On the philosophy of modeling—reply to a comment by M. R. DroopX

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/110088/1/lno19782320385.pd

The development of novel excipients for the stabilization of proteins against aggregation

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2011.Cataloged from PDF version of thesis.Includes bibliographical references (p. 121-129).Although protein based therapeutics is the fastest growing sector of the pharmaceutical industry, production costs remain incredibly high and rapid commercialization of new protein drug candidates are not being fully realized due to the presence of many barriers, namely the physical and chemical instabilities of proteins. Of these degradation pathways, protein aggregation is arguably the most common and troubling manifestation of protein instability, occurring in almost all phases of development. Protein aggregates are usually nonnative in structure, may exhibit reduced biological activity, and can remain soluble and/or precipitate from solution. In addition to reducing efficacy, if administered to a patient, aggregates can cause adverse reactions, such as immune response, sensitization, or even anaphylactic shock. Therefore, if even a small amount of aggregates form during formulation or storage, a product can be rendered unacceptable. Moreover, for the practical application of traditional and novel drug delivery techniques, protein based therapeutics must be formulated at relatively high concentrations and must remain stable for extended periods of time. The structural differences among various proteins are so significant, that the application of a universal stabilization strategy has not yet been successful, though the effects of common excipients are generally universal. The current approach toward stabilizing protein drugs against aggregation is by trial-and-error testing of different combinations of cosolutes (e.g. salts, sugars, surfactants, amino acids, etc.) using empirically derived heuristics. While ubiquitously used, this approach is inefficient and does not always enable the discovery of stable protein solution formulations. In response to this major problem, we have developed and tested a new class of excipients that has the potential for wide spread application as a universal stabilizer of protein therapeutics. When compared to other commonly used excipients, our novel excipients offer more than an order of magnitude improvement at suppressing the aggregation of a model protein. As a result, if used in formulations, the shelf life of a protein drug, at room or refrigerated temperatures, may be extended from a few weeks to several months or years. Furthermore, these excipients will likely be useful during production and purification for improving yield and lowering downstream purification costs.by Curtiss P. Schneider.Ph.D

Marine diatoms grown in chemostats under silicate or ammonium limitation. II. Transient response of Skeletonema costatum to a single addition of the limiting nutrient

Skeletonema costatum was grown at different steady-state growth rates in ammonium or silicate-limited chemostats. The culture was perturbed from its steady-state condition by a single addition of the limiting nutrients ammonium or silicate. The transient response was followed by measuring nutrient disappearance of the liliting perturbation experiment indicate that three distinct modes of uptake of the limiting nutrient can be distinguished; surge uptake ( V s ), internally controlled uptake ( V i ), and externally controlled uptake ( V e ). An interpretation of these three modes of uptake is given and their relation to control of uptake of the limiting nutrient is discussed. The uptake rates of the non-limiting nutrients were shown to be depressed during the surge of the uptake of the limiting nutrient. Kinetic uptake parameters, K s and V max , were obtained from data acquired during the externally controlled uptake segment, V e . The same V max value of 0. 12 h -1 , was obtained under either silicate or ammonium limitation. Estimates of K s were 0.4 μg-at NH 4 -N l -1 and 0.7 μg-at Si l -1 . Short-term 15 N uptake-rate measurements conducted on nitrogen-limited cultures appear to be a combination of V s or V i , or at lower substrate concentrations V s and V e . It is difficult to separate these different uptake modes in batch or tracer experiments, and ensuing problems in interpretation are discussed.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46629/1/227_2004_Article_BF00390940.pd

Stargardt macular dystrophy and therapeutic approaches

Stargardt macular dystrophy (Stargardt disease; STGD1; OMIM 248200) is the most prevalent inherited macular dystrophy. STGD1 is an autosomal recessive disorder caused by multiple pathogenic sequence variants in the large ABCA4 gene (OMIM 601691). Major advances in understanding both the clinical and molecular features, as well as the underlying pathophysiology, have culminated in many completed, ongoing and planned human clinical trials of novel therapies.The aims of this concise review are to describe (1) the detailed phenotypic and genotypic characteristics of the disease, multimodal imaging findings, natural history of the disease, and pathogenesis, (2) the multiple avenues of research and therapeutic intervention, including pharmacological, cellular therapies and diverse types of genetic therapies that have either been investigated or are under investigation and (3) the exciting novel therapeutic approaches on the translational horizon that aim to treat STGD1 by replacing the entire 6.8 kb ABCA4 open reading frame

Metal worker’s lung: spatial association with Mycobacterium avium

Background Outbreaks of hypersensitivity pneumonitis (HP) are not uncommon in workplaces where metal working fluid (MWF) is used to facilitate metal turning. Inhalation of microbe-contaminated MWF has been assumed to be the cause, but previous investigations have failed to establish a spatial relationship between a contaminated source and an outbreak. Objectives After an outbreak of five cases of HP in a UK factory, we carried out blinded, molecular-based microbiological investigation of MWF samples in order to identify potential links between specific microbial taxa and machines in the outbreak zone. Methods Custom-quantitative PCR assays, microscopy and phylogenetic analyses were performed on blinded MWF samples to quantify microbial burden and identify potential aetiological agents of HP in metal workers. Measurements and main results MWF from machines fed by a central sump, but not those with an isolated supply, was contaminated by mycobacteria. The factory sump and a single linked machine at the centre of the outbreak zone, known to be the workstation of the index cases, had very high levels of detectable organisms. Phylogenetic placement of mycobacterial taxonomic marker genes generated from these samples indicated that the contaminating organisms were closely related to Mycobacterium avium. Conclusions We describe, for the first time, a close spatial relationship between the abundance of a mycobacterium-like organism, most probably M. avium, and a localised outbreak of MWF-associated HP. The further development of sequence-based analytic techniques should assist in the prevention of this important occupational disease

Electron affinities of the first- and second- row atoms: benchmark ab initio and density functional calculations

A benchmark ab initio and density functional (DFT) study has been carried out on the electron affinities of the first- and second-row atoms. The ab initio study involves basis sets of $spdfgh$ and $spdfghi$ quality, extrapolations to the 1-particle basis set limit, and a combination of the CCSD(T), CCSDT, and full CI electron correlation methods. Scalar relativistic and spin-orbit coupling effects were taken into account. On average, the best ab initio results agree to better than 0.001 eV with the most recent experimental results. Correcting for imperfections in the CCSD(T) method improves the mean absolute error by an order of magnitude, while for accurate results on the second-row atoms inclusion of relativistic corrections is essential. The latter are significantly overestimated at the SCF level; for accurate spin-orbit splitting constants of second-row atoms inclusion of (2s,2p) correlation is essential. In the DFT calculations it is found that results for the 1st-row atoms are very sensitive to the exchange functional, while those for second-row atoms are rather more sensitive to the correlation functional. While the LYP correlation functional works best for first-row atoms, its PW91 counterpart appears to be preferable for second-row atoms. Among ``pure DFT'' (nonhybrid) functionals, G96PW91 (Gill 1996 exchange combined with Perdew-Wang 1991 correlation) puts in the best overall performance. The best results overall are obtained with the 1-parameter hybrid modified Perdew-Wang (mPW1) exchange functionals of Adamo and Barone [J. Chem. Phys. {\bf 108}, 664 (1998)], with mPW1LYP yielding the best results for first-row, and mPW1PW91 for second-row atoms. Indications exist that a hybrid of the type $a$ mPW1LYP + $(1-a)$ mPW1PW91 yields better results than either of the constituent functionals.Comment: Phys. Rev. A, in press (revised version, review of issues concerning DFT and electron affinities added

Ocean PHILLS hyperspectral imager : design, characterization, and calibration

The Ocean Portable Hyperspectral Imager for Low-Light Spectroscopy (Ocean PHILLS) is a hyperspectral imager specifically designed for imaging the coastal ocean. It uses a thinned, backside-illuminated CCD for high sensitivity and an all-reflective spectrograph with a convex grating in an Offner configuration to produce a nearly distortion-free image. The sensor, which was constructed entirely from commercially available components, has been successfully deployed during several oceanographic experiments in 1999–2001. Here we describe the instrument design and present the results of laboratory characterization and calibration. We also present examples of remote-sensing reflectance data obtained from the LEO-15 site in New Jersey that agrees well with ground-truth measurements.Keywords: Remote sensing, Ocean optics, Visible spectroscop

Novel Genetic Tools for Diaminopimelic Acid Selection in Virulence Studies of Yersinia pestis

Molecular studies of bacterial virulence are enhanced by expression of recombinant DNA during infection to allow complementation of mutants and expression of reporter proteins in vivo. For highly pathogenic bacteria, such as Yersinia pestis, these studies are currently limited because deliberate introduction of antibiotic resistance is restricted to those few which are not human treatment options. In this work, we report the development of alternatives to antibiotics as tools for host-pathogen research during Yersinia pestis infections focusing on the diaminopimelic acid (DAP) pathway, a requirement for cell wall synthesis in eubacteria. We generated a mutation in the dapA-nlpB(dapX) operon of Yersinia pestis KIM D27 and CO92 which eliminated the expression of both genes. The resulting strains were auxotrophic for diaminopimelic acid and this phenotype was complemented in trans by expressing dapA in single and multi-copy. In vivo, we found that plasmids derived from the p15a replicon were cured without selection, while selection for DAP enhanced stability without detectable loss of any of the three resident virulence plasmids. The dapAX mutation rendered Y. pestis avirulent in mouse models of bubonic and septicemic plague which could be complemented when dapAX was inserted in single or multi-copy, restoring development of disease that was indistinguishable from the wild type parent strain. We further identified a high level, constitutive promoter in Y. pestis that could be used to drive expression of fluorescent reporters in dapAX strains that had minimal impact to virulence in mouse models while enabling sensitive detection of bacteria during infection. Thus, diaminopimelic acid selection for single or multi-copy genetic systems in Yersinia pestis offers an improved alternative to antibiotics for in vivo studies that causes minimal disruption to virulence