Investigating the potential value of individual parameters of histological grading systems in a sheep model of cartilage damage : the modified Mankin method

A total histological grade does not necessarily distinguish between different manifestations of cartilage damage or degeneration. An accurate and reliable histological assessment method is required to separate normal and pathological tissue within a joint during treatment of degenerative joint conditions and to sub-classify the latter in meaningful ways. The Modified Mankin method may be adaptable for this purpose. We investigated how much detail may be lost by assigning one composite score/grade to represent different degenerative components of the osteoarthritic condition. We used four ovine injury models (sham surgery, anterior cruciate ligament/medial collateral ligament instability, simulated anatomic anterior cruciate ligament reconstruction and meniscal removal) to induce different degrees and potentially 'types' (mechanisms) of osteoarthritis. Articular cartilage was systematically harvested, prepared for histological examination and graded in a blinded fashion using a Modified Mankin grading method. Results showed that the possible permutations of cartilage damage were significant and far more varied than the current intended use that histological grading systems allow. Of 1352 cartilage specimens graded, 234 different manifestations of potential histological damage were observed across 23 potential individual grades of the Modified Mankin grading method. The results presented here show that current composite histological grading may contain additional information that could potentially discern different stages or mechanisms of cartilage damage and degeneration in a sheep model. This approach may be applicable to other grading systems.

Nitrate reduction over nanoscale zero-valent iron prepared by hydrogen reduction of goethite

Nitrate reduction with nanoscale zero-valent iron (NZVI) was reported as a potential technology to remove nitrate from nitrate-contaminated water. In this paper, nitrate reduction with NZVI prepared by hydrogen reduction of natural goethite (NZVI-N, -N represents natural goethite) and hydrothermal goethite (NZVI-H, -H represents hydrothermal goethite) was conducted. Besides, the effects of reaction time, nitrate concentration, iron-to-nitrate ratio on nitrate removal rate over NZVI-H and NZVI-N were investigated. To prove their excellent nitrate reduction capacities, NZVI-N and NZVI-H were compared with ordinary zero-valent iron (OZVI-N) through the static experiments. Based on all above investigations, the mechanism of nitrate reduction with NZVI-N was proposed. The result showed that reaction time, nitrate concentration, iron-to-nitrate ratio played an important role in nitrate reduction by NZVI-N and NZVI-H. Compared with OZVI, NZVI-N and NZVI-H showed little relationship with pH. And NZVI-N for nitrate composition offers a higher stability than NZVI-H because of the existence of Al-substitution. Furthermore, NZVI-N, prepared by hydrogen reduction of goethite, has higher activity for nitrate reduction and the products contain hydrogen, nitrogen, NH 4 +, a little nitrite, but no NOx, meanwhile NZVI-N was oxidized to Fe 2+. It is a relatively easy and cost-effective method for nitrate removal, so NZVI-N reducing nitrate has a great potential application in nitrate removal of groundwater. © 2012 Elsevier B.V.

Amphiphilic silicone archtectures via anaerobic thiol - ene chemistry

Despite broad application, few silicone-based surfactants of known structure or, therefore, surfactancy have been prepared because of an absence of selective routes and instability of silicones to acid and base. Herein the synthesis of a library of explicit silicone-poly(ethylene glycol) (PEG) materials is reported. Pure silicone fragments were generated by the B(C(6)F(5))(3)-catalyzed condensation of alkoxysilanes and vinyl-functionalized hydrosilanes. The resulting pure products were coupled to thiol-terminated PEG materials using photogenerated radicals under anaerobic conditions.

Surface plasmon hybridization and exciton coupling

We derive a semianalytical model to describe the interaction of a single photon emitter and a collection of arbitrarily shaped metal nanoparticles. The theory treats the metal nanoparticles classically within the electrostatic eigenmode method, wherein the surface plasmon resonances of collections of nanoparticles are represented by the hybridization of the plasmon modes of the noninteracting particles. The single photon emitter is represented by a quantum mechanical two-level system that exhibits line broadening due to a finite spontaneous decay rate. Plasmon-emitter coupling is described by solving the resulting Bloch equations. We illustrate the theory by studying model systems consisting of a single emitter coupled to one, two, and three nanoparticles, and we also compare the predictions of our model to published experimental data. ©2012 American Physical Society.

3D mesenchymal stem/stromal cell osteogenesis and autocrine signalling

Mesenchymal stem/stromal cells (MSC) are rapidly becoming a leading candidate for use in tissue regeneration, with first generation of therapies being approved for use in orthopaedic repair applications. Capturing the full potential of MSC will likely require the development of novel in vitro culture techniques and devices. Herein we describe the development of a straightforward surface modification of an existing commercial product to enable the efficient study of three dimensional (3D) human bone marrow-derived MSC osteogenic differentiation. Hundreds of 3D microaggregates, of either 42 or 168 cells each, were cultured in osteogenic induction medium and their differentiation was compared with that occurring in traditional two dimensional (2D) monolayer cultures. Osteogenic gene expression and matrix composition was significantly enhanced in the 3D microaggregate cultures. Additionally, BMP-2 gene expression was significantly up-regulated in 3D cultures at day 3 and 7 by approximately 25- and 30-fold, respectively. The difference in BMP-2 gene expression between 2D and 3D cultures was negligible in the more mature day 14 osteogenic cultures. These data support the notion that BMP-2 autocrine signalling is up-regulated in 3D MSC cultures, enhancing osteogenic differentiation. This study provides both mechanistic insight into MSC differentiation, as well as a platform for the efficient generation of microtissue units for further investigation or use in tissue engineering applications.

Absence of intersystem crossing in 1,4-didehydrobenzene

The rate of singlet-to-triplet intersystem crossing in 1,4-didehydrobenzene (the biradical produced as a reactive intermediate in the thermal cycloaromatization of enediynes), cannot be increased by the application of an external magnetic field. The rate of product formation and the distribution of stable products of 2,3-di-n-propyl-1,4-didehydrobenzene thermolysis is unchanged at magnetic flux densities in the range 0–2000 G and at 66 000 G. Similarly, the rate of thermolysis of an unsymmetrical enediyne is insensitive to magnetic field flux in the same range. This finding precludes the modulation of enediyne reaction rates in pharmaceutical and synthetic pursuits.

Alkyl radical geometry controls geminate cage recombination in alkylcobalamins

Abstract: The radical pair that results from photolysis of adenosylcob(II1)alamin (AdoCbl"') undergoes primary geminate recombination with a first-order rate constant of 1 x lo9 s-l. In contrast, methylcob(II1)alamin (CH3Cbl"') and aristeromicylcob(II1)alamin (AriCblII', the carbocyclic analogue of AdoCbl"' in which the ribofuranose ring oxygen has been replaced with a methylene group) does not undergo primary geminate recombination. The ribofwanose group enables a high rate of geminate recombination in the [Ado' Cbl"'] radical pair. This may be due to a stereoelectronic (p-anomeric) effect that maintains a pyramidal geometry at the 5'-carbon of the 5'-deoxyadenosyl radical, or it may be due to hindered rotation about the C4t-C5, bond such that /?-elimination to the olefin is prevented. Recombination in the geminate singlet radical pair is in competition with diffusive escape to form a solvent-separated radical pair. Hyperfine coupling from Co" promotes intersystem crossing to the triplet radical pair (Chagovetz, A. M.; Grissom, C. B. J. Am. Chem. SOC. 1993, 115, 12152). Recombination of the [CH3' Cbl"] radical pair is not prevented by a lack of intersystem crossing, as neither unlabeled or I3C-labeled CH3Cbl"' undergoes geminate recombination. There is only a small difference in the rate of diffusive recombination in the solvent cage for AdoCbl"', AriCbl"', and CH3Cbl"' following photolysis: 2.01 x 10" s-l, 2.20 x lo4 s-l, and 1.16 x lo4 s-l. The rate of diffusive recombination is limited by productive collisions and not by radical geometry or intersystem crossing. The CF3' radical that results from photolysis of (trifluoromethyl)cob(III)alamin (CF3Cbl"') maintains its pyramidal geometry and undergoes faster diffusive recombination in the solvent cage at 51 x lo4 s-l. The C-Co bond dissociation enthalpy in AriCbl"' is 37 f 1.4 kcaymol. The profound difference in geminate recombination rates for AdoCbl"' and CH3Cbl"' is consistent with their different biological roles as enzymatic cofactors: AdoCbl"' is an initiator of radical chain chemistry in the active site, whereas CH3Cbl"' is a methyl group donor in an S~2-type process.

A two-metal ion mechanism operates in the hammerhead ribozyme-mediated cleavage of an RNA substrate

Evidence for a two-metal ion mechanism for cleavage of the HH16 hammerhead ribozyme is provided by monitoring the rate of cleavage of the RNA substrate as a function of La3+ concentration in the presence of a constant concentration of Mg2+. We show that a bell-shaped curve of cleavage activation is obtained as La3+ is added in micromolar concentrations in the presence of 8 mM Mg2+, with a maximal rate of cleavage being attained in the presence of 3 microM La3+. These results show that two-metal ion binding sites on the ribozyme regulate the rate of the cleavage reaction and, on the basis of earlier estimates of the Kd values for Mg2+ of 3.5 mM and > 50 mM, that these sites bind La3+ with estimated Kd values of 0.9 and > 37.5 microM, respectively. Furthermore, given the very different effects of these metal ions at the two binding sites, with displacement of Mg2+ by La3+ at the stronger (relative to Mg2+) binding site activating catalysis and displacement of Mg2+ by La3+ at the weaker (relative to Mg2+) (relative to Mg2+) binding site inhibiting catalysis, we show that the metal ions at these two sites play very different roles. We argue that the metal ion at binding site 1 coordinates the attacking 2'-oxygen species in the reaction and lowers the pKa of the attached proton, thereby increasing the concentration of the attacking alkoxide nucleophile in an equilibrium process. In contrast, the role of the metal ion at binding site 2 is to catalyze the reaction by absorbing the negative charge that accumulates at the leaving 5'-oxygen in the transition state. We suggest structural reasons why the Mg(2+)-La3+ ion combination is particularly suited to demonstrating these different roles of the two-metal ions in the ribozyme cleavage reaction.

Observations on catalysis by hammerhead ribozymes are consistent with a two-divalent-metal-ion mechanism

Significant cleavage by hammerhead ribozymes requires activation by divalent metal ions. Several models have been proposed to account for the influence of metal ions on hammerhead activity. A number of recent papers have presented data that have been interpreted as supporting a one-metal-hydroxide-ion mechanism. In addition, a solvent deuterium isotope effect has been taken as evidence against a proton transfer in the rate-limiting step of the cleavage reaction. We propose that these data are more easily explained by a two-metal-ion mechanism that does not involve a metal hydroxide, but does involve a proton transfer in the rate-limiting step.

Inhibitors of human immunodeficiency virus type 1 reverse transcriptase target distinct phases of early reverse transcription

Early HIV-1 reverse transcription can be separated into initiation and elongation phases. Here we show, using PCR analysis of negative-strand strong-stop DNA [(−)ssDNA] synthesis in intact virus, that different reverse transcriptase (RT) inhibitors affect distinct phases of early natural endogenous reverse transcription (NERT). The effects of nevirapine on NERT were consistent with a mechanism of action including both specific and nonspecific binding events. The nonspecific component of this inhibition targeted the elongation reaction, whereas the specific effect seemed principally to be directed at very early events (initiation or the initiation-elongation switch). In contrast, foscarnet and the nucleoside analog ddATP inhibited both early and late (−)ssDNA synthesis in a similar manner. We also examined compounds that targeted other viral proteins and found that Ro24-7429 (a Tat antagonist) and rosmarinic acid (an integrase inhibitor) also directly inhibited RT. Our results indicate that NERT can be used to identify and evaluate compounds that directly target the reverse transcription complex.

Properties of the bovine viral diarrhoea virus replicase in extracts of infected MDBK cells

An assay for the bovine viral diarrhoea virus (BVDV) replicase was developed using extracts from BVDV-infected cells. The replicase activity was maximal approximately 8 h post-infection as measured by the generation of a genomic length radiolabelled RNA. Using a semi-denaturing gel system, three virus-specific in vitro radiolabelled nascent RNA species were identified. A fast-migrating RNA was demonstrated to be the double-stranded replicative form (RF). A second form was shown to be a partially single-stranded/partially doublestranded RNA, characteristic of the replicative intermediate (RI). A third form, which was often undetectable, migrated between the RF and RI and was probably genomic viral RNA. The optimal replicase activity was dependent on 5–10mM Mg2+ and although it was also active in 1–2mM Mn2+ it was inhibited at higher concentrations. The optimum KCl concentration for labelling of the RI and RF were different, suggestive of at least two distinct replicase activities. These results are supportive of a semi-conservative model of BVDV RNA replication.

Raman spectroscopic study of the minerals apophyllite-(KF) KCa4Si8O20F.8H2O and apophyllite-(KOH) KCa4Si8O20(F,OH).8H2O

Raman spectroscopy complimented with infrared spectroscopy has been used to study the variation in molecular structure of two minerals of the apophyllite mineral group, namely apophyllite-(KF)KCa4Si8O20F.8H2O and apophyllite-(KOH) KCa4Si8O20(F,OH).8H2O. apophyllite-(KF) and apophyllite-(KOH) are different minerals only because of the difference in the percentage of fluorine to hydroxyl ions. The Raman spectra are dominated by a very intense sharp peak at 1059 cm -1. A band at around 846 cm -1 is assigned to the water librational mode. It is proposed that the difference between apophyllite-(KF) and apophyllite-(KOH) is the observation of two Raman bands in the OH stretching region at around 3563 and 3625 cm -1. Multiple water stretching and bending modes are observed showing that there is much variation in hydrogen bonding between water and the silicate surfaces.