A quartz crystal microbalance method to quantify the size of hyaluronan and other glycosaminoglycans on surfaces.

Hyaluronan (HA) is a major component of peri- and extra-cellular matrices and plays important roles in many biological processes such as cell adhesion, proliferation and migration. The abundance, size distribution and presentation of HA dictate its biological effects and are also useful indicators of pathologies and disease progression. Methods to assess the molecular mass of free-floating HA and other glycosaminoglycans (GAGs) are well established. In many biological and technological settings, however, GAGs are displayed on surfaces, and methods to obtain the size of surface-attached GAGs are lacking. Here, we present a method to size HA that is end-attached to surfaces. The method is based on the quartz crystal microbalance with dissipation monitoring (QCM-D) and exploits that the softness and thickness of films of grafted HA increase with HA size. These two quantities are sensitively reflected by the ratio of the dissipation shift (ΔD) and the negative frequency shift (- Δf) measured by QCM-D upon the formation of HA films. Using a series of size-defined HA preparations, ranging in size from ~ 2 kDa tetrasaccharides to ~ 1 MDa polysaccharides, we establish a monotonic yet non-linear standard curve of the ΔD/ - Δf ratio as a function of HA size, which reflects the distinct conformations adopted by grafted HA chains depending on their size and surface coverage. We demonstrate that the standard curve can be used to determine the mean size of HA, as well as other GAGs, such as chondroitin sulfate and heparan sulfate, of preparations of previously unknown size in the range from 1 to 500 kDa, with a resolution of better than 10%. For polydisperse samples, our analysis shows that the process of surface-grafting preferentially selects smaller GAG chains, and thus reduces the average size of GAGs that are immobilised on surfaces comparative to the original solution sample. Our results establish a quantitative method to size HA and other GAGs grafted on surfaces, and also highlight the importance of sizing GAGs directly on surfaces. The method should be useful for the development and quality control of GAG-based surface coatings in a wide range of research areas, from molecular interaction analysis to biomaterials coatings.

Long-term monitoring of Vermont's forest soils: early trends and efforts to address innate variability.

Long-term monitoring of forest soils is necessary to understand the effects of continued environmental change, including climate change, atmospheric deposition of metals, and, in many regions, recovery from acidic precipitation. A monitoring program was initiated in 2002 at five protected forest sites, primarily Spodosol soils, in Vermont, northeastern USA. Every 5 years, ten soil pits were sampled from random subplots in a 50 × 50-m plot at each site. Samples were taken by genetic horizon and, to reduce variability and improve comparability, from four specific layers: the combined Oi/Oe layer, the combined Oa/A layer, the top 10 cm of the B horizon, and 60-70 cm below the soil surface (usually the C horizon). The samples were archived and a subset analyzed for carbon, nitrogen, and exchangeable cations. After four sampling campaigns, the average coefficients of variation (CVs) at each site had a broad range, 10.7% for carbon in the Oa/A horizon to 84.3% for exchangeable Ca2+ in the B horizon. An investigation of variability within the upper 10 cm of the B horizon across a 90-cm soil pit face showed similar CVs to the entire site, emphasizing the need for consistent and careful sampling. After 15 years, temporal trends were significant in the Oa/A and B horizons at two of the five sites, with one site showing an increase in carbon concentration in both layers along with increases in both exchangeable Ca2+ and Al3+ in the B horizon, perhaps linked to recovery from acidification. The monitoring program plans to continue at 5-year intervals for the next century.

The potential of memory enhancement through modulation of perineuronal nets.

With an increasingly aging global population, the incidence of neurological diseases such as dementia is set to increase to unmanageable levels, yet there are currently only symptomatic therapies available for treatment. The mechanisms underlying the development of some forms of dementia, such as Alzheimer's disease (AD), are not yet completely elucidated with several competing hypotheses existing. During the closure of the critical period in the brain, significant compositional changes occur to the neural extracellular matrix (ECM). Specifically, condensed mesh-like structures called perineuronal nets (PNNs) form around subsets of neurons and have a profound effect on axonal growth and limit neuronal plasticity. These PNNs act as a morphological checkpoint and can influence memory and cognition. Manipulating these important ECM structures may provide the key to reactivating plasticity and restoring memory, both of which are severely impaired in AD and other associated neurological diseases. This review explores the current understanding of how PNNs are manipulated and examines potential new methods for PNN modulation. LINKED ARTICLES: This article is part of a themed section on Therapeutics for Dementia and Alzheimer's Disease: New Directions for Precision Medicine. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.18/issuetoc.

CASSCF Calculations Reveal Competitive Chair (Pericyclic) and Boat (Pseudopericyclic) Transition States for the [3,3] Sigmatropic Rearrangement of Allyl Esters.

(10,8)CASPT2/6-31G**//(10,8)CASSCF/6-31G** and CCSD(T)/cc-pVDZ//(10,8)-CASSCF/6-31G** calculations have been performed on the potential surface for the [3,3] sigmatropic allyl ester rearrangements of cis-3-penten-2-yl acetate (16) to trans-3-penten-2-yl acetate (17) and 3-buten-2-yl acetate (21) to trans-2-buten-1-yl acetate (22). The results are compared to DFT (B3LYP/6-31G**) calculations on the known 16 → 17 rearrangement that reported it to be concerted and pseudopericyclic through a boat-shaped transition structure ( Birney, D. M. et al. J. Am. Chem. Soc. 2009 , 131 , 528 - 537 ). The CASSCF calculations, on the other hand, uncovered competitive concerted pathways for both the 16 → 17 and 21 → 22 rearrangements, though it was necessary to apply certain approximations in the former case. While one CASSCF pathway in each case involves a boat-shaped transition structure, similar to the one located through DFT calculations, the other pathway involves a chair-shaped transition structure. Preference for chair or boat is shown to be method dependent. Moreover, examination of the CASSCF active-space orbitals clearly confirms that the boat-shaped transition structures are pseudopericyclic but significantly also established that the chair-shaped transition structures are clearly pericyclic. Conclusions based on these results, and regarding our understanding of pericyclic vs pseudopericyclic reactions, are proffered.

CASSCF Computational Study of Pseudopericyclic Character in Electrocyclic Rearrangements Involving Heteroatoms.

The Complete Active Space Self-Consistent Field (CASSCF) computational method, with the 6-31G* basis set, was used to examine six electrocyclic rearrangements, each involving a 1,2,4,6-heptatetraene skeleton with two variously located oxygen and/or nitrogen heteroatoms, as a way to determine which, if any, are pseudopericyclic as opposed to pericyclic. Primarily through the close examination of the active space orbitals, but also considering transition structure geometries and activation energies, it was concluded that rearrangements 3 → 4, 5 → 6, 7 → 8, and 9 → 10 are pseudopericyclic with two orbital disconnections each, whereas the 13 → 14 and 15 → 16 rearrangements are pericyclic. Our conclusions agreed with those of others in two of the four cases that had been studied previously by density functional theory (3 → 4 and 7 → 8) but ran contrary to the previous conclusions that the 5 → 6 rearrangement is pericyclic and that the 15 → 16 rearrangement is pseudopericyclic. Our results are also compared and contrasted to previous similar ones of ours involving the 3 → 4 electrocyclization (essentially pericyclic), the 11 → 12 [3,3] sigmatropic rearrangement (pseudopericyclic), and similar [3,3] sigmatropic rearrangements (all pericyclic), and detailed rationales for these latest results are provided.

CASSCF molecular orbital calculations reveal a purely pseudopericyclic mechanism for a [3,3] sigmatropic rearrangement.

A comparative CASSCF/6-31G*-level computational study of the concerted [3,3] sigmatropic rearrangements of cis-1-iminyl-2-ketenylcyclopropane (15), cis-1-iminyl-2-propadienylcyclopropane (17), and cis-1-iminyl-2-keteniminylcyclopropane (19) to give products 16, 18, and 20, respectively, was conducted. Analysis of the active space MOs of TS(15-->16), TS(17-->18), and TS(19-->20) suggests that the 17 --> 18 and 19 --> 20 rearrangements are classically pericyclic, whereas the 15 --> 16 rearrangement is pseudopericyclic with two orbital disconnections-one involving the nitrogen lone-pair orbital and the other the carbonyl carbon of the ketene moiety. The novel TS(15-->16) was also found to have a highly planar, tight, geometry, whereas TS(17-->18) and TS(19-->20) were both shown to have the boat-shaped geometry expected for classically pericyclic [3,3] sigmatropic rearrangements. Results of calculations on the [3,3] sigmatropic rearrangements involving additional transition structures, TS(21-->22), TS(23-->24), TS(25-->26), TS(27-->28), TS(29-->30), and TS(31-->32), demonstrate the relative uniqueness of the pseudopericyclic one, TS(15-->16).

The Vermedx Diabetes Information System reduces healthcare utilization.

OBJECTIVE: To confirm the cost savings in a randomized clinical trial of the Vermedx Diabetes Information System (hereafter referred to as the Diabetes Information System [DIS]) in independently collected data using claims paid by a managed care insurer for patients with and without DIS participation. STUDY DESIGN: Longitudinal analysis of paid claims with concurrent and historical controls from October 2002 through October 2007. METHODS: Using locally weighted smoothing functions and linear regression analysis before and after commencement of the DIS, we compared the total claims paid per member per month for 153 patients using the DIS versus 870 control patients. RESULTS: For DIS patients, paid claims increased at a rate of $8.30 (95% confidence interval [CI], $1.12-$15.48) per month before the DIS started compared with -$3.92 (95% CI, -$9.50 to $1.67) after commencement of the DIS (P = .008). For control patients, the slope changed from $6.80 (95% CI, $3.78-$9.82) to $3.16 (95% CI, -$1.06 to $7.38) (P = .17). After commencement of the DIS, the slope of the claims in the DIS group is significantly lower than that of the control group (-$3.92 vs $3.16, P = .046). The mean estimated savings range from $504 per patient in year 1 of operations to $3563 in year 4. The cumulative net savings reach $8134 in 4 years. CONCLUSIONS: Participation in the DIS is associated with substantial reductions in claims paid, net of the costs of the intervention. The cost savings reported in the randomized clinical trial of the DIS are reproduced in an independent data set.

Secondary orbital effect in the electrocyclic ring closure of 7-Azahepta-1,2,4,6-tetraeneA CASSCF molecular orbital study.

Results of (10,9)CASSCF/6-31G* and B3LYP/6-31G* level calculations on the potential surface for the electrocyclic ring closure of E-7-azahepta-1,2,4,6-tetraene 3 to 1-aza-6-methylidenecyclohexa-2,4-diene ( 4) are reported, as well as parallel calculations on the electrocyclizations of hepta-1,2,4,6-tetraene 5, hexa-1,3,5-triene 7, Z and E-1-aza-1,3,5-hexatrienes 9 and 10, and Z-7-azahepta-1,2,4,6-tetraene 12 for purposes of careful comparison. The 3 --> 4 rearrangement has been studied computationally with density functional theory (DFT) by others, leading to disagreement over whether it is pseudopericyclic (de Lera, A. R.; Alvarez, R.; Lecea, B.; Torrado, A.; Cossío, F. P. Angew. Chem., Int. Ed. 2001, 40, 557-561; de Lera, A. R.; Cossío, F. P. Angew. Chem., Int. Ed. 2002, 41, 1150-1152) or pericyclic (Rodríguez-Otero, J.; Cabaleiro-Lago, E. Angew. Chem., Int. Ed. 2002, 41, 1147-1150). In accordance with disrotatory motion, the normal mode vectors for TS 3-->4 calculated at the (10,9)CASSCF/6-31G* level show a greater magnitude of rotation of the N1-H group relative to the N1-C2 bond being formed than in TS 3-->4 calculated at the B3LYP/6-31G* level. Furthermore, comparison of orbital correlation diagrams constructed entirely from localized complete active space (CAS) molecular orbitals (MOs) for the electrocyclizations of 3, 5, 7, 9, and 10 suggest that it is the highest occupied delocalized pi-MO of 3 that is primarily responsible for sigma-bond formation in 4, not the terminal allenyl pi-bond MO. However, there does appear to be a special secondary orbital effect role for the nitrogen lone-pair and hence the process is likely neither purely pericyclic nor pseudopericyclic.