Conversion of a polysaccharide to nitric oxide-releasing form. Dual-mechanism anticoagulant activity of diazeniumdiolated heparin

We describe heparin/diazeniumdiolate conjugates that generate nitric oxide (NO) at physiological pH. Like the heparin from which they were prepared, they inhibit thrombin-induced blood coagulation. Unlike heparin, they can also inhibit and reverse ADP-induced platelet aggregation (as expected for an NO-releasing agent), suggesting potential utility as dual-action antithrombotics.     

C-terminal functionalization of nylon-3 polymers: effects of C-terminal groups on antibacterial and hemolytic activities

Nylon-3 polymers contain β-amino-acid-derived subunits and can be viewed as higher homologues of poly(α-amino acids). This structural relationship raises the possibility that nylon-3 polymers offer a platform for development of new materials with a variety of biological activities, a prospect that has recently begun to receive experimental support. Nylon-3 homo- and copolymers can be prepared via anionic ring-opening polymerization of β-lactams, and use of an N-acyl-β-lactam as coinitiator in the polymerization reaction allows placement of a specific functional group, borne by the N-acyl-β-lactam, at the N-terminus of each polymer chain. Controlling the unit at the C-termini of nylon-3 polymer chains, however, has been problematic. Here we describe a strategy for specifying C-terminal functionality that is based on the polymerization mechanism. After the anionic ring-opening polymerization is complete, we introduce a new β-lactam, approximately 1 equiv relative to the expected number of polymer chains. Because the polymer chains bear a reactive imide group at their C-termini, this new β-lactam should become attached at this position. If the terminating β-lactam bears a distinctive functional group, that functionality should be affixed to most or all C-termini in the reaction mixture. We use the new technique to compare the impact of N- and C-terminal placement of a critical hydrophobic fragment on the biological activity profile of nylon-3 copolymers. The synthetic advance described here should prove to be generally useful for tailoring the properties of nylon-3 materials.     

A Quadrupole Dalton-based multi-attribute method for product characterization,process development,and quality control of therapeutic proteins

During manufacturing and storage process, therapeutic proteins are subject to various post-translational modifications (PTMs), such as isomerization, deamidation, oxidation, disulfide bond modifications and glycosylation. Certain PTMs may affect bioactivity, stability or pharmacokinetics and pharmacodynamics profile and are therefore classified as potential critical quality attributes (pCQAs). Identifying, monitoring and controlling these PTMs are usually key elements of the Quality by Design (QbD) approach. Traditionally, multiple analytical methods are utilized for these purposes, which is time consuming and costly. In recent years, multi-attribute monitoring methods have been developed in the biopharmaceutical industry. However, these methods combine high-end mass spectrometry with complicated data analysis software, which could pose difficulty when implementing in a quality control (QC) environment. Here we report a multi-attribute method (MAM) using a Quadrupole Dalton (QDa) mass detector to selectively monitor and quantitate PTMs in a therapeutic monoclonal antibody. The result output from the QDa-based MAM is straightforward and automatic. Evaluation results indicate this method provides comparable results to the traditional assays. To ensure future application in the QC environment, this method was qualified according to the International Conference on Harmonization (ICH) guideline and applied in the characterization of drug substance and stability samples. The QDa-based MAM is shown to be an extremely useful tool for product and process characterization studies that facilitates facile understanding of process impact on multiple quality attributes, while being QC friendly and cost-effective.     

Different signaling roles of two conserved residues in the cytoplasmic hairpin tip of Tsr, the Escherichia coli serine chemoreceptor

Bacterial chemoreceptors form ternary signaling complexes with the histidine kinase CheA through the coupling protein CheW. Receptor complexes in turn cluster into cellular arrays that produce highly sensitive responses to chemical stimuli. In Escherichia coli, receptors of different types form mixed trimer-of-dimers signaling teams through the tips of their highly conserved cytoplasmic domains. To explore the possibility that the hairpin loop at the tip of the trimer contact region might promote interactions with CheA or CheW, we constructed and characterized mutant receptors with amino acid replacements at the two nearly invariant hairpin charged residues of Tsr: R388, the most tip-proximal trimer contact residue, and E391, the apex residue of the hairpin turn. Mutant receptors were subjected to in vivo tests for the assembly and function of trimers, ternary complexes, and clusters. All R388 replacements impaired or destroyed Tsr function, apparently through changes in trimer stability or geometry. Large-residue replacements locked R388 mutant ternary complexes in the kinase-off (F, H) or kinase-on (W, Y) signaling state, suggesting that R388 contributes to signaling-related conformational changes in the trimer. In contrast, most E391 mutants retained function and all formed ternary signaling complexes efficiently. Hydrophobic replacements of any size (G, A, P, V, I, L, F, W) caused a novel phenotype in which the mutant receptors produced rapid switching between kinase-on and -off states, indicating that hairpin tip flexibility plays an important role in signal state transitions. These findings demonstrate that the receptor determinants for CheA and CheW binding probably lie outside the hairpin tip of the receptor signaling domain.     

Lidocaine block of cardiac sodium channels

Lidocaine block of cardiac sodium channels was studied in voltage-clamped rabbit purkinje fibers at drug concentrations ranging from 1 mM down to effective antiarrhythmic doses (5-20 μM). Dose-response curves indicated that lidocaine blocks the channel by binding one-to-one, with a voltage-dependent K(d). The half-blocking concentration varied from more than 300 μM, at a negative holding potential where inactivation was completely removed, to approximately 10 μM, at a depolarized holding potential where inactivation was nearly complete. Lidocaine block showed prominent use dependence with trains of depolarizing pulses from a negative holding potential. During the interval between pulses, repriming of I (Na) displayed two exponential components, a normally recovering component (τless than 0.2 s), and a lidocaine-induced, slowly recovering fraction (τ approximately 1-2 s at pH 7.0). Raising the lidocaine concentration magnified the slowly recovering fraction without changing its time course; after a long depolarization, this fraction was one-half at approximately 10 μM lidocaine, just as expected if it corresponded to drug-bound, inactivated channels. At less than or equal to 20 μM lidocaine, the slowly recovering fraction grew exponentially to a steady level as the preceding depolarization was prolonged; the time course was the same for strong or weak depolarizations, that is, with or without significant activation of I(Na). This argues that use dependence at therapeutic levels reflects block of inactivated channels, rather than block of open channels. Overall, these results provide direct evidence for the “modulated-receptor hypothesis” of Hille (1977) and Hondeghem and Katzung (1977). Unlike tetrodotoxin, lidocaine shows similar interactions with Na channels of heart, nerve, and skeletal muscle.     

Implementation of machine learning in the clinic: challenges and lessons in prospective deployment from the System for High Intensity EvaLuation During Radiation Therapy (SHIELD-RT) randomized controlled study

Background

Public Data Commons (PDC) have been highlighted in the scientific literature for their capacity to collect and harmonize big data. On the other hand, local data commons (LDC), located within an institution or organization, have been underrepresented in the scientific literature, even though they are a critical part of research infrastructure. Being closest to the sources of data, LDCs provide the ability to collect and maintain the most up-to-date, high-quality data within an organization, closest to the sources of the data. As a data provider, LDCs have many challenges in both collecting and standardizing data, moreover, as a consumer of PDC, they face problems of data harmonization stemming from the monolithic harmonization pipeline designs commonly adapted by many PDCs. Unfortunately, existing guidelines and resources for building and maintaining data commons exclusively focus on PDC and provide very little information on LDC.

Results

This article focuses on four important observations. First, there are three different types of LDC service models that are defined based on their roles and requirements. These can be used as guidelines for building new LDC or enhancing the services of existing LDC. Second, the seven core services of LDC are discussed, including cohort identification and facilitation of genomic sequencing, the management of molecular reports and associated infrastructure, quality control, data harmonization, data integration, data sharing, and data access control. Third, instead of commonly developed monolithic systems, we propose a new data sharing method for data harmonization that combines both divide-and-conquer and bottom-up approaches. Finally, an end-to-end LDC implementation is introduced with real-world examples.

Conclusions

Although LDCs are an optimal place to identify and address data quality issues, they have traditionally been relegated to the role of passive data provider for much larger PDC. Indeed, many LDCs limit their functions to only conducting routine data storage and transmission tasks due to a lack of information on how to design, develop, and improve their services using limited resources. We hope that this work will be the first small step in raising awareness among the LDCs of their expanded utility and to publicize to a wider audience the importance of LDC.

     

Misalignment-mediated DNA polymerase beta mutations: comparison of microsatellite and frame-shift error rates using a forward mutation assay

Mutations arising in microsatellite DNA are associated with neurological diseases and cancer. To elucidate the molecular basis of microsatellite mutation, we have determined the in vitro polymerase error frequencies at microsatellite sequences representative of those found in the human genome: [GT/CA](10), [TC/AG](11), and [TTCC/AAGG](9). DNA templates contained the microsatellites inserted in-frame into the 5' region of the herpes simplex virus thymidine kinase (HSV-tk) gene. Polymerase beta (polbeta) error frequencies were quantitated in microsatellite sequences, relative to frame-shift error frequencies in coding sequences, from the same DNA synthesis reaction. The polbeta error frequencies within the dinucleotide sequences were (2-9) x 10(-3), 14-72-fold higher than the ssDNA template frequencies. The polbeta error frequencies within the tetranucleotide sequences were (4-6) x 10(-3), a 4-13-fold increase over background. Strand biases were observed for the [TC/AG](11) and [TTCC/AAGG](9) alleles, in which more errors were produced when the purine strand served as a template. Mutations within each microsatellite included noncanonical base substitution events and single nucleotide deletions as well as the expected unit length changes. An exponential relationship was observed between the polymerase error frequency per site and both the number of repetitive units and total length of the allele. Our observations are consistent with the strand slippage model of microsatellite mutagenesis and demonstrate that DNA sequence and/or structural differences result in mutational strand biases. To our knowledge, this is the first direct quantitation of DNA polymerase errors in vitro using template microsatellite sequences.     

A mutation within the leucine-rich repeat domain of the Arabidopsis disease resistance gene RPS5 partially suppresses multiple bacterial and downy mildew resistance genes.

Recognition of pathogens by plants is mediated by several distinct families of functionally variable but structurally related disease resistance (R) genes. The largest family is defined by the presence of a putative nucleotide binding domain and 12 to 21 leucine-rich repeats (LRRs). The function of these LRRs has not been defined, but they are speculated to bind pathogen-derived ligands. We have isolated a mutation in the Arabidopsis RPS5 gene that indicates that the LRR region may interact with other plant proteins. The rps5-1 mutation causes a glutamate-to-lysine substitution in the third LRR and partially compromises the function of several R genes that confer bacterial and downy mildew resistance. The third LRR is relatively well conserved, and we speculate that it may interact with a signal transduction component shared by multiple R gene pathways.     

Effect of acid pH on the absorption spectra and photoreactions of bacteriorhodopsin.

Purple membranes (PM) from Halobacterium halobium were incorporated into 7.5% polyacrylamide gels to prevent aggregation which occurs in suspensions at low pH. At pH 7.0, the circular dichroism (CD) spectra and visible absorption spectra of light- and dark-adapted bacteriorhodopsin (bR558, respectively) and the flash photolysis cycle of bR568 in gels were essentially the same as those in PM suspensions. Titration of the gels with hydrochloric acid showed the transition to a form absorbing at 605 nm (bR605 acid) with pK = 2.9 and to a second form absorbing at 565 nm (bR565 acid) with pK = 0.5. Isosbestic points were seen for each transition in both light- and dark-adapted gels. In addition, a third isosbestic point was evident between pH 3.5 and 7. Visible CD spectra of bR568, bR605 acid, and bR565 acid all showed the bilobed pattern typical of bR568 in suspensions of PM. Flash kinetic spectrophotometry (with 40-microseconds time resolution) of bR605 acid and bR565 acid showed transient absorbance changes with at least one transiently blue-shifted form for each and an early red-shifted intermediate for bR565 acid. Chromophore extraction from membrane suspensions yielded all-trans-retinal for bR565 acid and a mixture of 13-cis and trans isomers for bR605 acid.