Development of Improved Spectrophotometric Assays for Biocatalytic Silyl Ether Hydrolysis

Reported herein is the development of assays for the spectrophotometric quantification of biocatalytic silicon−oxygen bond hydrolysis. Central to these assays are a series of chromogenic substrates that release highly absorbing phenoxy anions upon cleavage of the sessile bond. These substrates were tested with silicatein, an enzyme from a marine sponge that is known to catalyse the hydrolysis and condensation of silyl ethers. It was found that, of the substrates tested, tert-butyldimethyl(2-methyl-4-nitrophenoxy)silane provided the best assay performance, as evidenced by the highest ratio of enzyme catalysed reaction rate compared with the background (uncatalysed) reaction. These substrates were also found to be suitable for detailed enzyme kinetics measurements, as demonstrated by their use to determine the Michaelis−Menten kinetic parameters for silicatein

Regulation of mesenchymal stem cell morphology using hydrogel substrates with tunable topography and photoswitchable stiffness

Cell function can be directly influenced by the mechanical and structural properties of the extracellular environment. In particular, cell morphology and phenotype can be regulated via the modulation of both the stiffness and surface topography of cell culture substrates. Previous studies have highlighted the ability to design cell culture substrates to optimise cell function. Many such examples, however, employ photo-crosslinkable polymers with a terminal stiffness or surface profile. This study presents a system of polyacrylamide hydrogels, where the surface topography can be tailored and the matrix stiffness can be altered in situ with photoirradiation. The process allows for the temporal regulation of the extracellular environment. Specifically, the surface topography can be tailored via reticulation parameters to include creased features with control over the periodicity, length and branching. The matrix stiffness can also be dynamically tuned via exposure to an appropriate dosage and wavelength of light, thus, allowing for the temporal regulation of the extracellular environment. When cultured on the surface of the hydrogels, the morphology and alignment of immortalised human mesenchymal stem cells can be directly influenced through the tailoring of surface creases, while cell size can be altered via changes in matrix stiffness. This system offers a new platform to study cellular mechanosensing and the influence of extracellular cues on cell phenotype and function

Frataxin deficiency increases cyclooxygenase 2 and prostaglandins in cell and animal models of Friedreich's ataxia

© The Author 2014. Published by Oxford University Press This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.An inherited deficiency of the mitochondrial protein frataxin causes Friedreich's ataxia (FRDA); the mechanism by which this deficiency triggers neuro- and cardio-degeneration is unclear. Microarrays of neural tissue of animal models of the disease showed decreases in antioxidant genes, and increases in inflammatory genes. Cyclooxygenase (COX)-derived oxylipins are important mediators of inflammation. We measured oxylipin levels using tandem mass spectrometry and ELISAs in multiple cell and animal models of FRDA. Mass spectrometry revealed increases in concentrations of prostaglandins, thromboxane B2, 15-HETE and 11-HETE in cerebellar samples of knockin knockout mice. One possible explanation for the elevated oxylipins is that frataxin deficiency results in increased COX activity. While constitutive COX1 was unchanged, inducible COX2 expression was elevated over 1.35-fold (P < 0.05) in two Friedreich's mouse models and Friedreich's lymphocytes. Consistent with higher COX2 expression, its activity was also increased by 58% over controls. COX2 expression is driven by multiple transcription factors, including activator protein 1 and cAMP response element-binding protein, both of which were elevated over 1.52-fold in cerebella. Taken together, the results support the hypothesis that reduced expression of frataxin leads to elevation of COX2-mediated oxylipin synthesis stimulated by increases in transcription factors that respond to increased reactive oxygen species. These findings support a neuroinflammatory mechanism in FRDA, which has both pathomechanistic and therapeutic implications.The study was supported by NIH grants NS077777, EY012245 and AG025532 to G.A.C., and USDA-ARS Intramural Projects 5306-51530-019-00D and 1 U24 DK097154-01 to J.W.N. Funding to pay the Open Access publication charges for this article was provided by the NIH

Investigation of the neurovascular coupling in positive and negative BOLD responses in human brain at 7T

Decreases in stimulus-dependent blood oxygenation level dependent (BOLD) signal and their underlying neurovascular origins have recently gained considerable interest. In this study a multi-echo, BOLD-corrected vascular space occupancy (VASO) functional magnetic resonance imaging (fMRI) technique was used to investigate neurovascular responses during stimuli that elicit positive and negative BOLD responses in human brain at 7 T. Stimulus-induced BOLD, cerebral blood volume (CBV), and cerebral blood flow (CBF) changes were measured and analyzed in ‘arterial’ and ‘venous’ blood compartments in macro- and microvasculature. We found that the overall interplay of mean CBV, CBF and BOLD responses is similar for tasks inducing positive and negative BOLD responses. Some aspects of the neurovascular coupling however, such as the temporal response, cortical depth dependence, and the weighting between ‘arterial’ and ‘venous’ contributions, are significantly different for the different task conditions. Namely, while for excitatory tasks the BOLD response peaks at the cortical surface, and the CBV change is similar in cortex and pial vasculature, inhibitory tasks are associated with a maximum negative BOLD response in deeper layers, with CBV showing strong constriction of surface arteries and a faster return to baseline. The different interplays of CBV, CBF and BOLD during excitatory and inhibitory responses suggests different underlying hemodynamic mechanisms

The snomipede : a parallel platform for scanning near-field photolithography.

Using scanning near-field lithography (SNP), it is possible to pattern molecules at surfaces with a resolution as good as 9 nm [M. Montague, R. E. Ducker, K. S. L. Chong, R. J. Manning, F. J. M. Rutten, M. C. Davies and G. J. Leggett, Langmuir 23 (13), 7328–7337 (2007)]. However, in common with other scanning probe techniques, SNP has previously been considered a serial process, hindering its use in many applications. IBM’s “Millipede” addresses this problem by utilizing an array of local probes operating in parallel. Here, we describe the construction of two instruments (Snomipedes) that integrate near-field optical methods into the parallel probe paradigm and promise the integration of top–down and bottom–up fabrication methods over macroscopic areas. Both are capable of performing near-field lithography with 16 probes in parallel spanning approximately 2 mm. The instruments can work in both ambient and liquid environments, key to many applications in nanobiology. In both, separate control of writing is possible for each probe. We demonstrate the deprotection of self-assembled monolayers of alkylsilanes with photocleavable protecting groups and subsequent growth of nanostructured polymer brushes from these nanopatterned surfaces by atom-transfer radical polymerization

Sensitive and selective detection of DNA fragments associated with Ganoderma boninense by DNA-nanoparticle conjugate hybridisation

From Springer Nature via Jisc Publications RouterHistory: received 2020-05-04, accepted 2020-07-05, registration 2020-07-05, pub-electronic 2020-07-20, online 2020-07-20, pub-print 2020-10Publication status: PublishedFunder: Newton Fund; doi: http://dx.doi.org/10.13039/100010897; Grant(s): British Council 216196834Funder: Engineering and Physical Sciences Research Council; doi: http://dx.doi.org/10.13039/501100000266; Grant(s): EP/K024485/1Funder: Higher Education Funding Council for England; doi: http://dx.doi.org/10.13039/100011722; Grant(s): N8 Agrifood NetworkAbstract: A colourimetric assay for the detection of DNA fragments associated with the oil palm pathogen Ganoderma boninense and other fungi DNA is reported. The assay is based on the aggregation of DNA-nanoparticle conjugates in the presence of complementary DNA from the target organism. Here, various designs of DNA-nanoparticle conjugates were evaluated, and it was found that the best design gave a visually observable colour change with as little as 2 pmol of double-stranded DNA analyte even in the presence of a large excess of a mixture of non-complementary DNA. Overall, this label-free system is rapid, sensitive, selective, simple in design, and easy to carry out. It does not require specialist equipment or specialist training for the interpretation of the results, and therefore has the potential to be deployed for agricultural diagnostics in the field. Graphic abstract: Development of a colourimetric assay based on DNA-nanoparticle conjugates for the oil palm pathogen Ganoderma boninense

Biocatalytic Silylation: The Condensation of Phenols and Alcohols with Triethylsilanol

From MDPI via Jisc Publications RouterHistory: accepted 2021-07-20, pub-electronic 2021-07-22Publication status: PublishedFunder: Engineering and Physical Sciences Research Council; Grant(s): EP/S013539/1, EP/M506436/1Funder: Biotechnology and Biological Sciences Research Council; Grant(s): BB/L013649/1, BB/J014478/1, BB/M017702/1Funder: Tertiary Education Trust Fund; Grant(s): Graduate ScholarshipSilicatein-α (Silα), a hydrolytic enzyme derived from siliceous marine sponges, is one of the few enzymes in nature capable of catalysing the metathesis of silicon–oxygen bonds. It is therefore of interest as a possible biocatalyst for the synthesis of organosiloxanes. To further investigate the substrate scope of this enzyme, a series of condensation reactions with a variety of phenols and aliphatic alcohols were carried out. In general, it was observed that Silα demonstrated a preference for phenols, though the conversions were relatively modest in most cases. In the two pairs of chiral alcohols that were investigated, it was found that the enzyme displayed a preference for the silylation of the S-enantiomers. Additionally, the enzyme’s tolerance to a range of solvents was tested. Silα had the highest level of substrate conversion in the nonpolar solvents n-octane and toluene, although the inclusion of up to 20% of 1,4-dioxane was tolerated. These results suggest that Silα is a potential candidate for directed evolution toward future application as a robust and selective biocatalyst for organosiloxane chemistry

Biocatalytic Silylation: The Condensation of Phenols and Alcohols with Triethylsilanol

Silicatein-α (Silα), a hydrolytic enzyme derived from siliceous marine sponges, is one of the few enzymes in nature capable of catalysing the metathesis of silicon–oxygen bonds. It is therefore of interest as a possible biocatalyst for the synthesis of organosiloxanes. To further investigate the substrate scope of this enzyme, a series of condensation reactions with a variety of phenols and aliphatic alcohols were carried out. In general, it was observed that Silα demonstrated a preference for phenols, though the conversions were relatively modest in most cases. In the two pairs of chiral alcohols that were investigated, it was found that the enzyme displayed a preference for the silylation of the S-enantiomers. Additionally, the enzyme’s tolerance to a range of solvents was tested. Silα had the highest level of substrate conversion in the nonpolar solvents n-octane and toluene, although the inclusion of up to 20% of 1,4-dioxane was tolerated. These results suggest that Silα is a potential candidate for directed evolution toward future application as a robust and selective biocatalyst for organosiloxane chemistry

Nucleon and hadron structure changes in the nuclear medium and impact on observables

We review the effect of hadron structure changes in a nuclear medium using the quark-meson coupling (QMC) model, which is based on a mean field description of non-overlapping nucleon (or baryon) bags bound by the self-consistent exchange of scalar and vector mesons. This approach leads to simple scaling relations for the changes of hadron masses in a nuclear medium. It can also be extended to describe finite nuclei, as well as the properties of hypernuclei and meson-nucleus deeply bound states. It is of great interest that the model predicts a variation of the nucleon form factors in nuclear matter. We also study the empirically observed, Bloom-Gilman (quark-hadron) duality. Other applications of the model include subthreshold kaon production in heavy ion collisions, D and D-bar meson production in antiproton-nucleus collisions, and J/Psi suppression. In particular, the modification of the D and D-bar meson properties in nuclear medium can lead to a large J/Psi absorption cross section, which explains the observed J/Psi suppression in relativistic heavy ion collisions.Comment: 143 pages, 77 figures, references added, a review article accepted in Prog. Part. Nucl. Phy