Improved fuel cell and electrode designs for producing electricity from microbial degradation

A new one-compartment fuel cell was composed of a rubber bunged bottle with a center-inserted anode and a window-mounted cathode containing an internal, proton-permeable porcelain layer. This fuel cell design was less expensive and more practical than the conventional two-compartment system, which requires aeration and a ferricyanide solution in the cathode compartment. Three new electrodes containing bound electron mediators including a Mn(4+)-graphite anode, a neutral red (NR) covalently linked woven graphite anode, and an Fe(3+)-graphite cathode were developed that greatly enhanced electrical energy production (i.e., microbial electron transfer) over conventional graphite electrodes. The potentials of these electrodes measured by cyclic voltametry at pH 7.0 were (in volts): +0.493 (Fe(3+)-graphite); +0.15 (Mn(4+)-graphite); and -0.53 (NR-woven graphite). The maximal electrical productivities obtained with sewage sludge as the biocatalyst and using a Mn(4+)-graphite anode and a Fe(3+)-graphite cathode were 14 mA current, 0.45 V potential, 1,750 mA/m(2) current density, and 788 mW/m(2) of power density. With Escherichia coli as the biocatalyst and using a Mn(4+)-graphite anode and a Fe(3+)-graphite cathode, the maximal electrical productivities obtained were 2.6 mA current, 0.28 V potential, 325 mA/m(2) current density, and 91 mW/m(2) of power density. These results show that the amount of electrical energy produced by microbial fuel cells can be increased 1,000-fold by incorporating electron mediators into graphite electrodes. These results also imply that sewage sludge may contain unique electrophilic microbes that transfer electrons more readily than E. coli and that microbial fuel cells using the new Mn(4+)-graphite anode and Fe(3+)-graphite cathode may have commercial utility for producing low amounts of electrical power needed in remote locations.     

Linkage mapping of the primary disease locus for collie eye anomaly

Collie eye anomaly (cea) is a hereditary ocular disorder affecting development of the choroid and sclera segregating in several breeds of dog, including rough, smooth, and Border collies and Australian shepherds. The disease is reminiscent of the choroidal hypoplasia phenotype observed in humans in conjunction with craniofacial or renal abnormalities. In dogs, however, the clinical phenotype can vary significantly; many dogs exhibit no obvious clinical consequences and retain apparently normal vision throughout life, while severely affected animals develop secondary retinal detachment, intraocular hemorrhage, and blindness. We report genetic studies establishing that the primary cea phenotype, choroidal hypoplasia, segregates as an autosomal recessive trait with nearly 100% penetrance. We further report linkage mapping of the primary cea locus to a 3.9-cM region of canine chromosome 37 (LOD = 22.17 at theta = 0.076), in a region corresponding to human chromosome 2q35. These results suggest the presence of a developmental regulatory gene important in ocular embryogenesis, with potential implications for other disorders of ocular vascularization.     

Inhibition modifies the effects of slow calcium-activated potassium channels on epileptiform activity in a neuronal network model

Generation of epileptiform activity typically results from a change in the balance between network excitation and inhibition. Experimental evidence indicates that alterations of either synaptic activity or intrinsic membrane properties can produce increased network excitation. The slow Ca²⁺-activated K⁺ currents (sI_AHP) are important modulators of neuronal firing rate and excitability and have important established and potential roles in epileptogenesis. While the effects of changes in sI_AHP on individual neuronal excitability are readily studied and well established, the effects of such changes on network behavior are less well known. The experiments here utilize a defined small network model of multicompartment pyramidal cells and an inhibitory interneuron to study the effects of changes in sI_AHP on network behavior. The benefits of this model system include the ability to observe activity in all cells in a network and the effects of interactions of multiple simultaneous influences. In the model with no inhibitory interneuron, increasing sI_AHP results in progressively decreasing burst activity. Adding an inhibitory interneuron changes the observed effects; at modest inhibitory strengths, increasing sI_AHP in all network neurons actually results in increased network bursting (except at very high values). The duration of the burst activity is influenced by the length of delay in a feedback loop, with longer loops resulting in more prolonged bursting. These observations illustrate that the study of potential antiepileptogenic membrane effects must be extended to realistic networks. Network inhibition can dramatically alter the observations seen in pure excitatory networks.     

A deubiquitinating enzyme encoded by HSV-1 belongs to a family of cysteine proteases that is conserved across the family Herpesviridae

We have discovered a ubiquitin (Ub)-specific cysteine protease encoded within the N-terminal approximately 500 residues of the UL36 gene product, the largest (3164 aa) tegument protein of herpes simplex virus 1 (HSV-1). Enzymatic activity of this fragment, UL36USP, is detectable only after cleavage of UL36USP from full-length UL36 and occurs late during viral replication. UL36USP bears no homology to known deubiquitinating enzymes (DUBs) or Ub binding proteins. Sequence alignment of the large tegument proteins across the family Herpesviridae indicates conservation of key catalytic residues amongst these viruses. Recombinant UL36USP exhibits hydrolytic activity toward Ub-AMC and ubiquitinated branched peptides in vitro. In addition, recombinant UL36USP can cleave polyUb chains and appears to be specific for Lys48 linkages. Mutation of the active site cysteine residue (Cys65) to alanine abolishes this enzymatic activity. The lack of homology between UL36USP and eukaryotic DUBs makes this new family of herpesvirus ubiquitin-specific proteases attractive targets for selective inhibition.     

Assessing agricultural soil acidification and nutrient management in life cycle assessment

Purpose  

This paper describes part of the first detailed environmental life cycle assessment (LCA) of Australian red meat (beef and sheep meat) production. The study was intended to assist the methodological development of life cycle impact assessment by examining the feasibility of new indicators for natural resource management (NRM) issues relevant to soil management in agricultural LCA. This paper is intended to describe the NRM indicators directly related to agricultural soil chemistry.     

Superresolution imaging of multiple fluorescent proteins with highly overlapping emission spectra in living cells

Localization-based superresolution optical imaging is rapidly gaining popularity, yet limited availability of genetically encoded photoactivatable fluorescent probes with distinct emission spectra impedes simultaneous visualization of multiple molecular species in living cells. We introduce PAmKate, a monomeric photoactivatable far-red fluorescent protein, which facilitates simultaneous imaging of three photoactivatable proteins in mammalian cells using fluorescence photoactivation localization microscopy (FPALM). Successful probe identification was achieved by measuring the fluorescence emission intensity in two distinct spectral channels spanning only ∼100 nm of the visible spectrum. Raft-, non-raft-, and cytoskeleton-associated proteins were simultaneously imaged in both live and fixed fibroblasts coexpressing Dendra2-hemagglutinin, PAmKate-transferrin receptor, and PAmCherry1-β-actin fusion constructs, revealing correlations between the membrane proteins and membrane-associated actin structures.     

Excess of de novo deleterious mutations in genes associated with glutamatergic systems in nonsyndromic intellectual disability

Little is known about the genetics of nonsyndromic intellectual disability (NSID). We hypothesized that de novo mutations (DNMs) in synaptic genes explain an important fraction of sporadic NSID cases. In order to investigate this possibility, we sequenced 197 genes encoding glutamate receptors and a large subset of their known interacting proteins in 95 sporadic cases of NSID. We found 11 DNMs, including ten potentially deleterious mutations (three nonsense, two splicing, one frameshift, four missense) and one neutral mutation (silent) in eight different genes. Calculation of point-substitution DNM rates per functional and neutral site showed significant excess of functional DNMs compared to neutral ones. De novo truncating and/or splicing mutations in SYNGAP1, STXBP1, and SHANK3 were found in six patients and are likely to be pathogenic. De novo missense mutations were found in KIF1A, GRIN1, CACNG2, and EPB41L1. Functional studies showed that all these missense mutations affect protein function in cell culture systems, suggesting that they may be pathogenic. Sequencing these four genes in 50 additional sporadic cases of NSID identified a second DNM in GRIN1 (c.1679_1681dup/p.Ser560dup). This mutation also affects protein function, consistent with structural predictions. None of these mutations or any other DNMs were identified in these genes in 285 healthy controls. This study highlights the importance of the glutamate receptor complexes in NSID and further supports the role of DNMs in this disorder.     

Spectroscopic investigations of intermediates in the reaction of cytochrome P450BM3–F87G with surrogate oxygen atom donors

Rapid mixing of substrate-free ferric cytochrome P450_BM3–F87G with m-chloroperoxybenzoic acid (mCPBA) resulted in the sequential formation of two high-valent intermediates. The first was spectrally similar to compound I species reported previously for P450_CAM and CYP 119 using mCPBA as an oxidant, and it featured a low intensity Soret absorption band characterized by shoulder at 370 nm. This is the first direct observation of a P450 compound I intermediate in a type II P450 enzyme. The second intermediate, which was much more stable at pH values below 7.0, was characterized by an intense Soret absorption peak at 406 nm, similar to that seen with P450_CAM [T. Spolitak, J.H. Dawson, D.P. Ballou, J. Biol. Chem. 280 (2005) 20300–20309]. Double mixing experiments in which NADPH was added to the transient 406 nm-absorbing intermediate resulted in rapid regeneration of the resting ferric state, with the flavins of the flavoprotein domain in their reduced state. EPR results were consistent with this stable intermediate species being a cytochrome c peroxidase compound ES-like species containing a protein-based radical, likely localized on a nearby Trp or Tyr residue in the active site. Iodosobenzene, peracetic acid, and sodium m-periodate also generated the intermediate at 406 nm, but not the 370 nm intermediate, indicating a probable kinetic barrier to accumulating compound I in reactions with these oxidants. The P450 ES intermediate has not been previously reported using iodosobenzene or m-periodate as the oxygen donor.     

Chirally dimorphic male genitalia in praying mantids (Ciulfina: Liturgusidae)

Although male polymorphisms occur widely in nature and have received considerable recent attention from studies of alternative mating strategies, male genital polymorphisms are less well known. Here, we describe a dimorphism in the orientation of the male genitalic complex of the praying mantid genus Ciulfina. Populations of Ciulfina species vary in the proportion of males with dextral (right‐oriented) and sinistral (left‐oriented) genitalia, ranging from directional asymmetry (single orientation only) to apparent antisymmetry (equal proportions of both orientations). The proportion of dextral males varied between species (C. baldersoni: 46%; C. rentzi: 24%; C. klassi: 100%; C. biseriata: 83%) and between populations. We used elliptic Fourier analysis to quantify shape and size variation between the genitalia of dextral and sinistral males and determined that the two forms were mirror images of one another in two species. We found that the level of mechanical reproductive isolation between heterospecific populations of opposite genital orientation was no greater than that between heterospecific populations with the same orientation or of mixed orientation. Genital orientation therefore did not influence premating isolation between these species, despite complete postmating isolation. The geographic proximity of populations to heterospecifics also showed no particular pattern with respect to genital orientation. These results suggest that reversible trait asymmetry in Ciulfina is not driven by reproductive isolation, and add to the growing evidence against the species isolation hypothesis for rapid genital evolution. J. Morphol. 271:1176–1184, 2010. © 2010 Wiley‐Liss, Inc.