LINE-1 expression in cancer correlates with p53 mutation, copy number alteration, and S phase checkpoint

Retrotransposons are genomic DNA sequences that copy themselves to new genomic locations via RNA intermediates; LINE-1 is the only active and autonomous retrotransposon in the human genome. The mobility of LINE-1 is largely repressed in somatic tissues but is derepressed in many cancers, where LINE-1 retrotransposition is correlated with p53 mutation and copy number alteration (CNA). In cell lines, inducing LINE-1 expression can cause double-strand breaks (DSBs) and replication stress. Reanalyzing multiomic data from breast, ovarian, endometrial, and colon cancers, we confirmed correlations between LINE-1 expression, p53 mutation status, and CNA. We observed a consistent correlation between LINE-1 expression and the abundance of DNA replication complex components, indicating that LINE-1 may also induce replication stress in human tumors. In endometrial cancer, high-quality phosphoproteomic data allowed us to identify the DSB-induced ATM-MRN-SMC S phase checkpoint pathway as the primary DNA damage response (DDR) pathway associated with LINE-1 expression. Induction of LINE-1 expression in an in vitro model led to increased phosphorylation of MRN complex member RAD50, suggesting that LINE-1 directly activates this pathway

Low escape-rate genome safeguards with minimal molecular perturbation of Saccharomyces cerevisiae

As the use of synthetic biology both in industry and in academia grows, there is an increasing need to ensure biocontainment. There is growing interest in engineering bacterial- and yeast-based safeguard (SG) strains. First-generation SGs were based on metabolic auxotrophy; however, the risk of cross-feeding and the cost of growth-controlling nutrients led researchers to look for other avenues. Recent strategies include bacteria engineered to be dependent on nonnatural amino acids and yeast SG strains that have both transcriptional- and recombinational-based biocontainment. We describe improving yeast Saccharomyces cerevisiae-based transcriptional SG strains, which have near-WT fitness, the lowest possible escape rate, and nanomolar ligands controlling growth. We screened a library of essential genes, as well as the best-performing promoter and terminators, yielding the best SG strains in yeast. The best constructs were fine-tuned, resulting in two tightly controlled inducible systems. In addition, for potential use in the prevention of industrial espionage, we screened an array of possible "decoy molecules" that can be used to mask any proprietary supplement to the SG strain, with minimal effect on strain fitness

Sequence and Structural Convergence of Broad and Potent HIV Antibodies That Mimic CD4 Binding

Passive transfer of broadly neutralizing HIV antibodies can prevent infection, which suggests that vaccines that elicit such antibodies would be protective. Thus far, however, few broadly neutralizing HIV antibodies that occur naturally have been characterized. To determine whether these antibodies are part of a larger group of related molecules, we cloned 576 new HIV antibodies from four unrelated individuals. All four individuals produced expanded clones of potent broadly neutralizing CD4-binding-site antibodies that mimic binding to CD4. Despite extensive hypermutation, the new antibodies shared a consensus sequence of 68 immunoglobulin H (IgH) chain amino acids and arise independently from two related IgH genes. Comparison of the crystal structure of one of the antibodies to the broadly neutralizing antibody VRC01 revealed conservation of the contacts to the HIV spike

International Network for Comparison of HIV Neutralization Assays: The NeutNet Report II

BACKGROUND: Neutralizing antibodies provide markers for vaccine-induced protective immunity in many viral infections. By analogy, HIV-1 neutralizing antibodies induced by immunization may well predict vaccine effectiveness. Assessment of neutralizing antibodies is therefore of primary importance, but is hampered by the fact that we do not know which assay(s) can provide measures of protective immunity. An international collaboration (NeutNet) involving 18 different laboratories previously compared different assays using monoclonal antibodies (mAbs) and soluble CD4 (Phase I study). METHODS: In the present study (Phase II), polyclonal reagents were evaluated by 13 laboratories. Each laboratory evaluated nine plasmas against an 8 virus panel representing different genetic subtypes and phenotypes. TriMab, a mixture of three mAbs, was used as a positive control allowing comparison of the results with Phase I in a total of nine different assays. The assays used either uncloned virus produced in peripheral blood mononuclear cells (PBMCs) (Virus Infectivity Assays, VIA), or Env (gp160)-pseudotyped viruses (pseudoviruses, PSV) produced in HEK293T cells from molecular clones or from uncloned virus. Target cells included PBMC and genetically engineered cell lines in either single- or multiple-cycle infection format. Infection was quantified by using a range of assay read-outs including extra- or intra-cellular p24 antigen detection, luciferase, beta-galactosidase or green fluorescent protein (GFP) reporter gene expression. FINDINGS: Using TriMab, results of Phase I and Phase II were generally in agreement for six of the eight viruses tested and confirmed that the PSV assay is more sensitive than PBMC (p = 0.014). Comparisons with the polyclonal reagents showed that sensitivities were dependent on both virus and plasma. CONCLUSIONS: Here we further demonstrate clear differences in assay sensitivities that were dependent on both the neutralizing reagent and the virus. Consistent with the Phase I study, we recommend parallel use of PSV and VIA for vaccine evaluation

Modified Alvarado Scoring System as a diagnostic tool for Acute Appendicitis at Bugando Medical Centre, Mwanza, Tanzania

<p>Abstract</p> <p>Background</p> <p>Decision-making in patients with acute appendicitis poses a diagnostic challenge worldwide, despite much advancement in abdominal surgery. The Modified Alvarado Scoring System (MASS) has been reported to be a cheap and quick diagnostic tool in patients with acute appendicitis. However, differences in diagnostic accuracy have been observed if the scores were applied to various populations and clinical settings. The purpose of this study was to evaluate the diagnostic value of Modified Alvarado Scoring System in patients with acute appendicitis in our setting.</p> <p>Methods</p> <p>A cross-sectional study involving all patients suspected to have acute appendicitis at Bugando Medical Centre over a six-month period between November 2008 and April 2009 was conducted. All patients who met the inclusion criteria were consecutively enrolled in the study. They were evaluated on admission using the MASS to determine whether they had acute appendicitis or not. All patients underwent appendicectomy according to the hospital protocol. The decision to operate was the prerogative of the surgeon or surgical resident based on overall clinical judgment and not the MASS. The diagnosis was confirmed by histopathological examination. Data was collected using a pre-tested coded questionnaire and analyzed using SPSS statistical computer software.</p> <p>Results</p> <p>A total number of 127 patients were studied. Their ages ranged from eight to 76 years (mean 29.64 ± 12.97). There were 37 (29.1%) males and 90 (70.9%) females (M: F = 1:2.4). All patients in this study underwent appendicectomy. The perforation rate was 9.4%. Histopathological examination confirmed appendicitis in 85 patients (66.9%) and the remaining 42 patients had normal appendix giving a negative appendicectomy rate of 33.1% (26.8% for males and 38.3% for females). The sensitivity and specificity of MASS in this study were 94.1% (males 95.8% and females 88.3%) and 90.4% (males 92.9% and females 89.7%) respectively. The Positive Predictive Value and Negative Predictive Value were 95.2% (males 95.5% and females 90.6%) and 88.4% (males 89.3% and females 80.1%) respectively. The accuracy of MASS was 92.9% (males 91.5% and females 87.6%).</p> <p>Conclusion</p> <p>The study shows that use of MASS in patients suspected to have acute appendicitis provides a high degree of diagnostic accuracy and can be employed at Bugando Medical Centre to improve the diagnostic accuracy of acute appendicitis and subsequently reduces negative appendicectomy and complication rates. However, additional investigations may be required to confirm the diagnosis in case of atypical presentation.</p

An Analysis of the Sensitivity of Proteogenomic Mapping of Somatic Mutations and Novel Splicing Events in Cancer

Improvements in mass spectrometry (MS)-based peptide sequencing provide a new opportunity to determine whether polymorphisms, mutations, and splice variants identified in cancer cells are translated. Herein, we apply a proteogenomic data integration tool (QUILTS) to illustrate protein variant discovery using whole genome, whole transcriptome, and global proteome datasets generated from a pair of luminal and basal-like breast-cancer-patient-derived xenografts (PDX). The sensitivity of proteogenomic analysis for singe nucleotide variant (SNV) expression and novel splice junction (NSJ) detection was probed using multiple MS/MS sample process replicates defined here as an independent tandem MS experiment using identical sample material. Despite analysis of over 30 sample process replicates, only about 10% of SNVs (somatic and germline) detected by both DNA and RNA sequencing were observed as peptides. An even smaller proportion of peptides corresponding to NSJ observed by RNA sequencing were detected (<0.1%). Peptides mapping to DNA-detected SNVs without a detectable mRNA transcript were also observed, suggesting that transcriptome coverage was incomplete (∼80%). In contrast to germline variants, somatic variants were less likely to be detected at the peptide level in the basal-like tumor than in the luminal tumor, raising the possibility of differential translation or protein degradation effects. In conclusion, this large-scale proteogenomic integration allowed us to determine the degree to which mutations are translated and identify gaps in sequence coverage, thereby benchmarking current technology and progress toward whole cancer proteome and transcriptome analysis

Statistical Characterization of the Charge State and Residue Dependence of Low-Energy CID Peptide Dissociation Patterns

Data mining was performed on 28 330 unique peptide tandem mass spectra for which sequences were assigned with high confidence. By dividing the spectra into different sets based on structural features and charge states of the corresponding peptides, chemical interactions involved in promoting specific cleavage patterns in gas-phase peptides were characterized. Pairwise fragmentation maps describing cleavages at all Xxx-Zzz residue combinations for b and y ions reveal that the difference in basicity between Arg and Lys results in different dissociation patterns for singly charged Arg-and Lys-ending tryptic peptides. While one dominant protonation form (proton localized) exists for Arg-ending peptides, a heterogeneous population of different protonated forms or more facile interconversion of protonated forms (proton partially mobile) exists for Lys-ending peptides. Cleavage C-terminal to acidic residues dominates spectra from singly charged peptides that have a localized proton and cleavage N-terminal to Pro dominates those that have a mobile or partially mobile proton. When Pro is absent from peptides that have a mobile or partially mobile proton, cleavage at each peptide bond becomes much more prominent. Whether the above patterns can be found in b ions, y ions, or both depends on the location of the proton holder(s) in multiply protonated peptides. Enhanced cleavages C-terminal to branched aliphatic residues (Ile, Val, Leu) are observed in both b and y ions from peptides that have a mobile proton, as well as in y ions from peptides that have a partially mobile proton; enhanced cleavages Nterminal to these residues are observed in b ions from peptides that have a partially mobile proton. Statistical tools have been designed to visualize the fragmentation maps and measure the similarity between them. The pairwise cleavage patterns observed expand our knowledge of peptide gas-phase fragmentation behaviors and may be useful in algorithm development that employs improved models to predict fragment ion intensities. Low-energy collision-induced dissociation (CID) in mass spectrometry has been used extensively in peptide sequencing for protein identification and analysis of protein modifications. 1,2 Computer algorithms are employed to automate the spectral analysis and assign peptide sequences to the fragmentation spectra. 2-4 Numerous peptide fragmentation spectra are obtained everyday, but only a low percentage (e.g., 10-35%) of them are successfully identified by readily available algorithms. [5][6][7][8] One explanation for the high failure rate of current algorithms is the very simple fragmentation model on which they are based, which assumes that cleavage will occur mainly, if not exclusively, at the amide bonds between consecutive amino acid residues to produce ions of identical abundance (or intensity), regardless of the identity or the chemical property of the residues. 4,9 Although intensity patterns of the fragment ions from the same peptide under the same experimental settings are highly reproducible, current readily available algorithms only use the mass-to-charge (m/z) information from a tandem mass (MS/MS) spectrum and ignore the other information availablesthe relative intensity information. While this approach is sufficient to identify many peptides, many others yield fragmentation patterns that significantly deviate from a uniform fragmentation model. 8 A more detailed and chemically meaningful fragmentation model might improve algorithm performance and might include using different models for different candidate sequences. In addition, the gas-phase unimolecula

RAId_aPS: MS/MS analysis with multiple scoring functions and spectrum-specific statistics

Statistically meaningful comparison/combination of peptide identification results from various search methods is impeded by the lack of a universal statistical standard. Providing an E-value calibration protocol, we demonstrated earlier the feasibility of translating either the score or heuristic E-value reported by any method into the textbook-defined E-value, which may serve as the universal statistical standard. This protocol, although robust, may lose spectrum-specific statistics and might require a new calibration when changes in experimental setup occur. To mitigate these issues, we developed a new MS/MS search tool, RAId_aPS, that is able to provide spectrum-specific E-values for additive scoring functions. Given a selection of scoring functions out of RAId score, K-score, Hyperscore and XCorr, RAId_aPS generates the corresponding score histograms of all possible peptides using dynamic programming. Using these score histograms to assign E-values enables a calibration-free protocol for accurate significance assignment for each scoring function. RAId_aPS features four different modes: (i) compute the total number of possible peptides for a given molecular mass range, (ii) generate the score histogram given a MS/MS spectrum and a scoring function, (iii) reassign E-values for a list of candidate peptides given a MS/MS spectrum and the scoring functions chosen, and (iv) perform database searches using selected scoring functions. In modes (iii) and (iv), RAId_aPS is also capable of combining results from different scoring functions using spectrum-specific statistics. The web link is http://www.ncbi.nlm.nih.gov/CBBresearch/Yu/raid_aps/index.html. Relevant binaries for Linux, Windows, and Mac OS X are available from the same page.Comment: 34 pages, 10 figures, 1 supplementary information file (RAId_aPS_support.pdf). To view the supplementary file, please download and extract the gzipped tar source file listed under "Other formats

Modular Mass Spectrometric Tool for Analysis of Composition and Phosphorylation of Protein Complexes

The combination of high accuracy, sensitivity and speed of single and multiple-stage mass spectrometric analyses enables the collection of comprehensive sets of data containing detailed information about complex biological samples. To achieve these properties, we combined two high-performance matrix-assisted laser desorption ionization mass analyzers in one modular mass spectrometric tool, and applied this tool for dissecting the composition and post-translational modifications of protein complexes. As an example of this approach, we here present studies of the Saccharomyces cerevisiae anaphase-promoting complexes (APC) and elucidation of phosphorylation sites on its components. In general, the modular concept we describe could be useful for assembling mass spectrometers operating with both matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) ion sources into powerful mass spectrometric tools for the comprehensive analysis of complex biological samples