In Situ ATR-FTIR Studies on the ß-Sheet Formation of Native and Regenerated Bombyx mori Silk Material in Solution and Its Potential for Drug Releasing Coatings.

Dynamic attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy at both solutions and coatings of a semicrystalline silk material derived from Bombyx mori was applied to monitor the ß-sheet conformation, which is known to correlate with silk protein crystallinity. The secondary structure-sensitive Amide I band was analyzed. Two silk protein samples were studied: native-based silk buffer fibroin (NSF) was extracted from silk glands and regenerated silk fibroin (RSF) was extracted from degummed cocoons. Solutions of both NSF and RSF at 2 mg/mL featured low initial ß-sheet contents of 5-12%, which further increased to 47-53% after 24 h. RSF and NSF solutions at 23 mg/mL also featured low initial ß-sheet contents of 9-10%, which yet only slightly increased to 16-17% after 24 h. Coatings deposited from RSF solutions showed high surface integrity (Q > 99%) after rinsing in mineralized water, enabling interfacial drug delivery applications. RSF coatings were post-treated with either formic acid (FA) or pure methanol (MeOH) vapor to showcase inducibility of crystalline domains in RSF coatings. Such coatings were loaded with the model antibiotic drugs tetracycline (TCL) and streptomycin (STRP), and the sustained release of TCL was followed in contact with (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES) buffer. RSF/TCL coatings post-treated with formic acid (FA) vapor followed by methanol (MeOH) vapor showed a significantly lower (52%) initial burst of rather hydrophobic TCL compared to untreated RSF/TCL coatings (72%), while no such significant release difference was observed for hydrophilic STRP. This was rationalized by a specific interaction between nonpolar TCL and hydrophobic crystalline RSF domains.

Bioinspired calcium phosphate mineralization on Net-Shape-Nonwoven chitosan scaffolds stimulates human bone marrow stromal cell differentiation.

Chitosan fibers were processed using the Net-Shape-Nonwoven (NSN) technique in order to create porous scaffolds which were functionalized in two bioinspired ways: collagen type I coating and unique mineralization with organically modified hydroxyapatite (ormoHAP). While collagen is common to enhance cell attachment on surfaces, the electric-field assisted migration and deposition of ormoHAP on the surface of the NSN-scaffolds is a novel technique which enables sub-micrometer sized mineralization while maintaining the original pore structure. Microscopy revealed fast attachment and morphological adaptation of the cells on both, the pure and the functionalized NSN-scaffolds. Remarkably, the cell number of osteogenically induced hBMSC on ormoHAP-modified NSN-scaffolds increased 3.5-5 fold compared to pure NSN-scaffolds. Osteogenic differentiation of hBMSC/osteoblasts was highest on collagen-functionalized NSN-scaffolds. RT-PCR studies revealed gene expression of ALP, BSP II, and osteocalcin to be high for all NSN-scaffolds. Overall, the NSN-scaffold functionalization with collagen and ormoHAP improved attachment, proliferation, and differentiation of hBMSC and therefore revealed the remarkable potential of their application for the tissue engineering of bone.

Faba bean (Vicia faba) inclusion in dairy cow diets: Effect on nutrient digestion, rumen fermentation, nitrogen utilization, methane production, and milk performance.

The objective of this study was to determine the effect of replacing on isonitrogenous and isoenergetic basis soybean meal (SBM) and corn grain with ground or rolled faba bean (FB; Vicia faba major var. Baie-Saint-Paul) in dairy cow diets (17% of diet dry matter) on nutrient digestion, rumen fermentation, N utilization, methane production, and milk performance. For this purpose, 9 lactating cows were used in a replicated 3 × 3 Latin square design (35-d period) and fed (ad libitum) a total mixed ration (forage:concentrate ratio = 59:41 on a dry matter basis). In the concentrate portion, SBM and corn grain (control diet) were completely and partially replaced, respectively, with either ground or rolled FB. Ruminal degradability (in sacco) of crude protein was higher for ground FB (79.4%) compared with SBM (53.3%) and rolled FB (53.2%). Including FB in the diet did not affect dry matter intake, milk production, and milk composition. Experimental treatment had no effect on total volatile fatty acid concentration, acetate-to-propionate ratio, and protozoa numbers. Compared with cows fed the control diet, ruminal NH3 concentration increased and tended to increase for cows fed ground FB and rolled FB, respectively; however, we found no difference in ruminal NH3 concentration between the 2 processed FB. Apparent total-tract digestibility of crude protein was similar between cows fed the control diet and cows fed rolled FB and tended to increase for cows fed ground FB compared with cows fed the control diet. Feeding rolled FB decreased CP digestibility compared with feeding ground FB. Urinary and manure (feces + urine) N excretion (g/d or as a proportion of N intake) were not affected by the inclusion of FB in the diet. Enteric CH4 production was similar among the experimental diets. Results from this study show that including FB (17% of dietary dry matter) at the expense of SBM and corn grain in the diet had no effect on milk production, N excretion, and enteric CH4 production of dairy cows.

A unified statistical approach to deformation-based morphometry.

We present a unified statistical framework for analyzing temporally varying brain morphology using the 3D displacement vector field from a nonlinear deformation required to register a subject's brain to an atlas brain. The unification comes from a single model for structural change, rather than two separate models, one for displacement and one for volume changes. The displacement velocity field rather than the displacement itself is used to set up a linear model to account for temporal variations. By introducing the rate of the Jacobian change of the deformation, the local volume change at each voxel can be computed and used to measure possible brain tissue growth or loss. We have applied this method to detecting regions of a morphological change in a group of children and adolescents. Using structural magnetic resonance images for 28 children and adolescents taken at different time intervals, we demonstrate how this method works.

A novel gamma -secretase assay based on detection of the putative C-terminal fragment-gamma of amyloid beta protein precursor.

Alzheimer's disease is characterized by the deposits of the 4-kDa amyloid beta peptide (A beta). The A beta protein precursor (APP) is cleaved by beta-secretase to generate a C-terminal fragment, CTF beta, which in turn is cleaved by gamma-secretase to generate A beta. Alternative cleavage of the APP by alpha-secretase at A beta 16/17 generates the C-terminal fragment, CTFalpha. In addition to A beta, endoproteolytic cleavage of CTF alpha and CTF beta by gamma-secretase should yield a C-terminal fragment of 57-59 residues (CTF gamma). However, CTF gamma has not yet been reported in either brain or cell lysates, presumably due to its instability in vivo. We detected the in vitro generation of A beta as well as an approximately 6-kDa fragment from guinea pig brain membranes. We have provided biochemical and pharmacological evidence that this 6-kDa fragment is the elusive CTF gamma, and we describe an in vitro assay for gamma-secretase activity. The fragment migrates with a synthetic peptide corresponding to the 57-residue CTF gamma fragment. Three compounds previously identified as gamma-secretase inhibitors, pepstatin-A, MG132, and a substrate-based difluoroketone (t-butoxycarbonyl-Val-Ile-(S)-4-amino-3-oxo-2, 2-difluoropentanoyl-Val-Ile-OMe), reduced the yield of CTF gamma, providing additional evidence that the fragment arises from gamma-secretase cleavage. Consistent with reports that presenilins are the elusive gamma-secretases, subcellular fractionation studies showed that presenilin-1, CTF alpha, and CTF beta are enriched in the CTF gamma-generating fractions. The in vitro gamma-secretase assay described here will be useful for the detailed characterization of the enzyme and to screen for gamma-secretase inhibitors.

Cell-free assays for gamma-secretase activity.

The amyloid b-protein (Ab) deposited in Alzheimer's disease (AD) is a normally secreted proteolytic product of the amyloid b-protein precursor (APP). Generation of Ab from the APP requires two sequential proteolytic events: an initial b-secretase cleavage at the amino terminus of the Ab sequence followed by g-secretase cleavage at the carboxyl terminus of Ab. We describe the development of a robust in vitro assay for g-secretase cleavage by showing de novo Ab production in vitro and establish that this assay monitors authentic gamma-secretase activity by documenting the production of a cognate g-CTF, confirming the size of the Ab produced by mass spectrometry, and inhibiting cleavage in this system with multiple inhibitors that alter g-secretase activity in living cells. Using this assay, we demonstrate that the g-secretase activity 1) is tightly associated with the membrane, 2) can be solubilized, 3) has a pH optimum of 6.8 but is active from pH 6.0 to pH >8.4, and 4) ascertain that activities of the g-40 and g-42 are indeed pharmacologically distinct. These studies should facilitate the purification of the protease or proteases that are responsible for this unusual activity, which is a major therapeutic target for the treatment of AD.