to the long lifespan of senescent cells that are not cleared. Here, we show how combining a senescence-inducing inhibitor of the mitotic kinase Aurora A (AURKA) with an MDM2 antagonist activates p53 in senescent tumors harboring wild-type 53. In the model studied, this effect is accompanied by proliferation arrest, mitochondrial depolarization, apoptosis, and immune clearance of cancer cells by antitumor leukocytes in a manner reliant upon Ccl5, Ccl1, and Cxcl9. The AURKA/MDM2 combination therapy shows adequate bioavailability and low toxicity to the host. Moreover, the prominent response of patient-derived melanoma tumors to coadministered MDM2 and AURKA inhibitors offers a sound rationale for clinical evaluation. Taken together, our work provides a preclinical proof of concept for a combination treatment that leverages both senescence and immune surveillance to therapeutic ends.

Organocatalytic, diastereo- and enantioselective synthesis of nonsymmetric cis-stilbene diamines: a platform for the preparation of single-enantiomer cis-imidazolines for protein-protein inhibition.

The finding by scientists at Hoffmann-La Roche that cis-imidazolines could disrupt the protein-protein interaction between p53 and MDM2, thereby inducing apoptosis in cancer cells, raised considerable interest in this scaffold over the past decade. Initial routes to these small molecules (i.e., Nutlin-3) provided only the racemic form, with enantiomers being enriched by chromatographic separation using high-pressure liquid chromatography (HPLC) and a chiral stationary phase. Reported here is the first application of an enantioselective aza-Henry approach to nonsymmetric cis-stilbene diamines and cis-imidazolines. Two novel mono(amidine) organocatalysts (MAM) were discovered to provide high levels of enantioselection (>95% ee) across a broad range of substrate combinations. Furthermore, the versatility of the aza-Henry strategy for preparing nonsymmetric cis-imidazolines is illustrated by a comparison of the roles of aryl nitromethane and aryl aldimine in the key step, which revealed unique substrate electronic effects providing direction for aza-Henry substrate-catalyst matching. This method was used to prepare highly substituted cis-4,5-diaryl imidazolines that project unique aromatic rings, and these were evaluated for MDM2-p53 inhibition in a fluorescence polarization assay. The diversification of access to cis-stilbene diamine-derived imidazolines provided by this platform should streamline their further development as chemical tools for disrupting protein-protein interactions.

Nanoparticle-protein interactions: a thermodynamic and kinetic study of the adsorption of bovine serum albumin to gold nanoparticle surfaces.

Investigating the adsorption process of proteins on nanoparticle surfaces is essential to understand how to control the biological interactions of functionalized nanoparticles. In this work, a library of spherical and rod-shaped gold nanoparticles (GNPs) was used to evaluate the process of protein adsorption to their surfaces. The binding of a model protein (bovine serum albumin, BSA) to GNPs as a function of particle shape, size, and surface charge was investigated. Two independent comparative analytical methods were used to evaluate the adsorption process: steady-state fluorescence quenching titration and affinity capillary electrophoresis (ACE). Although under favorable electrostatic conditions kinetic analysis showed a faster adsorption of BSA to the surface of cationic GNPs, equilibrium binding constant determinations indicated that BSA has a comparable binding affinity to all of the GNPs tested, regardless of surface charge. BSA was even found to adsorb strongly to GNPs with a pegylated/neutral surface. However, these fluorescence titrations suffer from significant interference from the strong light absorption of the GNPs. The BSA-GNP equilibrium binding constants, as determined by the ACE method, were 10(5) times lower than values determined using spectroscopic titrations. While both analytical methods could be suitable to determine the binding constants for protein adsorption to NP surfaces, both methods have limitations that complicate the determination of protein-GNP binding constants. The optical properties of GNPs interfere with Ka determinations by static fluorescence quenching analysis. ACE, in contrast, suffers from material compatibility issues, as positively charged GNPs adhere to the walls of the capillary during analysis. Researchers seeking to determine equilibrium binding constants for protein-GNP interactions should therefore utilize as many orthogonal techniques as possible to study a protein-GNP system.

Preparation of (-)-Nutlin-3 using enantioselective organocatalysis at decagram scale.

Chiral nonracemic cis-4,5-bis(aryl)imidazolines have emerged as a powerful platform for the development of cancer chemotherapeutics, stimulated by the Hoffmann-La Roche discovery that Nutlin-3 can restore apoptosis in cells with wild-type p53. The lack of efficient methods for the enantioselective synthesis of cis-imidazolines, however, has limited their more general use. Our disclosure of the first enantioselective synthesis of (-)-Nutlin-3 provided a basis to prepare larger amounts of this tool used widely in cancer biology. Key to the decagram-scale synthesis described here was the discovery of a novel bis(amidine) organocatalyst that provides high enantioselectivity at warmer reaction temperature (-20 °C) and low catalyst loadings. Further refinements to the procedure led to the synthesis of (-)-Nutlin-3 in a 17 g batch and elimination of all but three chromatographic purifications.

Investigations on ß1,4-galactosyltransferase I using 6-sulfo-GlcNAc as an acceptor sugar substrate.

6-sulfate modified N-acetylglucosamine (6-sulfo-GlcNAc) is often found as part of many biologically important carbohydrate epitopes such as 6-sulfo-Le(X). In these epitopes, the 6-sulfo-GlcNAc moiety is extended by a galactose sugar in a ß1-4 linkage. The ß4GalT1 enzyme transfers galactose (Gal) from UDP-Gal to N-acetylglucosamine (GlcNAc) in the presence of manganese. Here we report that the ß4GalT1 enzyme transfers Gal to the 6-sulfo-GlcNAc and 4-methylumbelliferyl-6-sulfo-N-acetyl-ß-D-glucosaminide (6-sulfo-ßGlcNAc-MU) acceptor substrates, although with very low efficiency. To understand the effect that the 6-sulfate group on the GlcNAc acceptor has on the catalytic activity of the ß4GalT1 molecule, we have determined the crystal structure of the catalytic domain of bovine ß4GalT1 mutant enzyme M344H-ß4GalT1 complex with the 6-sulfo-GlcNAc molecule. In the crystal structure, the 6-sulfo-GlcNAc is bound to the protein in a way that is similar to the GlcNAc molecule. However, the 6-sulfate group engages in additional interactions with the hydrophobic region, residues 276-285, of the protein molecule, and this group is found wedged between the aromatic side chains of Phe-280 and Trp314 residues. Since the side chain of the Trp314 residue undergoes conformational changes during the catalytic cycle of the enzyme, molecular interaction between Trp314 and the 6-sulfate group might hinder this conformational change. Therefore, the lack of a favorable binding environment, together with hindrance to the conformational changes, might be responsible for the poor catalytic activity.

Chiral proton catalysis of secondary nitroalkane additions to azomethine: synthesis of a potent GlyT1 inhibitor.

The first enantioselective synthesis of a potent GlyT1 inhibitor is described. A 3-nitroazetidine donor is used in an enantioselective aza-Henry reaction catalyzed by a bis(amidine)-triflic acid salt organocatalyst, delivering the key intermediate with 92% ee. This adduct is reductively denitrated and converted to the target through a short sequence, thereby allowing assignment of the absolute configuration of the more potent enantiomer.

Catalytic, Enantioselective Synthesis of Stilbene cis-Diamines: A Concise Preparation of (-)-Nutlin-3, a Potent p53/MDM2 Inhibitor.

The first highly diastereo- and enantioselective additions of aryl nitromethane pronucleophiles to aryl aldimines are described. Identification of an electron rich chiral Bis(Amidine) catalyst for this aza-Henry variant was key to this development, leading ultimately to differentially protected cis-stilbene diamines in two steps. This method then became the lynchpin for an enantioselective synthesis of (-)-Nutlin-3 (Hoffmann-LaRoche), a potent cis-imidazoline small molecule inhibitor of p53-MDM2 used extensively as a probe of cell biology and currently in drug development.

Geometric restraint drives on- and off-pathway catalysis by the Escherichia coli menaquinol:fumarate reductase.

Complex II superfamily members catalyze the kinetically difficult interconversion of succinate and fumarate. Due to the relative simplicity of complex II substrates and their similarity to other biologically abundant small molecules, substrate specificity presents a challenge in this system. In order to identify determinants for on-pathway catalysis, off-pathway catalysis, and enzyme inhibition, crystal structures of Escherichia coli menaquinol:fumarate reductase (QFR), a complex II superfamily member, were determined bound to the substrate, fumarate, and the inhibitors oxaloacetate, glutarate, and 3-nitropropionate. Optical difference spectroscopy and computational modeling support a model where QFR twists the dicarboxylate, activating it for catalysis. Orientation of the C2-C3 double bond of activated fumarate parallel to the C(4a)-N5 bond of FAD allows orbital overlap between the substrate and the cofactor, priming the substrate for nucleophilic attack. Off-pathway catalysis, such as the conversion of malate to oxaloacetate or the activation of the toxin 3-nitropropionate may occur when inhibitors bind with a similarly activated bond in the same position. Conversely, inhibitors that do not orient an activatable bond in this manner, such as glutarate and citrate, are excluded from catalysis and act as inhibitors of substrate binding. These results support a model where electronic interactions via geometric constraint and orbital steering underlie catalysis by QFR.

Bifunctional asymmetric catalysis: amplification of Brønsted basicity can orthogonally increase the reactivity of a chiral Brønsted acid.

The reactivity of a series of symmetrical chiral Brønsted acids (polar ionic hydrogen-bond donors) follows the counterintuitive trend wherein the more Brønsted basic member is a more effective catalyst for the aza-Henry (nitro-Mannich) reaction. This new design element leads to a substantially more reactive catalyst for the aza-Henry reaction, one that can promote the addition of a secondary nitroalkane. Additionally, when an achiral Brønsted acid (TfOH) is used in slight excess of the neutral, chiral bisamidine ligand, diastereoselection can be optimized to levels generally greater than 15:1 while the enantioselection remains unchanged at generally >90% ee.