DNA barcoding should accompany taxonomy - the case of Cerebratulus spp (Nemertea)

Many issues in DNA barcoding need to be solved before it can reach its goal to become a general database for species identification. While species delimitations are more or less well established in several taxa, there are still many groups where this is not the case. Without the proper taxonomic background/knowledge and corroboration with other kinds of data, the DNA barcoding approach may fail to identify species accurately. The classification and taxonomy of phylum Nemertea (nemerteans, ribbon worms) are traditionally based on morphology, but are not corroborated by an increasing amount of genetic data when it comes to classification either into species or into higher taxa. The taxonomy of the phylum needs to be improved before the full potential of DNA barcoding can be utilized to make sure that valid Linnean names accompany the barcode sequences. We illustrate the problematic situation in the phylum Nemertea by a case study from the genus Cerebratulus.     

Influence of extraction solvent (buffer, methanol, acetone) and time on the quantification of indoIe-3-acetic acid in plants

The effect of extraction solvent and time on the measured indole-3-acetic acid (IAA) level was investigated in plant materials having different contents of lAA-conjugates, Tissues from pine ( Pinus sylvestris L.). tobacco ( Nicotiana tabacum L.), and maize ( Zea mays L.) were extracted for 1–9 h with Na-phosphate buffer (pH 7.5). 80% methanol and 70% acetone. IAA was measured by combined gas chromatography-selected ion minitoring-mass spectromctry (GC-SIM-MS) with [¹³C₆]-IAA as an internal standard.
Extraction of maize seedlings with buffer gave a higher estimate of free IAA than did extraction with methanol or acetone, which produced similar values. The increase in free IAA after buffer extraction was paralleled by a stoichiometric decrease in lAA-ester conjugates, indicating that free IAA was formed during buffer extraction by hydrolysis of these conjugates, which are abundant in maize seedlings. The amount of hydrolysis during a 1-h extraction period was estimated to be ca 3% of the total lAA-ester pool. However, in the pine extraxylary tissues and tobacco in-ternodes which lack a significant lAA-ester pool, buffer extraction resulted in the same IAA estimate as extraction with the organic solvents, but produced a cleaner extract. For all the plant materials investigated, a 1-h extraction period was sufficient for equilibrating the internal standard with the endogenous IAA pool.     

Structural basis of T-cell specificity and activation by the bacterial superantigen TSST-1

Superantigens (SAGs) bind simultaneously to major histocompatibility complex (MHC) and T-cell receptor (TCR) molecules, resulting in the massive release of inflammatory cytokines that can lead to toxic shock syndrome (TSS) and death. A major causative agent of TSS is toxic shock syndrome toxin-1 (TSST-1), which is unique relative to other bacterial SAGs owing to its structural divergence and its stringent TCR specificity. Here, we report the crystal structure of TSST-1 in complex with an affinity-matured variant of its wild-type TCR ligand, human T-cell receptor beta chain variable domain 2.1. From this structure and a model of the wild-type complex, we show that TSST-1 engages TCR ligands in a markedly different way than do other SAGs. We provide a structural basis for the high TCR specificity of TSST-1 and present a model of the TSST-1-dependent MHC-SAG-TCR T-cell signaling complex that is structurally and energetically unique relative to those formed by other SAGs. Our data also suggest that protein plasticity plays an exceptionally significant role in this affinity maturation process that results in more than a 3000-fold increase in affinity.     

Differential attention-dependent response modulation across cell classes in macaque visual area V4

The cortex contains multiple cell types, but studies of attention have not distinguished between them, limiting understanding of the local circuits that transform attentional feedback into improved visual processing. Parvalbumin-expressing inhibitory interneurons can be distinguished from pyramidal neurons based on their briefer action potential durations. We recorded neurons in area V4 as monkeys performed an attention-demanding task. We find that the distribution of action potential durations is strongly bimodal. Neurons with narrow action potentials have higher firing rates and larger attention-dependent increases in absolute firing rate than neurons with broad action potentials. The percentage increase in response is similar across the two classes. We also find evidence that attention increases the reliability of the neuronal response. This modulation is more than two-fold stronger among putative interneurons. These findings lead to the surprising conclusion that the strongest attentional modulation occurs among local interneurons that do not transmit signals between areas.     

Cell-specific chemotyping and multivariate imaging by combined FT-IR microspectroscopy and orthogonal projections to latent structures (OPLS) analysis reveals the chemical landscape of secondary xylem

Fourier‐transform infrared (FT‐IR) spectroscopy combined with microscopy enables chemical information to be acquired from native plant cell walls with high spatial resolution. Combined with a 64 × 64 focal plane array (FPA) detector, 4096 spectra can be simultaneously obtained from a 0.3 × 0.3 mm image; each spectrum represents a compositional and structural ‘fingerprint’ of all cell wall components. For optimal use and analysis of such a large amount of information, multivariate approaches are preferred. Here, FT‐IR microspectroscopy with FPA detection is combined with orthogonal projections to latent structures discriminant analysis (OPLS‐DA). This allows for: (i) the extraction of spectra from single cell types, (ii) identification and characterization of different chemotypes using the full spectral information, and (iii) further visualization of the pattern of identified chemotypes by multivariate imaging. As proof of concept, the chemotypes of Populus tremula xylem cell types are described. The approach revealed unknown features about chemical plasticity and patterns of lignin composition in wood fibers that would have remained hidden in the dataset with traditional data analysis. The applicability of the method to Arabidopsis xylem and its usefulness in mutant chemotyping is also demonstrated. The methodological approach is not limited to xylem tissues but can be applied to any plant organ/tissue also using other techniques such as Raman and UV microspectroscopy.     

Characterisation of source-separated household waste intended for composting

Large-scale composting of source-separated household waste has expanded in recent years in the Nordic countries. One problem can be low pH at the start of the process. Incoming biowaste at four composting plants was characterised chemically, physically and microbiologically. The pH of food waste ranged from 4.7 to 6.1 and organic acid concentration from 24 to 81 mmol kg(-1). The bacterial diversity in the waste samples was high, with all samples dominated by Gammaproteobacteria, particularly Pseudomonas and Enterobacteria (Escherichia coli, Klebsiella, Enterobacter). Lactic acid bacteria were also numerically important and are known to negatively affect the composting process because the lactic acid they produce lowers the pH, inhibiting other bacteria. The bacterial groups needed for efficient composting, i.e. Bacillales and Actinobacteria, were present in appreciable amounts. The results indicated that start-up problems in the composting process can be prevented by recycling bulk material and compost.     

Important processes during differentiation and early development of somatic embryos of Norway spruce as revealed by changes in global gene expression

The aim of this study has been to identify important processes that regulate early stages of embryo development in conifers. Somatic embryogenesis in Picea abies has become a model system for studying embryology in conifers, providing a well-characterized sequence of developmental stages, resembling zygotic embryogeny, which can be synchronized by specific treatments, making it possible to collect a large number of somatic embryos at specific developmental stages. We have used this model to analyze global changes in gene expression during early stages of embryo development by generating an expression profile of 12,536 complementary DNA clones. This has allowed us to identify molecular events regulating putative processes associated with pattern formation during the earliest stages of embryogenesis which have not been identified on the molecular level in conifers before. We recognize notable changes in the expression of genes involved in regulating auxin biosynthesis and auxin response, gibberellin-mediated signaling, signaling between the embryo and the female gametophyte, tissue specification including the formation of boundary regions, and the switch from embryonic to vegetative development. In addition, our results confirm the involvement of previously described processes, including stress, differentiation of a protoderm, and programmed cell death.     

Crystal structure of human peptidoglycan recognition protein S (PGRP-S) at 1.70 A resolution

Peptidoglycan recognition proteins (PGRPs) are pattern recognition receptors of the innate immune system that bind peptidoglycans (PGNs) of bacterial cell walls. These molecules, which are highly conserved from insects to mammals, contribute to host defense against infections by both Gram-positive and Gram-negative bacteria. Here, we present the crystal structure of human PGRP-S at 1.70A resolution. The overall structure of PGRP-S, which participates in intracellular killing of Gram-positive bacteria, is similar to that of other PGRPs, including Drosophila PGRP-LB and PGRP-SA and human PGRP-Ialpha. However, comparison with these PGRPs reveals important differences in both the PGN-binding site and a groove formed by the PGRP-specific segment on the opposite face of the molecule. This groove, which may constitute a binding site for effector or signaling proteins, is less hydrophobic and deeper in PGRP-S than in PGRP-IalphaC, whose PGRP-specific segments vary considerably in amino acid sequence. By docking a PGN ligand into the PGN-binding cleft of PGRP-S based on the known structure of a PGRP-Ialpha-PGN complex, we identified potential PGN-binding residues in PGRP-S. Differences in PGN-contacting residues and interactions suggest that, although PGRPs may engage PGNs in a similar mode, structural differences exist that likely regulate the affinity and fine specificity of PGN recognition.     

Characterization of T cell receptors engineered for high affinity against toxic shock syndrome toxin-1

Superantigens, including bacterial enterotoxins, are a family of proteins that bind simultaneously to MHC class II molecules and the Vbeta regions of T cell receptors. This cross-linking results in the activation of a large population of T cells that release massive amounts of inflammatory cytokines, ultimately causing a condition known as toxic shock syndrome. The staphylococcal superantigen toxic shock syndrome toxin-1 (TSST-1) is a causative agent of this disease, but its structure in complex with the cognate T cell receptor (human Vbeta2.1) has not been determined. To understand the molecular details of the interaction and to develop high affinity antagonists to TSST-1, we used directed evolution to generate a panel of high affinity receptors for TSST-1. Yeast display libraries of random and site-directed hVbeta2.1 mutants were selected for improved domain stability and for higher affinity binding to TSST-1. Stability mutations allowed the individual Vbeta domains to be expressed in a bacterial expression system. Affinity mutations were generated in CDR2 and FR3 residues, yielding improvements in affinity of greater than 10,000-fold (a K(D) value of 180 pmol). Alanine scanning mutagenesis of hVbeta2.1 wild-type and mutated residues allowed us to generate a map of the binding site for TSST-1 and to construct a docking model for the hVbeta2.1-TSST-1 complex. Our experiments suggest that the energetic importance of a single hVbeta2.1 wild-type residue likely accounts for the restriction of TSST-1 specificity to only this human Vbeta region. The high affinity mutants described here thus provide critical insight into the molecular basis of TSST-1 specificity and serve as potential leads toward the development of therapeutic agents for superantigen-mediated disease.