A high-throughput belowground plant diversity assay using next-generation sequencing of the trnL intron

Plant and Soil - Arbuscular mycorrhizal fungi (AMF) are ubiquitous in wetland habitats, but their diversity and distribution pattern in these habitats is poorly understood. Under varying...     

TBC1D14 regulates autophagy via the TRAPP complex and ATG9 traffic

Macroautophagy requires membrane trafficking and remodelling to form the autophagosome and deliver its contents to lysosomes for degradation. We have previously identified the TBC domain‐containing protein, TBC1D14, as a negative regulator of autophagy that controls delivery of membranes from RAB11‐positive recycling endosomes to forming autophagosomes. In this study, we identify the TRAPP complex, a multi‐subunit tethering complex and GEF for RAB1, as an interactor of TBC1D14. TBC1D14 binds to the TRAPP complex via an N‐terminal 103 amino acid region, and overexpression of this region inhibits both autophagy and secretory traffic. TRAPPC8, the mammalian orthologue of a yeast autophagy‐specific TRAPP subunit, forms part of a mammalian TRAPPIII‐like complex and both this complex and TBC1D14 are needed for RAB1 activation. TRAPPC8 modulates autophagy and secretory trafficking and is required for TBC1D14 to bind TRAPPIII. Importantly, TBC1D14 and TRAPPIII regulate ATG9 trafficking independently of ULK1. We propose a model whereby TBC1D14 and TRAPPIII regulate a constitutive trafficking step from peripheral recycling endosomes to the early Golgi, maintaining the cycling pool of ATG9 required for initiation of autophagy.     

Perspectives of surface plasmon resonance sensors for optimized biogas methanation

Biogas production is becoming significantly viable as an energy source for replacing fossil‐based fuels. The further development of the biogas production process could lead to significant improvements in its potential. Wastewater treatment currently accounts for 3% of the electrical energy load in developed countries, while it could be developed to provide a source of nitrogen and phosphorus, in addition to energy. The improvement of anaerobic digestion (AD) detection technologies is the cornerstone to reach higher methane productivities and develop fully automatized processes to decrease operational costs. New sensors are requested to automatically obtain a better interpretation of the complex and dynamical internal reactor environment. This will require detailed systematic detection in order to realize a near‐optimal production process. In this review, optical fiber‐based sensors will be discussed to assess their potential for use in AD. There is currently a disparity between the complexity of AD, and online detection. By improving the durability, sensitivity, and cost of dissolved H₂ (as well as H₂S, acetic acid, ammonia, and methane) sensor technology, further understanding of the AD process may allow the prevention of process failure. The emergence of surface plasmon resonance (SPR) sensing with optical fibers coupled with the H₂‐sensitive metal palladium, allows detection of dissolved hydrogen in liquid. By implementing these SPR sensors into AD, improvements to the biogas production process, even at small scales, may be achieved by guiding the process in the optimum direction, avoiding the collapse of the biological process. This review intends to assess the feasibility of online, cost‐effective, rapid, and efficient detection of dissolved H₂, as well as briefly assessing H₂S, acetic acid, ammonia, and methane in AD by SPR.     

How quantitative is metabarcoding: A meta‐analytical approach

Metabarcoding has been used in a range of ecological applications such as taxonomic assignment, dietary analysis and the analysis of environmental DNA. However, after a decade of use in these applications there is little consensus on the extent to which proportions of reads generated corresponds to the original proportions of species in a community. To quantify our current understanding, we conducted a structured review and meta‐analysis. The analysis suggests that a weak quantitative relationship may exist between the biomass and sequences produced (slope = 0.52 ± 0.34, p < 0.01), albeit with a large degree of uncertainty. None of the tested moderators, sequencing platform type, the number of species used in a trial or the source of DNA, were able to explain the variance. Our current understanding of the factors affecting the quantitative performance of metabarcoding is still limited: additional research is required before metabarcoding can be confidently utilized for quantitative applications. Until then, we advocate the inclusion of mock communities when metabarcoding as this facilitates direct assessment of the quantitative ability of any given study.     

Clinical safety and efficacy of NG440: a novel combination of rho iso-alpha acids from hops, rosemary, and oleanolic acid for inflammatory conditions

In this report, we examine the clinical safety and efficacy of NG440, a phytochemical-based antiinflammatory formula consisting of a combination of rho iso-alpha acids from hops, rosemary, and oleanolic acid. In a previous study, we demonstrated that NG440 significantly decreased pain by 50% in patients with osteoarthritis. Consistent with these data, results from a multicentre trial indicate that NG440 reduced pain scores in patients with joint discomfort, as measured by VAS (visual analog scale) methodology. As demonstrated in an ex vivo clinical study, these effects on pain relief may be due to reduced inflammatory cytokine production including lower prostaglandin E2 formation. Finally, strong data exist to suggest that NG440 is a safe formula for human consumption. Animal toxicity data revealed no adverse effects of NG440 at dosages < or =250 mg.kg-1.day-1 for 21 days. Furthermore, human trial data suggest that NG440 does not negatively impact cardiovascular and gastrointestinal markers normally affected by selective COX-2 enzyme inhibitors, including platelet function, blood pressure, blood cell count, or fecal calprotectin, a measure of gastrointestinal injury. In conclusion, NG440 may serve as a safe and efficacious alternative in some areas where specific COX-2 inhibitors have been traditionally used.     

Single-gene detection and karyotyping using small-target fluorescence in situ hybridization on maize somatic chromosomes

Combined with a system for identifying each of the chromosomes in a genome, visualizing the location of individual genetic loci by fluorescence in situ hybridization (FISH) would aid in assembling physical and genetic maps. Previously, large genomic clones have been successfully used as FISH probes onto somatic chromosomes but this approach is complicated in species with abundant repetitive elements. In this study, repeat-free portions of sequences that were anchored to particular chromosomes including genes, gene clusters, large cDNAs, and portions of BACs obtained from public databases were used to label the corresponding physical location using FISH. A collection of probes that includes at least one marker on each chromosome in the maize complement was assembled, allowing a small-target karyotyping system to be developed. This set provides the foundation onto which additional loci could be added to strengthen further the ability to perform chromosomal identification in maize and its relatives. The probes were demonstrated to produce signals in several wild relatives of maize, including Zea luxurians, Z. diploperennis, and Tripsacum dactyloides.     

Electron transport pathway for a Streptomyces cytochrome P450: cytochrome P450 105D5-catalyzed fatty acid hydroxylation in Streptomyces coelicolor A3(2)

Streptomyces and other bacterial actinomycete species produce many important natural products, including the majority of known antibiotics, and cytochrome P450 (P450) enzymes catalyze important biosynthetic steps. Relatively few electron transport pathways to P450s have been characterized in bacteria, particularly streptomycete species. One of the 18 P450s in Streptomyces coelicolor A3(2), P450 105D5, was found to bind fatty acids tightly and form hydroxylated products when electrons were delivered from heterologous systems. The six ferredoxin (Fdx) and four flavoprotein Fdx reductase (FDR) proteins coded by genes in S. coelicolor were expressed in Escherichia coli, purified, and used to characterize the electron transfer pathway. Of the many possibilities, the primary pathway was NADH --> FDR1 --> Fdx4 --> P450 105D5. The genes coding for FDR1, Fdx4, and P450 105D5 are located close together in the S. coelicolor genome. Several fatty acids examined were substrates, including those found in S. coelicolor extracts, and all yielded several products. Mass spectra of the products of lauric acid imply the 8-, 9-, 10-, and 11-hydroxy derivatives. Hydroxylated fatty acids were also detected in vivo in S. coelicolor. Rates of electron transfer between the proteins were measured; all steps were faster than overall hydroxylation and consistent with rates of NADH oxidation. Substrate binding, product release, and oxygen binding were relatively fast in the catalytic cycle; high kinetic deuterium isotope effects for all four lauric acid hydroxylations indicated that the rate of C-H bond breaking is rate-limiting in every case. Thus, an electron transfer pathway to a functional Streptomyces P450 has been established.     

Exploration of Ellsworth Subglacial Lake: a concept paper on the development, organisation and execution of an experiment to explore, measure and sample the environment of a West Antarctic subglacial lake

Antarctic subglacial lakes have, over the past few years, been hypothesised to house unique forms of life and hold detailed sedimentary records of past climate change. Testing this hypothesis requires in situ examinations. The direct measurement of subglacial lakes has been considered ever since the largest and best-known lake, named Lake Vostok, was identified as having a deep water-column. The Subglacial Antarctic Lake Environments (SALE) programme, set up by the Scientific Committee on Antarctic Research (SCAR) to oversee subglacial lakes research, state that prior exploration of smaller lakes would be a “prudent way forward”. Over 145 subglacial lakes are known to exist in Antarctica, but one lake in West Antarctica, officially named Ellsworth Subglacial Lake (referred to hereafter as Lake Ellsworth), stands out as a candidate for early exploration. A consortium of over 20 scientists from seven countries and 14 institutions has been assembled to plan the exploration of Lake Ellsworth. An eight-year programme is envisaged: 3 years for a geophysical survey, 2 years for equipment development and testing, 1 year for field planning and operation, and 2 years for sample analysis and data interpretation. The science experiment is simple in concept but complex in execution. Lake Ellsworth will be accessed using hot water drilling. Once lake access is achieved, a probe will be lowered down the borehole and into the lake. The probe will contain a series of instruments to measure biological, chemical and physical characteristics of the lake water and sediments, and will utilise a tether to the ice surface through which power, communication and data will be transmitted. The probe will pass through the water column to the lake floor. The probe will then be pulled up and out of the lake, measuring its environment continually as this is done. Once at the ice surface, any water samples collected will be taken from the probe for laboratory analysis (to take place over subsequent years). The duration of the science mission, from deployment of the probe to its retrieval, is likely to take between 24 and 36 h. Measurements to be taken by the probe will provide data about the following: depth, pressure, conductivity and temperature; pH levels; biomolecules (using life marker chips); anions (using a chemical analyzer); visualisation of the environment (using cameras and light sources); dissolved gases (using chromatography); and morphology of the lake floor and sediment structures (using sonar). After the probe has been retrieved, a sediment corer may be dropped into the lake to recover material from the lake floor. Finally, if time permits, a thermistor string may be left in the lake water to take time-dependent measurements of the lake’s water column over subsequent years. Given that the comprehensive geophysical survey of the lake will take place in two seasons during 2007–2009, a two-year instrument and logistic development phase from 2008 (after the lake’s bathymetry has been assessed) makes it possible that the exploration of Lake Ellsworth could take place at the beginning of the next decade.     

Structural analysis of P450 AmphL from Streptomyces nodosus provides insights into substrate selectivity of polyene macrolide antibiotic biosynthetic P450s

AmphL is a cytochrome P450 enzyme that catalyzes the C8 oxidation of 8-deoxyamphotericin B to the polyene macrolide antibiotic, amphotericin B. To understand this substrate selectivity, we solved the crystal structure of AmphL to a resolution of 2.0 Å in complex with amphotericin B and performed molecular dynamics (MD) simulations. A detailed comparison with the closely related P450, PimD, which catalyzes the epoxidation of 4,5-desepoxypimaricin to the macrolide antibiotic, pimaricin, reveals key catalytic structural features responsible for stereo- and regio-selective oxidation. Both P450s have a similar access channel that runs parallel to the active site I helix over the surface of the heme. Molecular dynamics simulations of substrate binding reveal PimD can “pull” substrates further into the P450 access channel owing to additional electrostatic interactions between the protein and the carboxyl group attached to the hemiketal ring of 4,5-desepoxypimaricin. This substrate interaction is absent in AmphL although the additional substrate -OH groups in 8-deoxyamphotericin B help to correctly position the substrate for C8 oxidation. Simulations of the oxy-complex indicates that these -OH groups may also participate in a proton relay network required for O₂ activation as has been suggested for two other macrolide P450s, PimD and P450eryF. These findings provide experimentally testable models that can potentially contribute to a new generation of novel macrolide antibiotics with enhanced antifungal and/or antiprotozoal efficacy.