Lessons from assembling UCEs: A comparison of common methods and the case of Clavinomia (Halictidae)

Sequence data assembly is a foundational step in high-throughput sequencing, with untold consequences for downstream analyses. Despite this, few studies have interrogated the many methods for assembling phylogenomic UCE data for their comparative efficacy, or for how outputs may be impacted. We study this by comparing the most commonly used assembly methods for UCEs in the under-studied bee lineage Nomiinae and a representative sampling of relatives. Data for 63 UCE-only and 75 mixed taxa were assembled with five methods, including ABySS, HybPiper, SPAdes, Trinity and Velvet, and then benchmarked for their relative performance in terms of locus capture parameters and phylogenetic reconstruction. Unexpectedly, Trinity and Velvet trailed the other methods in terms of locus capture and DNA matrix density, whereas SPAdes performed favourably in most assessed metrics. In comparison with SPAdes, the guided-assembly approach HybPiper generally recovered the highest quality loci but in lower numbers. Based on our results, we formally move Clavinomia to Dieunomiini and render Epinomia once more a subgenus of Dieunomia. We strongly advise that future studies more closely examine the influence of assembly approach on their results, or, minimally, use better-performing assembly methods such as SPAdes or HybPiper. In this way, we can move forward with phylogenomic studies in a more standardized, comparable manner.     

Human biomonitoring of 73 elements in blood,serum, erythrocytes and urine

BackgroundHuman biomonitoring studies of trace elements in biological fluids are mostly limited to a certain number of elements or biological materials. In this study, we describe the significant extension of a biomonitoring to 73 elements being present in concentration ranges from ng/L to g/L in clinically relevant specimens such as blood, serum, erythrocytes and urine.MethodsThe samples were collected from 102 occupationally non-exposed inhabitants of northern Germany. The elements were determined either by inductively coupled plasma tandem mass spectrometry (ICP-MS/MS) in the low concentration range or by inductively coupled plasma optical emission spectrometry (ICP-OES) for essential trace elements and electrolytes.ResultsMean values and selected percentiles of element concentrations are presented for all sample materials. From the results, we calculated the distribution of elements between plasma and blood cells. Application of ICP-MS/MS improves selectivity and accuracy in the determination of elements that are strongly spectrally interfered, such as Cr, Ge, Pd or Ti in blood samples.ConclusionsThis publication provides very valuable information for occupational or environmental hygienists, toxicologists and clinical chemists due to the particularly high number of determined elements and presented concentration ranges.     

<Emphasis Type="Italic">Anaconda</Emphasis>, a new class of transposon belonging to the <Emphasis Type="Italic">Mu</Emphasis> superfamily,has diversified by acquiring host genes during rice evolution

A new type of transposon, named Anaconda (Anac) has been found in rice (Oryza sativa). In this paper, we demonstrate that Anaconda elements have diversified by acquisition of host cellular genes, amplification of the elements, and substitution and deletion of short segments. We identified four Anaconda elements in studies of rice alternative oxidase (AOX) genes, and subsequently isolated an additional 23 elements based on the identity of their terminal inverted repeats (TIRs). The Anaconda elements have long TIRs (114–458 bp). They also have direct repeats of 9 or 10 bp in their flanking regions that are thought to have been generated upon transposition. These structural features reveal that the Anaconda elements belong to the Mu superfamily. The most prominent feature of the Anaconda elements is the high frequency with which they have acquired host cellular genes. Of the 27 elements found here, 19 appear to have sequences presumably derived from rice genes, for example, the genes for AOX1c (four elements), cytochrome P450 (five elements), l-asparaginase (five elements), and PCF8 (two elements). Four elements, AnacA1–A4, have both the AOX1c and P450 genes. One element, AnacB14, involves a gene similar to mudrA of maize MuDR. Database analyses revealed that the loci of 26 of the 27 Anaconda elements in the subspecies japonica are the same as those in the subspecies indica. This suggests that these elements were incorporated before the divergence of these two subspecies.     

A single-amino acid substitution in the sixth leucine-rich repeat of barley MLA6 and MLA13 alleviates dependence on RAR1 for disease resistance signaling

Interactions between barley and the powdery mildew pathogen, Blumeria graminis f. sp. hordei, (Bgh) are determined by unique combinations of host resistance genes, designated Mildew-resistance locus (Ml), and cognate pathogen avirulence genes. These interactions occur both dependent and independent of Rar1 (required for Mla12 resistance) and Sgt1 (Suppressor of G-two allele of skp1), which are differentially required for diverse plant disease-resistance pathways. We have isolated two new functional Mla alleles, Rar1-independent Mla7 and Rar1-dependent Mla10, as well as the Mla paralogs, Mla6-2 and Mla13-2. Utilizing the inherent diversity amongst Mla-encoded proteins, we identified the only two amino acids exclusively conserved in RAR1-dependent MLA6, MLA10, MLA12, and MLA13 that differ at the corresponding position in RAR1-independent MLA1 and MLA7. Two- and three-dimensional modeling places these residues on a predicted surface of the sixth leucine-rich repeat (LRR) domain at positions distinct from those within the beta-sheets hypothesized to determine resistance specificity. Site-directed mutagenesis of these residues indicates that RAR1 independence requires the presence of an aspartate at position 721, as mutation of this residue to a structurally similar, but uncharged, asparagine did not alter RAR1 dependence. These results demonstrate that a single-amino acid substitution in the sixth MLA LRR can alter host signaling but not resistance specificity to B. graminis.     

A Protease Activity Displaying Some Thrombin-like Characteristics in Conditioned Medium of Zinnia Mesophyll Cells Undergoing Tracheary Element Differentiation

The normal development of tracheary elements (TE) requires a selective degradation of the cytoplasm without loss of the extracellular wall that remains behind as the water-conducting units of xylem. Using zinnia-(Zinnia elegans L. cv. Green Envy) cultured mesophyll cells that synchronously transdifferentiate into TEs, extracellular and intracellular proteases, respectively, have been shown to both trigger death and to execute autolysis as the final component of a programmed cell death (PCD). We report here the appearance in the medium of an unusual proteolytic activity correlated with the PCD process just prior to the autolysis. The activity has a pH optimum of 5.5–6.0 and displays some thrombin characteristics. This protease activity has 1) a 10-fold higher affinity towards a thrombin-specific chromogenic substrate than toward a trypsin-specific chromogenic substrate; 2) a 1000-fold lower sensitivity to soybean trypsin inhibitor (STI) compared to trypsin; and 3) limited ability to cleave the protease-activated receptor-1, the native thrombin substrate. However, the addition of partially purified fraction containing the thrombin-like protease activity to the medium of PCD-competent cells does not prematurely trigger PCD, and the thrombin-specific peptide inhibitor phenylalanine-proline-aspartic acid-chloromethylketone fails to inhibit PCD or tracheary element (TE) formation. This suggests that this protease activity may play a role within the cells in execution of the autolysis or in the collapse of the tonoplast rather than as an extracellular proteolytic activity participating in the chain of events leading to cell death. Online publication: 7 April 2005     

A family of evolution-entropy hybrid methods for ranking protein residues by importance

In order to identify the amino acids that determine protein structure and function it is useful to rank them by their relative importance. Previous approaches belong to two groups; those that rely on statistical inference, and those that focus on phylogenetic analysis. Here, we introduce a class of hybrid methods that combine evolutionary and entropic information from multiple sequence alignments. A detailed analysis in insulin receptor kinase domain and tests on proteins that are well-characterized experimentally show the hybrids' greater robustness with respect to the input choice of sequences, as well as improved sensitivity and specificity of prediction. This is a further step toward proteome scale analysis of protein structure and function.     

Expressed gene clusters associated with cellular sensitivity and resistance towards anti-viral and anti-proliferative actions of interferon

Interferons (IFN) are multi-functional proteins that induce a large number of genes which mediate many biological processes including host defense, cell growth control, signaling, and metabolism. Bioinformatics analysis of the 3'-untranslated regions of IFN-stimulated genes (ISGs) showed that the AU-rich elements (ARE) exist in approximately 10% of the mRNA induced by IFN. The human epithelial cell lines, WISH and 293, and the human B cell lines, Daudi and RPMI 1788, were assessed for their response to type-I IFN. Due to their differential response to the anti-viral and anti-proliferative action of IFN-alpha, they were used as cellular models for genome wide ARE-gene expression. The anti-viral and anti-proliferative actions of IFN-alpha were substantially more potent against WISH and Daudi cells than 293 and RPMI 1788 cells, respectively. These results correlated with the Stat1-driven gene expression as assessed by monitoring the expression of Stat1-mediated IFN-inducible 6-16 mRNA. Interferons were able to induce a significant proportion of common and distinct ARE-genes, but the patterns of expression were different and dependent on the type of the cell, type of IFN, and status of the cellular sensitivity to IFN. Clustering algorithms generated two informative expressed gene clusters that were selectively associated with cellular sensitivity and resistance to the anti-viral and anti-proliferative action of IFN. Use of rationally designed microarray experiments in IFN biology yielded informative clusters that may provide candidate genes for diagnostic or for evaluation of therapeutic possibilities.     

A Structural-informatics approach for tracing beta-sheets: building pseudo-C(alpha) traces for beta-strands in intermediate-resolution density maps

We report the development of two computational methods to assist density map interpretation at intermediate resolutions: sheettracer for building pseudo-C(alpha) models of beta-sheets, and a deconvolution method for enhancing features attributed to major secondary structural elements. Sheettracer is tightly coupled with sheetminer, which was developed to locate sheet densities in intermediate-resolution density maps. The results from sheetminer are used as inputs to sheettracer, which employs a multi-step ad hoc morphological analysis of sheet densities to trace individual strands of beta-sheets. The methods were tested on simulated density maps from 12 protein crystal structures that represent a reasonably complete sampling of sheet morphology. The sheet-tracing results were quantitatively assessed in terms of sensitivity, specificity and rms deviations. Furthermore, sheettracer and the deconvolution method were rigorously tested on experimental maps of the lambda2 protein of reovirus at resolutions of 7.6A and 11.8A. Our results clearly demonstrate the capability of sheettracer in building pseudo-C(alpha) models of beta-sheets in intermediate-resolution density maps and the power of the deconvolution method in enhancing the performance of sheettracer. These computational methods, along with other related ones, should facilitate recognition and analysis of folding motifs from experimental data at intermediate resolutions.     

Identification of regulatory cis-acting elements for alternative splicing of presenilin 2 exon 5 under hypoxic stress conditions

An alternatively spliced form of the presenilin 2 (PS2) gene lacking exon 5 (PS2V) was found in human brains with sporadic Alzheimer's disease. PS2V was induced by hypoxic stress in human neuroblastoma SK-N-SH cells, indicating that hypoxic stress affects the splicing machineries for PS2 exon 5. Here, we identified the critical cis-acting element (sec 2) on the PS2 pre-mRNA responsible for the aberrant splicing of PS2 exon 5 under hypoxic stress conditions. The element was composed of 23 nucleotides in exon 5 and RNA structural analyses showed a stem-loop structure in this sequence. Treatment with an antisense oligonucleotide directed toward the cis-acting element caused an increase in exon 5 inclusion. These results indicate that the sec 2 identified in this study is a novel regulatory element for exon 5 splicing under stress conditions and that trans-acting factors could specifically bind to the element to skip exon 5 of PS2.