In vivo and in vitro anaerobic mating in Candida albicans

Candida albicans cells of opposite mating types are thought to conjugate during infection in mammalian hosts, but paradoxically, the mating-competent opaque state is not stable at mammalian body temperatures. We found that anaerobic conditions stabilize the opaque state at 37 degrees C, block production of farnesol, and permit in vitro mating at 37 degrees C at efficiencies of up to 84%. Aerobically, farnesol prevents mating because it kills the opaque cells necessary for mating, and as a corollary, farnesol production is turned off in opaque cells. These in vitro observations suggest that naturally anaerobic sites, such as the efficiently colonized gastrointestinal (GI) tract, could serve as niches for C. albicans mating. In a direct test of mating in the mouse GI tract, prototrophic cells were obtained from auxotrophic parent cells, confirming that mating will occur in this organ. These cells were true mating products because they were tetraploid, mononuclear, and prototrophic, and they contained the heterologous hisG marker from one of the parental strains.     

Bacteria on leaves: a previously unrecognised source of N2O in grazed pastures

Nitrous oxide (N₂O) emissions from grazed pastures are a product of microbial transformations of nitrogen and the prevailing view is that these only occur in the soil. Here we show this is not the case. We have found ammonia-oxidising bacteria (AOB) are present on plant leaves where they produce N₂O just as in soil. AOB (Nitrosospira sp. predominantly) on the pasture grass Lolium perenne converted 0.02–0.42% (mean 0.12%) of the oxidised ammonia to N₂O. As we have found AOB to be ubiquitous on grasses sampled from urine patches, we propose a ‘plant'' source of N₂O may be a feature of grazed grassland.In terms of climate forcing, nitrous oxide (N₂O) is the third most important greenhouse gas (Blunden and Arndt, 2013). Agriculture is the largest source of anthropogenic N₂O (Reay et al., 2012) with about 20% of agricultural emissions coming from grassland grazed by animals (Oenema et al., 2005).Grazed grassland is a major source of N₂O because grazers harvest nitrogen (N) from plants across a wide area but recycle it back onto the pasture, largely as urine, in patches of very high N concentration. The N in urine patches is often in excess of what can be used by plants resulting in losses through leaching as nitrate, as N₂O and through volatilisation as ammonia (NH₃) creating a high NH₃ environment in the soil and plant canopy; an important point that we will return to later. The established wisdom is that N₂O is generated exclusively by soil-based microbes such as ammonia-oxidising bacteria (AOB). This soil biology is represented in models designed to simulate N₂O emissions and the soil is a target for mitigation strategies such as the use of nitrification inhibitors.We have previously shown that pasture plants can emit N₂O largely through acting as a conduit for emissions generated in the soil, which are themselves controlled to some degree by the plant (Bowatte et al., 2014). In this case the origin of the emission is still the soil microbes. However, AOB have been found on the leaves of plants, for example, Norway spruce (Papen et al., 2002; Teuber et al., 2007) and weeds in rice paddies (Bowatte et al., 2006), prompting us to ask whether AOB might be present on the leaves of pasture species and contribute to N₂O emissions as they do in soil.We looked for AOB on plants in situations where NH₃ concentrations were likely to be high, choosing plants from urine patches in grazed pastures and plants from pastures surrounding a urea fertiliser manufacturing plant. DNA was extracted from the leaves (including both the surface and apoplast) and the presence of AOB tested using PCR. AOB were present in all the species we examined—the grasses Lolium perenne, Dactylis glomerata, Anthoxanthum odoratum, Poa pratensis, Bromus wildenowii and legumes Trifolium repens and T. subterraneum.To measure whether leaf AOB produce N₂O, we used intact plants of ryegrass (L. perenne) lifted as cores from a paddock that had been recently grazed by adult sheep. The cores were installed in a chamber system designed to allow sampling of above- and belowground environments separately (Bowatte et al., 2014). N₂O emissions were measured from untreated (control) plants and from plants where NH₃ was added to the aboveground chamber and leaves were either untreated or sterilised by wiping twice with paper towels soaked in 1% hypoclorite (Sturz et al., 1997) and then with sterile water. We tested for the presence and abundance of AOB on the leaves by extracting DNA and using PCR and real-time PCR targeting the ammonia monoxygenase A (amoA) gene, which is characteristic of AOB. AOB identity was established using cloning and DNA sequencing. Further details of these experiments can be found in the Supplementary Information.The addition of NH₃ to untreated plants significantly stimulated N₂O emissions (P<0.001) compared with the controls; by contrast, the plants with sterilised leaves produced significantly less N₂O than controls (P<0.001) even with NH₃ added (Figure 1) providing strong evidence for emissions being associated with bacteria on the leaves. Control plants did emit N₂O suggesting there was either sufficient NH₃ available for bacterially generated emissions and/or other plant-based mechanisms were involved (Bowatte et al., 2014).Open in a separate windowFigure 1Effect of an elevated NH₃ atmosphere and surface sterilisation of leaves on leaf N₂O emissions measured over 1-h periods on three occasions during the day. Values are means (s.e.m.), where n=7.The major AOB species identified was Nitrosospira strain III7 that has been previously shown to produce N₂O (Jiang and Bakken, 1999). We measured 10⁹ AOB cells per m² ryegrass leaf, assuming a specific leaf area of 250 cm² g⁻¹ leaf.The rate of production of N₂O (0.1–0.17 mg N₂O-N per m² leaf area per hour) can be translated to a field situation using the leaf area index (LAI)—1 m² leaf per m² ground would be an LAI of 1. LAI in a pasture can vary from <1 to >6 depending on the management (for example, Orr et al., 1988). At LAI of 1, the AOB leaf emission rate would equate to a N₂O emission rate of about 0.1–0.3 mg N₂O-N per m² ground per hour. By comparison, the emission rates measured after dairy cattle urine (650 kg N ha⁻¹) was applied to freely and poorly drained soil were 0.024–1.55 and 0.048–3.33 mg N₂O-N per m² ground per hour, respectively (Li and Kelliher, 2005).The fraction of the NH₃ that was converted to N₂O by the leaf AOB was 0.02–0.42% (mean 0.12%). The mean value is close to that measured for Nitrosospira strains including strain III7 isolated from acidic, loamy and sandy soils where values ranged from 0.07 to 0.10% (Jiang and Bakken, 1999). This is good evidence that the AOB on leaves have the capacity to produce N₂O at the same rate as AOB in soils. We do not suggest that leaf AOB will produce as much N₂O as soil microbes; however, because leaf AOB have access to a source of substrate—volatilised NH₃—that is unavailable to soil microbes and may constitute 26% (Laubach et al., 2013) to 40% (Carran et al., 1982) of the N deposited in the urine, N₂O emissions from these aboveground AOB are additional to soil emissions. Further research is required to identify the situations in which leaf AOB contribute to total emissions and to quantify this contribution.     

Generation and characterization of the first inhibitory antibody targeting tumour-associated carbonic anhydrase XII

The carbonic anhydrases (CAs) constitute a family of almost ubiquitous enzymes of significant importance for many physiological and pathological processes. CAs reversely catalyse the conversion of CO₂ + H₂O to HCO₃ ⁻ and H⁺, thereby contributing to the regulation of intracellular pH. Above all, CAs are of key importance for cells that perform glycolysis that inevitably leads to the intracellular accumulation of lactate. CA XII is a plasma membrane-associated isoform of the enzyme, which is induced by hypoxia and oestrogen and, consequently, expressed at high levels on various types of cancer and, intriguingly, on cancer stem cells. The enzyme is directly involved in tumour progression, and its inhibition has an anti-tumour effect. Apart from its role in carcinogenesis, the enzyme contributes to various other diseases like glaucoma and arteriosclerotic plaques, among others. CA XII is therefore regarded as promising target for specific therapies. We have now generated the first monoclonal antibody (6A10) that binds to the catalytic domain of CA XII on vital tumour cells and inhibits CA XII enzyme activity at nanomolar concentrations and thus much more effective than acetazolamide. In vitro results demonstrate that inhibition of CA XII by 6A10 inhibits the growth of tumour cells in 3-dimensional structures. In conclusion, we generated the first specific and efficient biological inhibitor of tumour-associated CA XII. This antibody may serve as a valuable tool for in vivo diagnosis and adjuvant treatment of different types of cancer.     

A telemedicine instrument for remote evaluation of tremor: design and initial applications in fatigue and patients with Parkinson's Disease

Introduction  

A novel system that combines a compact mobile instrument and Internet communications is presented in this paper for remote evaluation of tremors. The system presents a high potential application in Parkinson's disease and connects to the Internet through a TCP/IP protocol. Tremor transduction is carried out by accelerometers, and the data processing, presentation and storage were obtained by a virtual instrument. The system supplies the peak frequency (fp), the amplitude (Afp) and power in this frequency (Pfp), the total power (Ptot), and the power in low (1-4 Hz) and high (4-7 Hz) frequencies (Plf and Phf, respectively).     

Crystal Structure of Dicamba Monooxygenase: A Rieske Nonheme Oxygenase that Catalyzes Oxidative Demethylation

Dicamba (3,6-dichloro-2-methoxybenzoic acid) is a widely used herbicide that is efficiently degraded by soil microbes. These microbes use a novel Rieske nonheme oxygenase, dicamba monooxygenase (DMO), to catalyze the oxidative demethylation of dicamba to 3,6-dichlorosalicylic acid (DCSA) and formaldehyde. We have determined the crystal structures of DMO in the free state, bound to its substrate dicamba, and bound to the product DCSA at 2.10-1.75 Å resolution. The structures show that the DMO active site uses a combination of extensive hydrogen bonding and steric interactions to correctly orient chlorinated, ortho-substituted benzoic-acid-like substrates for catalysis. Unlike other Rieske aromatic oxygenases, DMO oxygenates the exocyclic methyl group, rather than the aromatic ring, of its substrate. This first crystal structure of a Rieske demethylase shows that the Rieske oxygenase structural scaffold can be co-opted to perform varied types of reactions on xenobiotic substrates.     

Tpl2 transduces CD40 and TNF signals that activate ERK and regulates IgE induction by CD40

Macrophages from Tpl2 knockout (Tpl2(-/-)) mice exhibit a defect in ERK activation by lipopolysaccharide (LPS). This impairs the nucleocytoplasmic transport of the tumor necrosis factor alpha (TNF-alpha) mRNA and prevents the induction of TNF-alpha by LPS. As a result, Tpl2(-/-) mice are resistant to LPS/D-galactosamine-induced shock. We demonstrate that Tpl2 is essential for ERK signals transduced by members of the TNF receptor superfamily, such as CD40 and the TNF receptor 1. Thus, ERK activation was impaired in Tpl2(-/-) B cells and macrophages stimulated with agonistic CD40 antibody or TNF-alpha, whereas the induction of other mitogen-activated protein kinases, such as JNK and p38, and the activation of NF-kappaB were unaffected. Tpl2 was recruited to a CD40/TRAF6 complex in response to CD40 stimulation. Moreover, TRAF6, which when overexpressed activates ERK, failed to do so in Tpl2(-/-) cells. The selective signaling defect resulting from the inactivation of Tpl2 allowed us to demonstrate that CD40-mediated ERK activation contributes to immunoglobulin production but is not essential for B-cell proliferation.     

Chemical synthesis of cholesteryl beta-D-galactofuranoside and -pyranoside

Two isomeric cholesteryl galactosides, cholesteryl beta-D-galactofuranoside and -pyranoside, have been synthesized by the Koenigs-Knorr reaction. Glycosylation of cholesterol with 2,3,5,6-tetra-O-benzoyl-D-galactofuranosyl bromide, followed by Zemplén saponification with sodium methoxide, gave cholesteryl beta-D-galactofuranoside. By using 2,3,4,6-tetra-O-acetyl-D-galactopyranosyl bromide as the glycosyl donor, followed by alkaline hydrolysis, cholesteryl beta-D-galactopyranoside was obtained. The title compounds were characterized by their IR spectra and by their (1)H and (13)C NMR spectra. Structure considerations of the two cholesteryl galactosides correlated with data in the literature, thus confirming that cholesteryl beta-D-galactopyranoside is an antigenic lipid of Lyme disease agent, Borrelia burgdorferi.