LINE-1 ORF1p as a candidate biomarker in high grade serous ovarian carcinoma.

Long interspersed element 1 (LINE-1) open reading frame 1 protein (ORF1p) expression is a common feature of many cancer types, including high-grade serous ovarian carcinoma (HGSOC). Here, we report that ORF1p is not only expressed but also released by ovarian cancer and primary tumor cells. Immuno-multiple reaction monitoring-mass spectrometry assays showed that released ORF1p is confidently detectable in conditioned media, ascites, and patients' plasma, implicating ORF1p as a potential biomarker. Interestingly, ORF1p expression is detectable in fallopian tube (FT) epithelial precursors of HGSOC but not in benign FT, suggesting that ORF1p expression in an early event in HGSOC development. Finally, treatment of FT cells with DNA methyltransferase inhibitors led to robust expression and release of ORF1p, validating the regulatory role of DNA methylation in LINE-1 repression in non-tumorigenic tissue.

Linking ammonium nitrate-aluminum (AN-AL) post-blast residues to pre-blast explosive materials using isotope ratio and trace elemental analysis for source attribution.

Forensic science practitioners are often called upon to attribute crimes using trace evidence, such as explosive remnants, with the ultimate goal of associating a crime with a suspect or suspects in order to prevent further attacks. The explosive charge is an attractive component for attribution in crimes involving explosives as there are limited pathways for acquisition. However, there is currently no capability to link an explosive charge to its source via post-blast trace residues using isotope ratios or trace elements. Here, we sought to determine if pre-blast attribution signatures are preserved after detonation and can be subsequently recovered and detected. A field study was conducted to recover samples of post-blast explosives from controlled detonations of ammonium nitrate-aluminum (AN-Al), which were then analyzed via isotope ratio mass spectrometry (IRMS) and inductively coupled plasma-mass spectrometry (ICP-MS) for quantitation and profiling of isotopes ratio and trace element signatures, respectively. Oxygen and nitrogen isotope ratios from AN-Al yielded some of the most promising results with considerable overlap within one standard deviation of the reference between the spreads of pre- and post-blast data. Trace element results from AN-Al support the findings in the isotope ratio data, with 26 elements detected in both pre- and post-blast samples, and several elements including B, Cd, Cr, Ni, Sn, V, and Zn showing considerable overlap. These preliminary results provide a proof-of-concept for the development of forensic examinations that can attribute signatures from post-blast debris to signatures in pre-blast explosive materials for use in future investigations.

Highly accurate classification of biological spores by culture medium for forensic attribution using multiple chemical signature types and machine learning.

Future proliferation of biological expertise and new technology may increasingly lower the difficulty to produce biological organisms for misuse. Rapid attribution of a biological attack is needed to quickly identify the person or lab responsible and prevent additional attacks by enabling the apprehension of suspects. Here, triplicate batches of Bacillus anthracis Sterne strain (BaSt) spores were grown in a total of seven amateur and professional media. Multiple orthogonal analytical signatures (peptides, metabolites, lipids by fatty acid methyl ester (FAME) analysis, bulk organic profile, and trace elements) were collected from the BaSt spores. The proteomics and metabolomics analyses identified promising attribution signature compounds that are unique to each of the seven production methods. In addition, while each of the signature types showed varying degrees of value individually for attributing BaSt spores to the culture medium used to prepare them, fusing results from all five signatures types to increase sourcing robustness and using a random forest sourcing algorithm yielded 100% hold-one-batch-out cross-validation classification accuracy and an average relative source probability for the correct source 5.5× higher than the most probable incorrect source. These preliminary results provide a proof-of-concept for the development of forensic examinations that can attribute biological agents to production methods for use in future investigations.

Automated Microchromatography Enables Multiplexing of Immunoaffinity Enrichment of Peptides to Greater than 150 for Targeted MS-Based Assays.

Immunoaffinity enrichment of peptides coupled with analysis by stable isotope dilution multiple reaction mass spectrometry has been shown to have analytical performance and detection limits suitable for many biomarker verification studies and biological applications. Prior studies have shown that antipeptide antibodies can be multiplexed up to 50 in a single assay without significant loss of performance. Achieving higher multiplex levels is relevant to all studies involving precious biological material as this minimizes the amount of sample that must be consumed to measure a given set of analytes and reduces the assay cost per analyte. Here we developed automated methods employing the Agilent AssayMAP Bravo microchromatography platform and used these methods to characterize the performance of immunoaffinity enrichment of peptides up to multiplex levels of 172. Median capture efficiency for the target peptides remained high (88%) even at levels of 150-plex and declined to 70% at 172-plex compared to antibody performance observed at standard lower multiplex levels (n = 25). Subsequently, we developed and analytically characterized a multiplexed immuno-multiple reaction monitoring-mass spectrometry (immuno-MRM-MS) assay (n = 110) and applied it to measure candidate protein biomarkers of cardiovascular disease in plasma of patients undergoing planned myocardial infarction. The median lower limit of detection of all peptides was 71.5 amol/µL (nM), and the coefficient of variation (CV) was less than 15% at the lower limit of quantification. The results demonstrate that high multiplexed immuno-MRM-MS assays are readily achievable using the optimized sample processing and peptide capture methods described here.

Discrimination against major groove adducts by Y-family polymerases of the DinB subfamily.

Y-family DNA polymerases bypass DNA adducts in a process known as translesion synthesis (TLS). Y-family polymerases make contacts with the minor groove side of the DNA substrate at the nascent base pair. The Y-family polymerases also contact the DNA major groove via the unique little finger domain, but they generally lack contacts with the major groove at the nascent base pair. Escherichia coli DinB efficiently and accurately copies certain minor groove guanosine adducts. In contrast, we previously showed that the presence in the DNA template of the major groove-modified base 1,3-diaza-2-oxophenothiazine (tC) inhibits the activity of E. coli DinB. Even when the DNA primer is extended up to three nucleotides beyond the site of the tC analog, DinB activity is strongly inhibited. These findings prompted us to investigate discrimination against other major groove modifications by DinB and its orthologs. We chose a set of pyrimidines and purines with modifications in the major groove and determined the activity of DinB and several orthologs with these substrates. DinB, human pol kappa, and Sulfolobus solfataricus Dpo4 show differing specificities for the major groove adducts pyrrolo-dC, dP, N(6)-furfuryl-dA, and etheno-dA. In general, DinB was least efficient for bypass of all of these major groove adducts, whereas Dpo4 was most efficient. DinB activity was essentially completely inhibited by the presence of etheno-dA, while pol kappa activity was strongly inhibited. All three of these DNA polymerases were able to bypass N(6)-furfuryl-dA with modest efficiency, with DinB being the least efficient. We also determined that the R35A variant of DinB enhances bypass of N(6)-furfuryl-dA but not etheno-dA. In sum, we find that whereas DinB is specific for bypass of minor groove adducts, it is specifically inhibited by major groove DNA modifications.

Multiple strategies for translesion synthesis in bacteria.

Damage to DNA is common and can arise from numerous environmental and endogenous sources. In response to ubiquitous DNA damage, Y-family DNA polymerases are induced by the SOS response and are capable of bypassing DNA lesions. In Escherichia coli, these Y-family polymerases are DinB and UmuC, whose activities are modulated by their interaction with the polymerase manager protein UmuD. Many, but not all, bacteria utilize DinB and UmuC homologs. Recently, a C-family polymerase named ImuC, which is similar in primary structure to the replicative DNA polymerase DnaE, was found to be able to copy damaged DNA and either carry out or suppress mutagenesis. ImuC is often found with proteins ImuA and ImuB, the latter of which is similar to Y­family polymerases, but seems to lack the catalytic residues necessary for polymerase activity. This imuAimuBimuC mutagenesis cassette represents a widespread alternative strategy for translesion synthesis and mutagenesis in bacteria. Bacterial Y­family and ImuC DNA polymerases contribute to replication past DNA damage and the acquisition of antibiotic resistance.