Disparate molecular evolution of two types of repetitive DNAs in the genome of the grasshopper Eyprepocnemis plorans

Wide arrays of repetitive DNA sequences form an important part of eukaryotic genomes. These repeats appear to evolve as coherent families, where repeats within a family are more similar to each other than to other orthologous representatives in related species. The continuous homogenization of repeats, through selective and non-selective processes, is termed concerted evolution. Ascertaining the level of variation between repeats is crucial to determining which evolutionary model best explains the homogenization observed for these sequences. Here, for the grasshopper Eyprepocnemis plorans, we present the analysis of intragenomic diversity for two repetitive DNA sequences (a satellite DNA (satDNA) and the 45S rDNA) resulting from the independent microdissection of several chromosomes. Our results show different homogenization patterns for these two kinds of paralogous DNA sequences, with a high between-chromosome structure for rDNA but no structure at all for the satDNA. This difference is puzzling, considering the adjacent localization of the two repetitive DNAs on paracentromeric regions in most chromosomes. The disparate homogenization patterns detected for these two repetitive DNA sequences suggest that several processes participate in the concerted evolution in E. plorans, and that these mechanisms might not work as genome-wide processes but rather as sequence-specific ones.     

A New in Vivo Cross-linking Mass Spectrometry Platform to Define Protein–Protein Interactions in Living Cells

Protein–protein interactions (PPIs) are fundamental to the structure and function of protein complexes. Resolving the physical contacts between proteins as they occur in cells is critical to uncovering the molecular details underlying various cellular activities. To advance the study of PPIs in living cells, we have developed a new in vivo cross-linking mass spectrometry platform that couples a novel membrane-permeable, enrichable, and MS-cleavable cross-linker with multistage tandem mass spectrometry. This strategy permits the effective capture, enrichment, and identification of in vivo cross-linked products from mammalian cells and thus enables the determination of protein interaction interfaces. The utility of the developed method has been demonstrated by profiling PPIs in mammalian cells at the proteome scale and the targeted protein complex level. Our work represents a general approach for studying in vivo PPIs and provides a solid foundation for future studies toward the complete mapping of PPI networks in living systems.Protein–protein interactions (PPIs)¹ play a key role in defining protein functions in biological systems. Aberrant PPIs can have drastic effects on biochemical activities essential to cell homeostasis, growth, and proliferation, and thereby lead to various human diseases (1). Consequently, PPI interfaces have been recognized as a new paradigm for drug development. Therefore, mapping PPIs and their interaction interfaces in living cells is critical not only for a comprehensive understanding of protein function and regulation, but also for describing the molecular mechanisms underlying human pathologies and identifying potential targets for better therapeutics.Several strategies exist for identifying and mapping PPIs, including yeast two-hybrid, protein microarray, and affinity purification mass spectrometry (AP-MS) (2–5). Thanks to new developments in sample preparation strategies, mass spectrometry technologies, and bioinformatics tools, AP-MS has become a powerful and preferred method for studying PPIs at the systems level (6–9). Unlike other approaches, AP-MS experiments allow the capture of protein interactions directly from their natural cellular environment, thus better retaining native protein structures and biologically relevant interactions. In addition, a broader scope of PPI networks can be obtained with greater sensitivity, accuracy, versatility, and speed. Despite the success of this very promising technique, AP-MS experiments can lead to the loss of weak/transient interactions and/or the reorganization of protein interactions during biochemical manipulation under native purification conditions. To circumvent these problems, in vivo chemical cross-linking has been successfully employed to stabilize protein interactions in native cells or tissues prior to cell lysis (10–16). The resulting covalent bonds formed between interacting partners allow affinity purification under stringent and fully denaturing conditions, consequently reducing nonspecific background while preserving stable and weak/transient interactions (12–16). Subsequent mass spectrometric analysis can reveal not only the identities of interacting proteins, but also cross-linked amino acid residues. The latter provides direct molecular evidence describing the physical contacts between and within proteins (17). This information can be used for computational modeling to establish structural topologies of proteins and protein complexes (17–22), as well as for generating experimentally derived protein interaction network topology maps (23, 24). Thus, cross-linking mass spectrometry (XL-MS) strategies represent a powerful and emergent technology that possesses unparalleled capabilities for studying PPIs.Despite their great potential, current XL-MS studies that have aimed to identify cross-linked peptides have been mostly limited to in vitro cross-linking experiments, with few successfully identifying protein interaction interfaces in living cells (24, 25). This is largely because XL-MS studies remain challenging due to the inherent difficulty in the effective MS detection and accurate identification of cross-linked peptides, as well as in unambiguous assignment of cross-linked residues. In general, cross-linked products are heterogeneous and low in abundance relative to non-cross-linked products. In addition, their MS fragmentation is too complex to be interpreted using conventional database searching tools (17, 26). It is noted that almost all of the current in vivo PPI studies utilize formaldehyde cross-linking because of its membrane permeability and fast kinetics (10–16). However, in comparison to the most commonly used amine reactive NHS ester cross-linkers, identification of formaldehyde cross-linked peptides is even more challenging because of its promiscuous nonspecific reactivity and extremely short spacer length (27). Therefore, further developments in reagents and methods are urgently needed to enable simple MS detection and effective identification of in vivo cross-linked products, and thus allow the mapping of authentic protein contact sites as established in cells, especially for protein complexes.Various efforts have been made to address the limitations of XL-MS studies, resulting in new developments in bioinformatics tools for improved data interpretation (28–32) and new designs of cross-linking reagents for enhanced MS analysis of cross-linked peptides (24, 33–39). Among these approaches, the development of new cross-linking reagents holds great promise for mapping PPIs on the systems level. One class of cross-linking reagents containing an enrichment handle have been shown to allow selective isolation of cross-linked products from complex mixtures, boosting their detectability by MS (33–35, 40–42). A second class of cross-linkers containing MS-cleavable bonds have proven to be effective in facilitating the unambiguous identification of cross-linked peptides (36–39, 43, 44), as the resulting cross-linked products can be identified based on their characteristic and simplified fragmentation behavior during MS analysis. Therefore, an ideal cross-linking reagent would possess the combined features of both classes of cross-linkers. To advance the study of in vivo PPIs, we have developed a new XL-MS platform based on a novel membrane-permeable, enrichable, and MS-cleavable cross-linker, Azide-A-DSBSO (azide-tagged, acid-cleavable disuccinimidyl bis-sulfoxide), and multistage tandem mass spectrometry (MSⁿ). This new XL-MS strategy has been successfully employed to map in vivo PPIs from mammalian cells at both the proteome scale and the targeted protein complex level.     

Resequencing the Mycobacterium avium subsp. paratuberculosis K10 Genome: Improved Annotation and Revised Genome Sequence

We report the resequencing and revised annotation of the Mycobacterium avium subsp. paratuberculosis K10 genome. A total of 90 single-nucleotide errors and a 51-bp indel in the original K10 genome were corrected, and the whole genome annotation was revised. Correction of these sequencing errors resulted in 28 frameshift alterations. The amended genome sequence is accessible via the supplemental section of study SRR060191 in the NCBI Sequence Read Archive and will serve as a valuable reference genome for future studies.The American bovine isolate K10 remains the only Mycobacterium avium subsp. paratuberculosis genome to be fully sequenced and published to date (1). Although this 4.8-Mbp genome likely contains some assembly errors (3), it has provided, and will continue to provide, an invaluable resource for Mycobacterium research. The assembly errors were identified through optical mapping of related M. avium subsp. paratuberculosis strain ATCC 19698, which revealed a 648-kb inversion around the origin of replication and two additional copies of the insertion sequences IS1311 and IS_MAP03 (3). These findings were subsequently validated via PCR, Southern blotting, and (Sanger) sequence analysis in ATCC 19698 and were also confirmed to be present in K10 (3). We designate this interim corrected genome M. avium subsp. paratuberculosis K10′. To further improve this resource, we undertook a resequencing project of the original M. avium subsp. paratuberculosis K10 genome.Whole-genome sequencing was performed on the Illumina GAIIx platform using one flow cell lane with 36-cycle paired-end chemistry. Reads were variably trimmed at the 3′ end based on the Illumina Read Segment Quality Indicator (Illumina manual), and read pairs containing ambiguous bases were removed. Read mapping onto the K10′ genome sequence was performed using SHRiMP (ver. 1.3.2) (2), and single-nucleotide polymorphisms and indels (deletion and insertion polymorphisms [DIPs]) were called using Nesoni (ver. 0.29; Monash University Victorian Bioinformatics Consortium) with default parameters. Read mapping determined that the data set comprised an average sequence coverage of 72.6 across the K10′ genome. This high sequence coverage allowed differences between K10\K10′ and the resequenced version of the genome, designated K10", to be identified with high confidence.Ninety single-nucleotide differences and one 51-bp indel were identified in the K10" genome. As confirmation that these differences are likely to represent errors in the original genome sequence, we have also detected these polymorphisms in two additional bovine M. avium subsp. paratuberculosis genomes recently sequenced and assembled within our laboratory (data not shown). Seven of the 90 differences and the 51-bp indel were subjected to PCR and Sanger sequencing for verification. All of the polymorphisms were confirmed to be present in K10" compared to the original genome sequence.Thirty-six single-nucleotide deletions and four nucleotide insertions were identified in K10" compared to the reference. These DIPs resulted in 27 frameshift mutations of protein coding loci. As a consequence of these frameshifts, one complete coding sequence (CDS) feature was removed (MAPK_3751), one novel CDS was created (MAPK_2081b), and one pseudogene was repaired (MAPK_4158-4159). In almost all of the other cases, the frameshifts resulted in proteins which more closely resembled their orthologs in M. avium subsp. hominissuis and M. intracellulare. Other frameshifts of biological interest include the truncation of a PPE family protein (MAPK_1173) and the extension of an MCE (mammalian cell entry) family protein (MAPK_4086). Compared to the reference, K10" also had a 51-bp indel within a possible MCE family protein (MAPK_1575). This indel consisted of an 11-bp deletion (bases 2436510 to 2436520 in the original K10 sequence) and an insertion of 51 bp. The resulting protein sequence now more closely resembles orthologs of the MCE family in other Mycobacterium spp. In conclusion, the fact that so many of the amended bases have resulted in revised coding regions indicates the underlying importance of this exercise.     

Effects of d-amphetamine on the behavior of pigeons exposed to the peak procedure

The effects of d-amphetamine on pigeons' key-pecking under the peak interval (PI) procedure were investigated in two experiments. In experiment I the effects of doses of d-amphetamine from 0.75 to 3.0 mg/kg on responding under PI 30 and 45 s were studied for 10 successive days. Reductions in peak time and wait time were observed at both PI values and an increase in the width was found at PI 30 s. There was no evidence of tolerance. In experiment II, pigeons exposed to a PI 45 s schedule were administered doses of D-amphetamine of 1.5 and 3.0 mg/kg for 30 successive days. Reductions in peak time and wait time were found here. Evidence of tolerance was found in wait time, peak time and width of the distribution at the higher dose. In both experiments a rate-dependent effect of the drug was found in the portion of each peak trial before the time that food was delivered on reinforced trials; this effect was weaker after the customary time of food delivery. The rate-dependent effect for responses before food time, combined with little effect of the drug on responses after food time, is shown by simulation to be sufficient to account for the reduction in peak time, without the need to appeal to an internal clock mechanism.     

Identification of small-molecule inhibitors of protein kinase B (PKB/AKT) in an AlphaScreenTM high-throughput screen

Protein kinase B (PKB/AKT) has been identified as a promising cancer drug target downstream of PI3 kinase. To find novel inhibitors of PKB/AKT kinase activity for progression as anticancer agents, the authors have used a high-throughput screen based on AlphaScreentrade mark technology. A known kinase inhibitor, the isoquinoline H8, was used as a positive control with mean inhibition in the screen of 43.4% +/- 13.1%. The performance of the screen was highly acceptable with Z' and Z factors of 0.83 +/- 0.07 and 0.75 +/- 0.04, respectively. A number of confirmed hits ( approximately 0.1% hit rate) were identified from 63,500 compounds screened. Five compounds have previously been described as PKB inhibitors, demonstrating the ability of the assay to find authentic inhibitors of the enzyme. Five hits had the potential to interfere with the assay signal and were deemed to be false positives. Two compounds were nonspecific inhibitors of PKB as enzyme inhibition in a filter-based assay was markedly reduced in the presence of 0.01% Triton X100. The authors now include an interference assay during hit confirmation procedures and check compound activity in the presence of Triton X100 in an attempt to eliminate nonspecific aggregators at an early stage.     

In vivo analysis in Drosophila reveals differential requirements of contact residues in Axin for interactions with GSK3β or β-catenin

Proper regulation of the Wingless/Wnt signaling pathway is essential for normal development. The scaffolding protein Axin plays a key role in this process through interactions with Drosophila Shaggy and Armadillo. In the current studies, we used a yeast two-hybrid assay to identify ten amino acids in Axin that are critical for in vitro interaction with Shaggy and two for interaction with Armadillo. We then generated five Axin variants in which individual putative contact amino acids were mutated and compared their activity, as assayed by rescue of axin null mutant flies, to that of Axin lacking the entire Shaggy (AxinΔSgg) or Armadillo (AxinΔArm) binding domain. Although we expected these mutants to function identically to Axin in which the entire binding domain was deleted, we instead observed a spectrum of phenotypic rescue. Specifically, two point mutants within the Shaggy binding domain showed loss of activity similar to that of AxinΔSgg and dominantly interfered with complex function, whereas a third mutant allele, AxinK446E, retained most function. Two Axin point mutants within the Armadillo binding domain were weak alleles and retained most function. These findings demonstrate the importance of in vivo verification of the role of specific amino acids within a protein.     

Preliminary analysis to estimate the spatial distribution of benefits of P load reduction: Identifying the spatial influence of phosphorus loading from the Maumee River (USA) in western Lake Erie

Since the early 2000s, Lake Erie has been experiencing annual cyanobacterial blooms that often cover large portions of the western basin and even reach into the central basin. These blooms have affected several ecosystem services provided by Lake Erie to surrounding communities (notably drinking water quality). Several modeling efforts have identified the springtime total bioavailable phosphorus (TBP) load as a major driver of maximum cyanobacterial biomass in western Lake Erie, and on this basis, international water management bodies have set a phosphorus (P) reduction goal. This P reduction goal is intended to reduce maximum cyanobacterial biomass, but there has been very limited effort to identify the specific locations within the western basin of Lake Erie that will likely experience the most benefits. Here, we used pixel‐specific linear regression to identify where annual variation in spring TBP loads is most strongly associated with cyanobacterial abundance, as inferred from satellite imagery. Using this approach, we find that annual TBP loads are most strongly associated with cyanobacterial abundance in the central and southern areas of the western basin. At the location of the Toledo water intake, the association between TBP load and cyanobacterial abundance is moderate, and in Maumee Bay (near Toledo, Ohio), the association between TBP and cyanobacterial abundance is no better than a null model. Both of these locations are important for the delivery of specific ecosystem services, but this analysis indicates that P load reductions would not be expected to substantially improve maximum annual cyanobacterial abundance in these locations. These results are preliminary in the sense that only a limited set of models were tested in this analysis, but these results illustrate the importance of identifying whether the spatial distribution of management benefits (in this case P load reduction) matches the spatial distribution of management goals (reducing the effects of cyanobacteria on important ecosystem services).     

PDL241, a novel humanized monoclonal antibody,reveals CD319 as a therapeutic target for rheumatoid arthritis

Introduction

Targeting the CD20 antigen has been a successful therapeutic intervention in the treatment of rheumatoid arthritis (RA). However, in some patients with an inadequate response to anti-CD20 therapy, a persistence of CD20^- plasmablasts is noted. The strong expression of CD319 on CD20^- plasmablast and plasma cell populations in RA synovium led to the investigation of the potential of CD319 as a therapeutic target.

Methods

PDL241, a novel humanized IgG₁ monoclonal antibody (mAb) to CD319, was generated and examined for its ability to inhibit immunoglobulin production from plasmablasts and plasma cells generated from peripheral blood mononuclear cells (PBMC) in the presence and absence of RA synovial fibroblasts (RA-SF). The in vivo activity of PDL241 was determined in a human PBMC transfer into NOD scid IL-2 gamma chain knockout (NSG) mouse model. Finally, the ability of PDL241 to ameliorate experimental arthritis was evaluated in a collagen-induced arthritis (CIA) model in rhesus monkeys.

Results

PDL241 bound to plasmablasts and plasma cells but not naïve B cells. Consistent with the binding profile, PDL241 inhibited the production of IgM from in vitro PBMC cultures by the depletion of CD319⁺ plasmablasts and plasma cells but not B cells. The activity of PDL241 was dependent on an intact Fc portion of the IgG₁ and mediated predominantly by natural killer cells. Inhibition of IgM production was also observed in the human PBMC transfer to NSG mouse model. Treatment of rhesus monkeys in a CIA model with PDL241 led to a significant inhibition of anti-collagen IgG and IgM antibodies. A beneficial effect on joint related parameters, including bone remodeling, histopathology, and joint swelling was also observed.

Conclusions

The activity of PDL241 in both in vitro and in vivo models highlights the potential of CD319 as a therapeutic target in RA.