Subchronic feeding, allergenicity, and genotoxicity safety evaluations of single strain bacterial protein.

Microbial proteins are potentially important alternatives to animal protein. A safety assessment was conducted on a Clostridium protein which can serve as a high-quality protein source in human food. A battery of toxicity studies was conducted comprising a 14-day dose-range finding dietary study in rats, 90-day dietary study in rats and in vitro genotoxicity studies. The allergenic potential was investigated by bioinformatics analysis. In the 90-day feeding study, rats were fed diets containing 0, 5.0, 7.5, and 10% Clostridium protein. The Clostridium protein-containing diets were well-tolerated and no adverse effects on the health or growth were observed. Significant reductions in neutrophil counts were observed in all female rats compared to controls, which were slightly outside of reference ranges. These effects were not deemed to be adverse due to the absence of comparable findings in male rats and high physiological variability of measured values within groups. A No-Observed-Adverse-Effect-Level (NOAEL) of at least 10% Clostridium protein, the highest dose tested and corresponding to 5,558 and 6,671 mg/kg body weight/day for male and female rats, respectively, was established. No evidence of genotoxicity was observed and the allergenic potential was low. These results support the use of Clostridium protein as a food ingredient.

Non-destructive determination of functionalized polyelectrolyte placement in layer-by-layer films by IR ellipsometry.

Layer-by-layer (LbL) assembly facilitates controlled coatings on a variety of surfaces with the ability to manipulate the composition through the thickness by selection of the complementary pairs. However, the characterization of these composition profiles tends to be destructive and requires significant compositional differences that can limit their utility. Here, we demonstrate the ability to non-destructively quantify the depth dependence of the allyl content associated with the selective incorporation of poly(sodium acrylate-co-allylacrylamide) (84 : 16 mol : mol) (allyl-PAA) in LbL films based on the assembly of poly(diallyldimethylammonium chloride) (PDAC)/poly(acrylic acid) (PAA) and PDAC/allyl-PAA. Although the atomic composition of the film is not dramatically influenced by the change between PAA and allyl-PAA, the absorption in the IR near 1645 cm-1 by the allyl group provides sufficient optical contrast to distinguish the LbL components with spectroscopic ellipsometry. The use of IR spectroscopic ellipsometry can determine the thickness of layers that contain allyl-PAA and also gradients that develop due to re-arrangements during the LbL process. With multiple films fabricated simultaneously, the location of the gradient between the 1st and 2nd series of multilayers (e.g., first PDAC/PAA bilayers and then PDAC/allyl-PAA bilayers) can be readily assessed. The results from a variety of different multilayer architectures indicate that the gradient is located within the thickness expected for the 1st deposited bilayer stack (PDAC/PAA or PDAC/allyl-PAA). These results are indicative of a dynamic dissolution-deposition process (in- and out- diffusion) during the fabrication of these LbL films. These results provide additional evidence into the mechanisms for exponential growth in LbL assemblies. The ability to quantify a gradient with the low contrast system examined indicates that spectroscopic IR ellipsometry should be able to non-destructively determine compositional gradients for most polymer films where such gradients exist.

Mechanisms of Directional Polymer Crystallization.

Zone annealing, a directional crystallization technique originally used for the purification of semiconductors, is applied here to crystalline polymers. Tight control over the final lamellar orientation and thickness of semicrystalline polymers can be obtained by directionally solidifying the material under optimal conditions. It has previously been postulated by Lovinger and Gryte that, at steady state, the crystal growth rate of a polymer undergoing zone annealing is equal to the velocity at which the sample is drawn through the temperature gradient. These researchers further implied that directional crystallization only occurs below a critical velocity, when crystal growth rate dominates over nucleation. Here, we perform an analysis of small-angle X-ray scattering, differential scanning calorimetry, and cross-polarized optical microscopy of zone-annealed poly(ethylene oxide) to examine these conjectures. Our long period data validate the steady-state ansatz, while an analysis of Herman's orientation function confirms the existence of a transitional region around a critical velocity, v crit, where there is a coexistence of oriented and isotropic domains. Below v crit, directional crystallization is achieved, while above v crit, the mechanism more closely resembles that of conventional isotropic isothermal crystallization.

Effect of Adjacent Hydrophilic Polymer Thin Films on Physical Aging and Residual Stress in Thin Films of Poly(butylnorbornene-ran-hydroxyhexafluoroisopropyl norbornene).

The properties of thin supported polymer films can be dramatically impacted by the substrate upon which it resides. A simple way to alter the properties of the substrate (chemistry, rigidity, dynamics) is by coating it with an immiscible polymer. Here we describe how ultrathin (ca. 2 nm) hydrophilic polymer layers of poly(acrylic acid), PAA, and poly(styrenesulfonate), PSS, impact the aging behavior and the residual stress in thin films of poly(butylnorbornene-ran-hydroxyhexafluoroisopropyl norbornene), BuNB-r-HFANB. The aging rate decreases as the film thickness (h) is decreased, but the extent of this change depends on the adjacent layer. Even for the thickest films (h>500 nm), there is a decrease in the aging rate at 100 °C when BuNB-r-HFANB is in contact with PSS. In an effort to understand the origins of these differences in the aging behavior, the elastic modulus and residual stress (σR) in the films were determined by wrinkling as a function of aging time. The change in the elastic modulus during aging does not appear to be directly correlated with the densification or expansion of the films, but the aging rates appear to roughly scale as hσR 1/3. These results illustrate that the physical aging of thin polymer films can be altered by adjacent polymers.

Loss of Sarm1 does not suppress motor neuron degeneration in the SOD1G93A mouse model of amyotrophic lateral sclerosis.

Axon degeneration occurs in all neurodegenerative diseases, but the molecular pathways regulating axon destruction during neurodegeneration are poorly understood. Sterile Alpha and TIR Motif Containing 1 (Sarm1) is an essential component of the prodegenerative pathway driving axon degeneration after axotomy and represents an appealing target for therapeutic intervention in neurological conditions involving axon loss. Amyotrophic lateral sclerosis (ALS) is characterized by rapid, progressive motor neuron degeneration and muscle atrophy, causing paralysis and death. Patient tissue and animal models of ALS show destruction of upper and lower motor neuron cell bodies and loss of their associated axons. Here, we investigate whether loss of Sarm1 can mitigate motor neuron degeneration in the SOD1G93A mouse model of ALS. We found no change in survival, behavioral, electrophysiogical or histopathological outcomes in SOD1G93A mice null for Sarm1. Blocking Sarm1-mediated axon destruction alone is therefore not sufficient to suppress SOD1G93A-induced neurodegeneration. Our data suggest the molecular pathways driving axon loss in ALS may be Sarm1-independent or involve genetic pathways that act in a redundant fashion with Sarm1.

Using Drosophila models of Huntington's disease as a translatable tool.

The Huntingtin (Htt) protein is essential for a wealth of intracellular signaling cascades and when mutated, causes multifactorial dysregulation of basic cellular processes. Understanding the contribution to each of these intracellular pathways is essential for the elucidation of mechanisms that drive pathophysiology. Using appropriate models of Huntington's disease (HD) is key to finding the molecular mechanisms that contribute to neurodegeneration. While mouse models and cell lines expressing mutant Htt have been instrumental to HD research, there has been a significant contribution to our understating of the disease from studies utilizing Drosophila melanogaster. Flies have an Htt protein, so the endogenous pathways with which it interacts are likely conserved. Transgenic flies engineered to overexpress the human mutant HTT gene display protein aggregation, neurodegeneration, behavioral deficits and a reduced lifespan. The short life span of flies, low cost of maintaining stocks and genetic tools available for in vivo manipulation make them ideal for the discovery of new genes that are involved in HD pathology. It is possible to do rapid genome wide screens for enhancers or suppressors of the mutant Htt-mediated phenotype, expressed in specific tissues or neuronal subtypes. However, there likely remain many yet unknown genes that modify disease progression, which could be found through additional screening approaches using the fly. Importantly, there have been instances where genes discovered in Drosophila have been translated to HD mouse models.

Exome-wide rare variant analysis identifies TUBA4A mutations associated with familial ALS.

Exome sequencing is an effective strategy for identifying human disease genes. However, this methodology is difficult in late-onset diseases where limited availability of DNA from informative family members prohibits comprehensive segregation analysis. To overcome this limitation, we performed an exome-wide rare variant burden analysis of 363 index cases with familial ALS (FALS). The results revealed an excess of patient variants within TUBA4A, the gene encoding the Tubulin, Alpha 4A protein. Analysis of a further 272 FALS cases and 5,510 internal controls confirmed the overrepresentation as statistically significant and replicable. Functional analyses revealed that TUBA4A mutants destabilize the microtubule network, diminishing its repolymerization capability. These results further emphasize the role of cytoskeletal defects in ALS and demonstrate the power of gene-based rare variant analyses in situations where causal genes cannot be identified through traditional segregation analysis.

The curette prep: a modification of the traditional scabies preparation.

An accurate diagnosis of scabies is critical for proper treatment of this common infestation. In our clinic, we have developed a modification of the traditional method of performing a scabies preparation, called the curette prep, that substitutes a disposable curette for a scalpel blade when obtaining skin scrapings for examination. The major advantages of this technique are greater acceptability and safety for pediatric patients.

Configurationally restricted bismacrocyclic CXCR4 receptor antagonists.

A zinc(II) containing configurationally restricted analogue of bismacrocyclic cyclam-type CXCR4 chemokine receptor antagonists has been synthesized and shown to adopt only one configuration in solution. The single crystal X-ray structure reveals favorable binding to acetate via a bidentate chelation that can be related to the proposed interaction with aspartate on the receptor protein surface. The zinc(II) complex is highly active against HIV infection in vitro.

Reactivity of a 1:1 copper-oxygen complex: isolation of a Cu(II)-o-iminosemiquinonato species.

While a 1:1 Cu-O2 adduct is generally unreactive with organic substrates, phosphines displace O2 via an associative process and added Cu(I) leads to a novel internal ligand oxidation to yield a Cu(II)-o-iminosemiquinone complex.

Ultrastable complexes for in vivo use: a bifunctional chelator incorporating a cross-bridged macrocycle.

The synthesis, copper(II) complexation and biotin conjugation of a bifunctional chelator incorporating a cross-bridged macrocycle are described.

A manganese superoxide dismutase mimic based on cis,cis-1,3,5-triaminocyclohexane.

A novel manganese(iii) complex with a water soluble cis,cis-1,3,5-triaminocyclohexane-based ligand was synthesised and shown to exhibit superoxide dismutase activity.

Metabolites of a blocked chloramphenicol producer.

Addition of p-aminophenylalanine (4), an advanced biosynthetic precursor of the antibiotic chloramphenicol (5), to a Streptomyces venezuelae pabAB mutant (VS629) restored chloramphenicol production and led to formation of the non-chlorinated analogue corynecin II (6) and four acetanilide derivatives: p-(acetylamino)phenylalanine (7), p-(acetylamino)benzyl alcohol (13), p-(acetylamino)benzoic acid (14), and p-(acetylamino)phenol (acetaminophen, 16). Metabolite structures were deduced from NMR and MS-MS data and established by chromatographic and spectroscopic comparisons with authentic samples. Reference compound 13 was synthesized by reducing the acid chloride of 14. Shunt pathways are proposed to account for the formation of the metabolites from p-aminophenylalanine.

Snapshots of dioxygen activation by copper: the structure of a 1:1 Cu/O(2) adduct and its use in syntheses of asymmetric Bis(mu-oxo) complexes.

The X-ray structure of a 1:1 Cu/O(2) adduct revealed side-on (eta(2)) O(2) coordination. Density functional calculations corroborated the structure, indicated a significant contribution of a Cu(III)-(O(2)(2-)) resonance form, and provided insights into the key bonding interactions. Reaction of a 1:1 adduct supported by a slightly different beta-diketiminate ligand with Cu(I) reagents resulted in the formation of novel asymmetric bis(mu-oxo) complexes that were identified by EPR, UV-vis, and Raman spectroscopy, as well as by an X-ray structure in one instance.

Isolation of 3' -O-acetylchloramphenicol: a possible intermediate in chloramphenicol biosynthesis.

3' -O-acetylchloramphenicol, commonly formed from chloramphenicol by resistant bacteria, has been isolated from the antibiotic-producing organism. Biosynthetic experiments suggest that it is a protected intermediate in chloramphenicol biosynthesis, implicating acetylation as a self-resistance mechanism in the producing organism.

A new species of endophytic Balansia from Veracruz, Mexico.

A new graminicolous species of Clavicipitaceae, Balansia brunnans sp. nov., has been found to infect Panicum xalapénse. Staining of living host tissues indicates the presence of intercellular endophytic mycelium. Stromata develop just below the nodes on the culms. Balansia brunnans is comparable to Balansia aristidae, B. discoidea, B. gaduae, B. nigricans, and B. strangulans in development of stromata on culms and possession of an endophytic mycelial stage. Among the differences between Balansia brunnans and other comparable species is that it possesses a brown perithecial stroma, whereas comparable species have black perithecial stromata. A key is provided to distinguish B. brunnans from similar species.