Development and characterization in vitro of a catalase-based biosensor for hydrogen peroxide monitoring

There is increasing evidence that hydrogen peroxide (H₂O₂) may act as a neuromodulator in the brain, as well as contributing to neurodegeneration in diseased states, such as Parkinson's disease. The ability to monitor changes in endogenous H₂O₂ in vivo with high temporal resolution is essential in order to further elucidate the roles of H₂O₂ in the central nervous system. Here, we describe the in vitro characterization of an implantable catalase-based H₂O₂ biosensor. The biosensor comprises two amperometric electrodes, one with catalase immobilized on the surface and one without enzyme (blank). The analytical signal is then the difference between the two electrodes. The H₂O₂ sensitivity of various designs was compared, and ranged from 0 to 56 ± 4 mA cm⁻² M⁻¹. The most successful design incorporated a Nafion^® layer followed by a poly-o-phenylenediamine (PPD) polymer layer. Catalase was adsorbed onto the PPD layer and then cross-linked with glutaraldehyde. The ability of the biosensors to exclude interference from ascorbic acid, and other interference species found in vivo, was also tested. A variety of the catalase-based biosensor designs described here show promise for in vivo monitoring of endogenous H₂O₂ in the brain.     

Simultaneous detection of four nitrofuran metabolites in honey using a multiplexing biochip screening assay

A chemiluminescence-based biochip array sensing technique has been developed and applied to the screening of honey samples for residues of banned nitrofuran antibiotics. Using a multiplex approach, metabolites of the four main nitrofuran antibiotics could be simultaneously detected. Individual antibodies specific towards the metabolites were spotted onto biochips. A competitive assay format, with chemiluminescent response, was employed. The method was validated in accordance with EU legislation (2002/657/EC, 2002), and assessed by comparison with UHPLC-MS/MS testing of 134 honey samples of worldwide origin. A similar extraction method, based on extraction of the analytes on Oasis? SPE cartridges, followed by derivatisation with nitrobenzaldehyde and partition into ethyl acetate, was used for both screening and LC-MS/MS methods. The biochip array method was capable of detecting all four metabolites below the reference point for action of 1 μg kg(-1). The detection capability was below 0.5 μg kg(-1) for the metabolites AHD, AOZ and AMOZ; it was below 0.9 μg kg(-1) for SEM. IC(50) values ranged from 0.14 μg kg(-1) (AMOZ) to 2.19 μg kg(-1) (SEM). This biosensor method possesses the potential to be a fit-for-purpose screening technique in the arena of food safety technology.     

Secondary structure and dynamics of an intrinsically unstructured linker domain

The transient secondary structure and dynamics of an intrinsically unstructured linker domain from the 70 kDa subunit of human replication protein A was investigated using solution state NMR. Stable secondary structure, inferred from large secondary chemical shifts, was observed for a segment of the intrinsically unstructured linker domain when it is attached to an N-terminal protein interaction domain. Results from NMR relaxation experiments showed the rotational diffusion for this segment of the intrinsically unstructured linker domain to be correlated with the N-terminal protein interaction domain. When the N-terminal domain is removed, the stable secondary structure is lost and faster rotational diffusion is observed. The large secondary chemical shifts were used to calculate phi and psi dihedral angles and these dihedral angles were used to build a backbone structural model. Restrained molecular dynamics were performed on this new structure using the chemical shift based dihedral angles and a single NOE distance as restraints. In the resulting family of structures a large, solvent exposed loop was observed for the segment of the intrinsically unstructured linker domain that had large secondary chemical shifts.     

Roles of SIRT1 in the acute and restorative phases following induction of inflammation

Endotoxin is a potent inducer of systemic inflammatory responses in human and rodents. Here, we show that in vivo endotoxin triggers a rapid and transient decline in ATP concentration in human peripheral blood leukocytes and murine peripheral blood leukocytes and liver, which is associated with a brief increase in expression of the autophagy indicator LC3-II. In both of these tissues, the ATP concentration reaches a nadir, and autophagy is induced between 2 and 4 h post-endotoxin infusion, and homeostasis is restored within 12 h. Mouse liver SIRT1 and AMP-activated protein kinase (AMPK) protein expression levels decline precipitously within 10 min and remain below detection levels for up to 12 h post-endotoxin administration. In marked contrast, the expression of HIF-1α is induced within 90 min and remains elevated for up to 12 h. The ATP recovery is delayed, and the increases in both HIF-1α expression and autophagy are prolonged in endotoxin-challenged SIRT1 liver knock-out mice. Resveratrol prevents the decline in ATP concentration and SIRT1 expression, as well as the increase in HIF-1α expression and autophagy in liver of endotoxin-challenged wild type mice but not in SIRT1 liver knock-out mice. These results provide novel insight into the state of both cellular bioenergetics and metabolic networks during the acute phase of systemic inflammation and suggest a role for SIRT1 in acute metabolic decline, as well as the restoration of metabolic homeostasis during an inflammatory challenge.     

Microsatellite markers for the native Texas perennial grass, Panicum hallii (Poaceae)

? Premise of the study: We developed microsatellites for Panicum hallii for studies of gene flow, population structure, breeding experiments, and genetic mapping. ? Methods and Results: Next-generation (454) genomic sequence data were used to design markers. Eighteen robust markers were discovered, 15 of which were polymorphic across six accessions of P. hallii var. hallii. Fourteen of the markers cross-amplified in a P. capillare accession. For the 15 polymorphic markers, the total number of alleles per locus ranged from two to 26 (mean: 11.0) across six populations (11-19 individuals per population). Observed heterozygosity (mean: 0.031) was 13.7 times lower than the expected heterozygosity (mean: 0.426). ? Conclusions: The deficit of heterozygous individuals is consistent with P. hallii having a high rate of self-fertilization. These markers will be useful for studies in P. hallii and related species.     

Mapping of ionomic traits in Mimulus guttatus reveals Mo and Cd QTLs that colocalize with MOT1 homologues

Natural variation in the regulation of the accumulation of mineral nutrients and trace elements in plant tissues is crucial to plant metabolism, development, and survival across different habitats. Studies of the genetic basis of natural variation in nutrient metabolism have been facilitated by the development of ionomics. Ionomics is a functional genomic approach for the identification of the genes and gene networks that regulate the elemental composition, or ionome, of an organism. In this study, we evaluated the genetic basis of divergence in elemental composition between an inland annual and a coastal perennial accession of Mimulus guttatus using a recombinant inbred line (RIL) mapping population. Out of 20 elements evaluated, Mo and Cd were the most divergent in accumulation between the two accessions and were highly genetically correlated in the RILs across two replicated experiments. We discovered two major quantitative trait loci (QTL) for Mo accumulation, the largest of which consistently colocalized with a QTL for Cd accumulation. Interestingly, both Mo QTLs also colocalized with the two M. guttatus homologues of MOT1, the only known plant transporter to be involved in natural variation in molybdate uptake.     

How to build a mammalian super-predator

Questions surrounding the biology of large fossil predators that differ markedly from living forms have long intrigued palaeobiologists. Among such taxa few have excited more interest than sabertooth cats, whose distinctive hypertrophied canines are suggestive of killing behaviors and feeding ecologies that may have departed widely from those of extant carnivores. Moreover, considerable variation among sabertooth species is further suggestive of intriguing differences within the group. Behavior and ecology in another large, extinct mammalian carnivore, the Australian marsupial lion (Thylacoleo carnifex), has also proven contentious. In this study, we assemble a wide range of cranio-dental and postcranial indices in a dataset including machairodont sabertooths, T. carnifex and an extensive sample of extant taxa in order to examine the palaeobiology of these charismatic fossil carnivores. Results of multivariate analyses point to significant relationships between behavior and overall body proportions in extant mammalian carnivores. Postcranial morphologies of two American dirk-tooth species of sabertooth (Smilodon) depart greatly from those of living felids and group most closely with bears among living placentals. Scimitar-tooth species of Homotherium and Machairodus cluster with modern pantherine cats. The marsupial lion groups with Smilodon. If these latter two phylogenetically disparate clades do represent a specialized, robust ecomorph adapted to predation on large prey, then it is a body plan that might be effectively identified on the basis of a handful of ‘bear-like’ postcranial features in combination with a more typically ‘felid-like’ carnassialization of the cheektooth row.