A reappraisal of orders and families within the subclass Chaetothyriomycetidae (Eurotiomycetes,Ascomycota)

The subclass Chaetothyriomycetidae (Eurotiomycetes, Ascomycota) is an assemblage of ecologically diverse species, ranging from mutualistic lichenised fungi to human opportunistic pathogens. Recent contributions from molecular studies have changed our understanding of the composition of this subclass. Among others, ant-associated fungi, deep-sea fungi and bryophilous fungi were also shown to belong to this group of ascomycetes. The delimitation of orders and families within this subclass has not previously been re-assessed using a broad phylogenetic study and the phylogenetic position of some taxa such as the lichenised family Celotheliaceae or the Chaetothyrialean bryophilous fungi is still unclear. In our study, we assemble new and published sequences from 132 taxa and reconstruct phylogenetic relationships using four markers (nuLSU, nuSSU, mtSSU and RPB1). Results highlight several shortfalls in the current classification of this subclass, mainly due to un-assigned paraphyletic taxa. The family Epibryaceae is therefore described to circumscribe a previously un-assigned lineage. Celotheliales ad int. is suggested for the lineage including the lichen genus Celothelium and various plant pathogens. The delimitation of the family Trichomeriaceae is also broadened to include the genus Knufia and some anamorphic taxa. As defined here, Chaetothyriomycetidae includes four orders (Celotheliales ad int., Chaetothyriales, Pyrenulales, and Verrucariales) and ten families (Adelococcaceae, Celotheliaceae, Chaetothyriaceae, Cyphellophoraceae, Epibryaceae fam. nov., Herpotrichiellaceae, Pyrenulaceae, Requienellaceae, Trichomeriaceae, and Verrucariaceae).     

Reassessment of the taxonomic position of Iranocypris typhlops Bruun & Kaiser, 1944 (Actinopterygii,Cyprinidae)

The Iranian cave barb (Iranocypris typhlops Bruun & Kaiser, 1944) is a rare and endemic species of the family Cyprinidae known from a single locality in the Zagros Mountains, western Iran. This species is “Vulnerable” according to the IUCN Red List and is one of the top four threatened freshwater fish species in Iran. Yet, the taxonomic position of I. typhlops is uncertain. We examined phylogenetic relationships of this species with other species of the family Cyprinidae based on the mitochondrial cytochrome b gene. Our results show that I. typhlops is monophyletic and is sister taxon of a cluster formed by Garra rufa (Heckel, 1843) and Garra barreimiae (Fowler & Steinitz, 1956) within a clade that includes other species of the genus Garra. Based on previous molecular and morphological studies, as well as our new results, we recommend that I. typhlops should be transferred to the genus Garra Hamilton, 1822.     

Scanning electron microscopic assessment on surface morphology of preserved human amniotic membrane after gamma sterilisation

Human amniotic membrane that has been processed and sterilised by gamma irradiation is widely used as a biological dressing in surgical applications. The morphological structure of human amniotic membrane was studied under scanning electron microscopy (SEM) to assess effects of gamma radiation on human amniotic membrane following different preservation methods. The amniotic membrane was preserved by either air drying or submerged in glycerol before gamma irradiated at 15, 25 and 35 kGy. Fresh human amniotic membrane, neither preserved nor irradiated was used as the control. The surface morphology of glycerol preserved amnion was found comparable to the fresh amniotic membrane. The cells of the glycerol preserved was beautifully arranged, homogonous in size and tended to round up. The cell structure in the air dried preserved amnion seemed to be flattened and dehydrated. The effects of dehydration on intercellular channels and the microvilli on the cell surface were clearly seen at higher magnifications (10,000×). SEM revealed that the changes of the cell morphology of the glycerol preserved amnion were visible at 35 kGy while the air dried already changed at 25 kGy. Glycerol preservation method is recommended for human amniotic membrane as the cell morphological structure is maintained and radiation doses lower than 25 kGy for sterilization did not affect the appearance of the preserved amnion.     

Genetically encoded impairment of neuronal KCC2 cotransporter function in human idiopathic generalized epilepsy

The KCC2 cotransporter establishes the low neuronal Cl⁻ levels required for GABA_A and glycine (Gly) receptor-mediated inhibition, and KCC2 deficiency in model organisms results in network hyperexcitability. However, no mutations in KCC2 have been documented in human disease. Here, we report two non-synonymous functional variants in human KCC2, R952H and R1049C, exhibiting clear statistical association with idiopathic generalized epilepsy (IGE). These variants reside in conserved residues in the KCC2 cytoplasmic C-terminus, exhibit significantly impaired Cl⁻-extrusion capacities resulting in less hyperpolarized Gly equilibrium potentials (E_Gly), and impair KCC2 stimulatory phosphorylation at serine 940, a key regulatory site. These data describe a novel KCC2 variant significantly associated with a human disease and suggest genetically encoded impairment of KCC2 functional regulation may be a risk factor for the development of human IGE.     

CompareSVM: supervised,Support Vector Machine (SVM) inference of gene regularity networks

Background

Predication of gene regularity network (GRN) from expression data is a challenging task. There are many methods that have been developed to address this challenge ranging from supervised to unsupervised methods. Most promising methods are based on support vector machine (SVM). There is a need for comprehensive analysis on prediction accuracy of supervised method SVM using different kernels on different biological experimental conditions and network size.

Results

We developed a tool (CompareSVM) based on SVM to compare different kernel methods for inference of GRN. Using CompareSVM, we investigated and evaluated different SVM kernel methods on simulated datasets of microarray of different sizes in detail. The results obtained from CompareSVM showed that accuracy of inference method depends upon the nature of experimental condition and size of the network.

Conclusions

For network with nodes (<200) and average (over all sizes of networks), SVM Gaussian kernel outperform on knockout, knockdown, and multifactorial datasets compared to all the other inference methods. For network with large number of nodes (~500), choice of inference method depend upon nature of experimental condition. CompareSVM is available at http://bis.zju.edu.cn/CompareSVM/.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-014-0395-x) contains supplementary material, which is available to authorized users.     

Confetti: A Multiprotease Map of the HeLa Proteome for Comprehensive Proteomics

Bottom-up proteomics largely relies on tryptic peptides for protein identification and quantification. Tryptic digestion often provides limited coverage of protein sequence because of issues such as peptide length, ionization efficiency, and post-translational modification colocalization. Unfortunately, a region of interest in a protein, for example, because of proximity to an active site or the presence of important post-translational modifications, may not be covered by tryptic peptides. Detection limits, quantification accuracy, and isoform differentiation can also be improved with greater sequence coverage. Selected reaction monitoring (SRM) would also greatly benefit from being able to identify additional targetable sequences. In an attempt to improve protein sequence coverage and to target regions of proteins that do not generate useful tryptic peptides, we deployed a multiprotease strategy on the HeLa proteome. First, we used seven commercially available enzymes in single, double, and triple enzyme combinations. A total of 48 digests were performed. 5223 proteins were detected by analyzing the unfractionated cell lysate digest directly; with 42% mean sequence coverage. Additional strong-anion exchange fractionation of the most complementary digests permitted identification of over 3000 more proteins, with improved mean sequence coverage. We then constructed a web application (https://proteomics.swmed.edu/confetti) that allows the community to examine a target protein or protein isoform in order to discover the enzyme or combination of enzymes that would yield peptides spanning a certain region of interest in the sequence. Finally, we examined the use of nontryptic digests for SRM. From our strong-anion exchange fractionation data, we were able to identify three or more proteotypic SRM candidates within a single digest for 6056 genes. Surprisingly, in 25% of these cases the digest producing the most observable proteotypic peptides was neither trypsin nor Lys-C. SRM analysis of Asp-N versus tryptic peptides for eight proteins determined that Asp-N yielded higher signal in five of eight cases.Mass-spectrometry based proteomics provides various tools to detect and quantify changes in protein expression or post-translational modifications (PTMs).¹ In bottom-up proteomics, these analyses typically involve using peptides derived from the tryptic digestion of proteins. Although trypsin is a robust enzyme and provides peptides suitable for mass spectrometry, not all sequences are detectable by this approach (1). Sequences may be missed because of the limited number and uneven distribution of lysine and arginine residues throughout a protein sequence. Tryptic coverage of interesting regions of sequence, such as trans-membrane domains that may contain notable PTMs, is often incomplete (2). Sequence coverage greater than that offered by trypsin is a requirement for many studies (3).Missing sequence coverage can also adversely affect analysis by selected reaction monitoring (SRM). Although SRM has emerged in recent years as a highly sensitive and accurate method for protein detection and quantification (4), it is sometimes hampered by the limited number of targetable peptides (primarily tryptic peptides) available in public databases. Improving amino acid sequence coverage would provide more targets for SRM assay development, facilitating protein quantification and the ability to target specific isoforms or sequence regions of interest.Fractionation is commonly employed to increase protein identifications and improve sequence coverage, but introduces a number of complexities. Separation of proteins or peptides significantly increases the number of samples to analyze and the amount of data to process. Species may be present in multiple fractions or in different fractions in different runs, which makes quantitative analysis with techniques like SRM difficult. However, SRM has sufficient sensitivity that peptides identified in fractionated discovery experiments are often targetable in whole lysate (5).One approach to increase sequence coverage without fractionation or purification is to use proteases other than trypsin for digestion (6, 7). In recent years, there has been a surge in the use of alternative proteases to improve sequence coverage. Biringer et al. demonstrated in 2006 that combining the MS data from tryptic and Glu-C digestions of human cerebrospinal fluid (CSF) resulted in increased protein identifications. Sequence coverage also improved versus individual enzyme digests, though this was shown only for the 38 most confidently identified proteins (8). In 2010, Swaney et al. expanded the multi-enzyme approach to five specific proteases (trypsin, Lys-C, Arg-C, Asp-N, and Glu-C) and showed that although this method only modestly increases the number of protein IDs, it significantly increases the average sequence coverage (from 24.5% to 43.4%) (9). The most comprehensive coverage of a human cell line to date was reported by Nagaraj et al., in which in-depth proteomics with two levels of prefractionation and analysis using trypsin, Lys-C, and Glu-C was carried out for the HeLa cell line. A total of 10,255 proteins and 166,420 peptides were identified (10). However, none of these studies investigated the use of consecutive enzymatic digestion on a sample.The Mann laboratory recently introduced a strategy, using consecutive digestion in conjunction with filter-aided sample preparation (FASP), for two-step and three-step digestions with various combinations of trypsin, Lys-C, Glu-C, Arg-C, and Asp-N (11). The consecutive use of Lys-C and trypsin enabled the identification of up to 40% more proteins and phosphorylation sites in comparison to trypsin alone. However, a systematic study of all common commercially available proteases for comprehensive mapping of the human proteome has not yet been performed.These prior studies have clearly shown the ability of tandem and parallel protease digestion to improve protein ID and sequence coverage. However, their focus has been either to improve the number of protein identifications or to improve the sequence coverage of few targets. In an effort to provide a resource for targeting as much of the amino acid sequence in a human cell line as possible, we conducted a comprehensive study in which seven commercially available enzymes were used individually and in combination. First, we digested HeLa lysate with a total of 48 single, double, and triple enzyme combinations. Across these combinations we detected 5223 proteins with an average of 42% sequence coverage by analyzing the total cell lysate digest without fractionation. We then selected the best five complementary digests for each of Orbitrap elite collision induced dissociation (CID) and Q exactive higher-energy CID (HCD) analyses. A strong-anion exchange fractionation strategy was applied to these best digests, from which we were able to identify 8470 proteins with 40.3% mean sequence coverage. Combining all digests, both unfractionated and SAX, gave 8539 proteins with 44.7% mean coverage. These data are now publically available (https://proteomics.swmed.edu/confetti) and can be queried using a simple web interface to discover the enzyme or combination of enzymes required to yield a peptide spanning a certain region of interest on a protein.Finally, we performed a proof-of-concept experiment to demonstrate that SRM assays using nontryptic peptides are viable, and in some cases more sensitive than tryptic assays. Though tryptic peptides are generally sufficient for protein quantification by SRM we believe there will be increased use of nontryptic SRM as coverage of specific regions of sequence becomes more important. For example, bio-marker studies considering the presence of specific PTMs rather than general protein abundance are increasingly common. Truly comprehensive PTM studies require access to the nontryptic proteome.     

The correlation of cathodic peak potentials of vitamin K3 derivatives and their calculated electron affinities : The role of hydrogen bonding and conformational changes

2-methyl-1,4-naphtoquinone 1 (vitamin K₃, menadione) derivatives with different substituents at the 3-position were synthesized to tune their electrochemical properties. The thermodynamic midpoint potential (E_1/2) of the naphthoquinone derivatives yielding a semi radical naphthoquinone anion were measured by cyclic voltammetry in the aprotic solvent dimethoxyethane (DME). Using quantum chemical methods, a clear correlation was found between the thermodynamic midpoint potentials and the calculated electron affinities (E_A). Comparison of calculated and experimental values allowed delineation of additional factors such as the conformational dependence of quinone substituents and hydrogen bonding which can influence the electron affinities (E_A) of the quinone. This information can be used as a model to gain insight into enzyme-cofactor interactions, particularly for enzyme quinone binding modes and the electrochemical adjustment of the quinone motif.     

Visualization, documentation, analysis, and communication of large-scale gene regulatory networks

Genetic regulatory networks (GRNs) are complex, large-scale, and spatially and temporally distributed. These characteristics impose challenging demands on software tools for building GRN models, and so there is a need for custom tools. In this paper, we report on our ongoing development of BioTapestry, an open source, freely available computational tool designed specifically for building GRN models. We also outline our future development plans, and give some examples of current applications of BioTapestry.