Heterologous expression and structural characterization of two low pH laccases from a biopulping white-rot fungus Physisporinus rivulosus

The lignin-degrading, biopulping white-rot fungus Physisporinus rivulosus secretes several laccases of distinct features such as thermostability, extremely low pH optima and thermal activation for oxidation of phenolic substrates. Here we describe the cloning, heterologous expression and structural and enzymatic characterisation of two previously undescribed P. rivulosus laccases. The laccase cDNAs were expressed in the methylotrophic yeast Pichia pastoris either with the native or with Saccharomyces cerevisiae α-factor signal peptide. The specific activity of rLac1 and rLac2 was 5 and 0.3 μkat/μg, respectively. However, mutation of the last amino acid in the rLac2 increased the specific laccase activity by over 50-fold. The recombinant rLac1 and rLac2 enzymes demonstrated low pH optima with both 2,6-dimethoxyphenol (2,6-DMP) and 2,2′-azino-bis(3-ethylbenzathiazoline-6-sulfonate). Both recombinant laccases showed moderate thermotolerance and thermal activation at +60 °C was detected with rLac1. By homology modelling, it was deduced that Lac1 and Lac2 enzymes demonstrate structural similarity with the Trametes versicolor and Trametes trogii laccase crystal structures. Comparison of the protein architecture at the reducing substrate-binding pocket near the T1-Cu site indicated the presence of five amino acid substitutions in the structural models of Lac1 and Lac2. These data add up to our previous reports on laccase production by P. rivulosus during biopulping and growth on Norway spruce. Heterologous expression of the novel Lac1 and Lac2 isoenzymes in P. pastoris enables the detailed study of their properties and the evaluation of their potential as oxidative biocatalysts for conversion of wood lignin, lignin-like compounds and soil-polluting xenobiotics.     

Dietary choice for a balanced nutrient intake increases the mean and reduces the variance in the reproductive performance of male and female cockroaches

Sexual selection may cause dietary requirements for reproduction to diverge across the sexes and promote the evolution of different foraging strategies in males and females. However, our understanding of how the sexes regulate their nutrition and the effects that this has on sex‐specific fitness is limited. We quantified how protein (P) and carbohydrate (C) intakes affect reproductive traits in male (pheromone expression) and female (clutch size and gestation time) cockroaches (Nauphoeta cinerea). We then determined how the sexes regulate their intake of nutrients when restricted to a single diet and when given dietary choice and how this affected expression of these important reproductive traits. Pheromone levels that improve male attractiveness, female clutch size and gestation time all peaked at a high daily intake of P:C in a 1:8 ratio. This is surprising because female insects typically require more P than males to maximize reproduction. The relatively low P requirement of females may reflect the action of cockroach endosymbionts that help recycle stored nitrogen for protein synthesis. When constrained to a single diet, both sexes prioritized regulating their daily intake of P over C, although this prioritization was stronger in females than males. When given the choice between diets, both sexes actively regulated their intake of nutrients at a 1:4.8 P:C ratio. The P:C ratio did not overlap exactly with the intake of nutrients that optimized reproductive trait expression. Despite this, cockroaches of both sexes that were given dietary choice generally improved the mean and reduced the variance in all reproductive traits we measured relative to animals fed a single diet from the diet choice pair. This pattern was not as strong when compared to the single best diet in our geometric array, suggesting that the relationship between nutrient balancing and reproduction is complex in this species.     

On the Reversibility and Fragility of Sodium Metal Electrodes

Metallic sodium is receiving renewed interest as a battery anode material because the metal is earth‐abundant, inexpensive, and offers a high specific storage capacity (1166 mAh g^?1 at ?2.71 V vs the standard hydrogen potential). Unlike metallic lithium, the case for Na as the anode in rechargeable batteries has already been demonstrated on a commercial scale in high‐temperature Na||S and Na||NiCl₂ secondary batteries, which increases interest. The reversibility of room temperature sodium anodes is investigated in galvanostatic plating/stripping reactions using in situ optical visualization and galvanostatic polarization measurements. It is discovered that electronic disconnection of mossy metallic Na deposits (“orphaning”) is a dominant source of anode irreversibility in liquid electrolytes. The disconnection is shown by means of direct visualization studies to be triggered by a root‐breakage process during the stripping cycle. As a further step toward electrode designs that are able to accommodate the fragile Na deposits, electrodeposition of Na is demonstrated in nonplanar electrode architectures, which provide continuous and morphology agnostic access to the metal at all stages of electrochemical cycling. On this basis, nonplanar Na electrodes are reported, which exhibit exceptionally high levels of reversibility (Coulombic efficiency >99.6% for 1 mAh cm^?2 Na throughput) in room‐temperature, liquid electrolytes.     

Wolbachia infection can bias estimates of intralocus sexual conflict

Males and females share most of their genome and develop many of the same traits. However, each sex frequently has different optimal values for these shared traits, creating intralocus sexual conflict. This conflict has been observed in wild and laboratory populations of insects and affects important evolutionary processes such as sexual selection, the maintenance of genetic variation, and possibly even speciation. Given the broad impacts of intralocus conflict, accurately detecting and measuring it is important. A common way to detect intralocus sexual conflict is to calculate the intersexual genetic correlation for fitness, with negative values suggesting conflict. Here, we highlight a potential confounder of this measure—cytoplasmic incompatibility caused by the intracellular parasite Wolbachia. Infection with Wolbachia can generate negative intersexual genetic correlations for fitness in insects, suggestive of intralocus sexual conflict. This is because cytoplasmic incompatibility reduces the fitness of uninfected females mated to infected males, while uninfected males will not suffer reductions in fitness if they mate with infected females and may even be fitter than infected males. This can lead to strong negative intersexual genetic correlations for fitness, mimicking intralocus conflict. We illustrate this issue using simulations and then present Drosophila simulans data that show how reproductive incompatibilities caused by Wolbachia infection can generate signals of intralocus sexual conflict. Given that Wolbachia infection in insect populations is pervasive, but populations usually contain both infected and uninfected individuals providing scope for cytoplasmic incompatibility, this is an important consideration for sexual conflict research but one which, to date, has been largely underappreciated.     

Phylogeny of the Celastraceae inferred from phytochrome B gene sequence and morphology

Phylogenetic relationships within Celastraceae were inferred using a simultaneous analysis of 61 morphological characters and 1123 base pairs of phytochrome B exon 1 from the nuclear genome. No gaps were inferred, and the gene tree topology suggests that the primers were specific to a single locus that did not duplicate among the lineages sampled. This region of phytochrome B was most useful for examining relationships among closely related genera. Fifty-one species from 38 genera of Celastraceae were sampled. The Celastraceae sensu lato (including Hippocrateaceae) were resolved as a monophyletic group. Loesener's subfamilies and tribes of Celastraceae were not supported. The Hippocrateaceae were resolved as a monophyletic group nested within a paraphyletic Celastraceae sensu stricto. Goupia was resolved as more closely related to Euphorbiaceae, Corynocarpaceae, and Linaceae than to Celastraceae. Plagiopteron (Flacourtiaceae) was resolved as the sister group of Hippocrateoideae. Brexia (Brexiaceae) was resolved as closely related to Elaeodendron and Pleurostylia. Canotia was resolved as the sister group of Acanthothamnus within Celastraceae. Perrottetia and Mortonia were resolved as the sister group of the rest of the Celastraceae. Siphonodon was resolved as a derived member of Celastraceae. Maytenus was resolved as three disparate groups, suggesting that this large genus needs to be recircumscribed.     

Costimulation blockade, busulfan, and bone marrow promote titratable macrochimerism, induce transplantation tolerance, and correct genetic hemoglobinopathies with minimal myelosuppression.

Mixed hemopoietic chimerism has the potential to correct genetic hemological diseases (sickle cell anemia, thalassemia) and eliminate chronic immunosuppressive therapy following organ transplantation. To date, most strategies require either recipient conditioning (gamma-irradiation, depletion of the peripheral immune system) or administration of "mega" doses of bone marrow to facilitate reliable engraftment. Although encouraging, many issues remain that may restrict or prevent clinical application of such strategies. We describe an alternative, nonirradiation based strategy using a single dose of busulfan, costimulation blockade, and T cell-depleted donor bone marrow, which promotes titratable macrochimerism and a reshaping of the T cell repertoire. Chimeras exhibit robust donor-specific tolerance, evidenced by acceptance of fully allogeneic skin grafts and failure to generate donor-specific proliferative responses in an in vivo graft-versus-host disease model of alloreactivity. In this model, donor cell infusion and costimulation blockade without busulfan were insufficient for tolerance induction as donor-specific IFN-gamma-producing T cells re-emerged and skin grafts were rejected at approximately 100 days. When applied to a murine beta-thalassemia model, this approach allows for the normalization of hemologic parameters and replacement of the diseased red cell compartment. Such a protocol may allow for clinical application of mixed chimerism strategies in patients with end-stage organ disease or hemoglobinopathies.     

Mechanisms of GDF-5 action during skeletal development

Mutations in GDF-5, a member of the TGF-beta superfamily, result in the autosomal recessive syndromes brachypod (bp) in mice and Hunter-Thompson and Grebe-type chondrodysplasias in humans. These syndromes are all characterised by the shortening of the appendicular skeleton and loss or abnormal development of some joints. To investigate how GDF-5 controls skeletogenesis, we overexpressed GDF-5 during chick limb development using the retrovirus, RCASBP. This resulted in up to a 37.5% increase in length of the skeletal elements, which was predominantly due to an increase in the number of chondrocytes. By injecting virus at different stages of development, we show that GDF-5 can increase both the size of the early cartilage condensation and the later developing skeletal element. Using in vitro micromass cultures as a model system to study the early steps of chondrogenesis, we show that GDF-5 increases chondrogenesis in a dose-dependent manner. We did not detect changes in proliferation. However, cell suspension cultures showed that GDF-5 might act at these stages by increasing cell adhesion, a critical determinant of early chondrogenesis. In contrast, pulse labelling experiments of GDF-5-infected limbs showed that at later stages of skeletal development GDF-5 can increase proliferation of chondrocytes. Thus, here we show two mechanisms of how GDF-5 may control different stages of skeletogenesis. Finally, our data show that levels of GDF-5 expression/activity are important in controlling the size of skeletal elements and provides a possible explanation for the variation in the severity of skeletal defects resulting from mutations in GDF-5.     

Electrorotation studies of baby hamster kidney fibroblasts infected with herpes simplex virus type 1

The dielectric properties of baby hamster kidney fibroblast (BHK(C-13)) cells have been measured using electrorotation before and after infection with herpes simplex virus type 1 (HSV-1). The dielectric properties and morphology of the cells were investigated as a function of time after infection. The mean specific capacitance of the uninfected cells was 2.0 microF/cm2, reducing to a value of 1. 5 microF/cm2 at 12 h after infection. This change was interpreted as arising from changes in the cell membrane morphology coupled with alterations in the composition of the cell membrane as infection progressed. The measured changes in the cell capacitance were correlated with alterations in cellular morphology determined from scanning electron microscope (SEM) images. Between 9 and 12 h after infection the internal permittivity of the cell exhibited a rapid change, reducing in value from 75epsilono to 58epsilono, which can be correlated with the generation of large numbers of Golgi-derived membrane vesicles and enveloped viral capsids. The data are discussed in relation to the known life cycle of HSV-1 and indicate that electrorotation can be used to observe dynamic changes in both the dielectric and morphological properties of virus-infected cells. Calculations of the dielectrophoretic spectrum of uninfected and infected cells have been performed, and the results show that cells in the two states could be separated using appropriate frequencies and electrode arrays.     

Regulation of intracellular polyamine biosynthesis and transport by NO and cytokines TNF-alpha and IFN-gamma

Nitric oxide (NO)has been described to exert cytostatic effects on cellularproliferation; however the mechanisms responsible for these effectshave yet to be fully resolved. Polyamines, conversely, are requiredcomponents of cellular proliferation. In experimental models ofinflammation, a relationship between these two pathways has beensuggested by the temporal regulation of a common precursor, arginine.This study was undertaken to determine the effects NO and the NOsynthase (NOS)-inducing cytokines, tumor necrosis factor- (TNF-)and interferon- (IFN-), exert on polyamine regulation. Thetransformed kidney proximal tubule cell line, MCT, maintains highconstitutive levels of the first polyamine biosynthetic enzyme, ornithine decarboxylase (ODC). NO donors markedly suppressed ODC activity in MCT and all other cell lines examined. TNF- and IFN- induction of NO generation resulted in suppressed ODC activity, aneffect prevented by the inducible NOS inhibitorL-N⁶-(1-iminoethyl)lysine(L-NIL). Dithiothreitol reversalof NO-mediated ODC suppression supports nitrosylation as the mechanismof inactivation. We also evaluated polyamine uptake, inasmuch asinhibition of ODC can result in a compensatory induction of polyaminetransporters. Administration of NO donors, or TNF- and IFN-,suppressed[³H]putrescine uptake,thereby preventing transport-mediated reestablishment of intracellularpolyamine levels. This study demonstrates the capacity of NO andinflammatory cytokines to regulate both polyamine biosynthesis and transport.