Scientific reports, 2019
Antimicrobial peptides (AMPs) exhibit broad-spectrum antimicrobial activity, and have promise as new therapeutic agents. While the adult North American bullfrog (Rana [Lithobates] catesbeiana) is a prolific source of high-potency AMPs, the aquatic tadpole represents a relatively untapped source for new AMP discovery. The recent publication of the bullfrog genome and transcriptomic resources provides an opportune bridge between known AMPs and bioinformatics-based AMP discovery. The objective of the present study was to identify novel AMPs with therapeutic potential using a combined bioinformatics and wet lab-based approach. In the present study, we identified seven novel AMP precursor-encoding transcripts expressed in the tadpole. Comparison of their amino acid sequences with known AMPs revealed evidence of mature peptide sequence conservation with variation in the prepro sequence. Two mature peptide sequences were unique and demonstrated bacteriostatic and bactericidal activity against Mycobacteria but not Gram-negative or Gram-positive bacteria. Nine known and seven novel AMP-encoding transcripts were detected in premetamorphic tadpole back skin, olfactory epithelium, liver, and/or tail fin. Treatment of tadpoles with 10 nM 3,5,3'-triiodothyronine for 48 h did not affect transcript abundance in the back skin, and had limited impact on these transcripts in the other three tissues. Gene mapping revealed considerable diversity in size (1.6-15 kbp) and exon number (one to four) of AMP-encoding genes with clear evidence of alternative splicing leading to both prepro and mature amino acid sequence diversity. These findings verify the accuracy and utility of the bullfrog genome assembly, and set a firm foundation for bioinformatics-based AMP discovery.
The analysis of cell-free circulating tumor DNA (ctDNA) is potentially a less invasive, more dynamic assessment of cancer progression and treatment response than characterizing solid tumor biopsies. Standard isolation methods require separation of plasma by centrifugation, a time-consuming step that complicates automation. To address these limitations, we present an automatable magnetic bead-based ctDNA isolation method that eliminates centrifugation to purify ctDNA directly from peripheral blood (PB). To develop and test our method, ctDNA from cancer patients was purified from PB and plasma. We found that allelic fractions of somatic single-nucleotide variants from target gene capture libraries were comparable, indicating that the PB ctDNA purification method may be a suitable replacement for the plasma-based protocols currently in use.
BMC geriatrics, 2019
Super-Seniors are healthy, long-lived individuals who were recruited at age 85 years or older with no history of cancer, cardiovascular disease, diabetes, dementia, or major pulmonary disease. In a 10-year follow-up, we aimed to determine whether surviving Super-Seniors showed compression of morbidity, and to test whether the allele frequencies of longevity-associated variants in APOE and FOXO3 were more extreme in such long-term survivors.
Journal of proteome research, 2019
Optimizing the quality of proteomics data collected from a mass spectrometer (MS) requires careful selection of acquisition parameters and proper assessment of instrument performance. Software tools capable of extracting a broad set of information from raw files, including meta, scan, quantification, and identification data, are needed to provide guidance for MS system management. In this work, direct extraction and utilization of these data is demonstrated using RawTools, a standalone tool for extracting meta and scan data directly from raw MS files generated on Thermo Orbitrap instruments. RawTools generates summarized and detailed plain text outputs after parsing individual raw files, including scan rates and durations, duty cycle characteristics, precursor and reporter ion quantification, and chromatography performance. RawTools also contains a diagnostic module that includes an optional "preview" database search for facilitating informed decision-making related to optimization of MS performance based on a variety of metrics. RawTools has been developed in C# and utilizes the Thermo RawFileReader library and thus can process raw MS files with high speed and high efficiency on all major operating systems (Windows, MacOS, Linux). To demonstrate the utility of RawTools, the extraction of meta and scan data from both individual and large collections of raw MS files was carried out to identify problematic characteristics of instrument performance. Taken together, the combined rich feature-set of RawTools with the capability for interrogation of MS and experiment performance makes this software a valuable tool for proteomics researchers.
In its third edition, the Vancouver Autophagy Symposium presented a platform for vibrant discussion on the differential roles of macroautophagy/autophagy in disease. This one-day symposium was held at the BC Cancer Research Centre in Vancouver, BC, bringing together experts in cell biology, protein biochemistry and medicinal chemistry across several different disease models and model organisms. The Vancouver Autophagy Symposium featured 2 keynote speakers that are well known for their seminal contributions to autophagy research, Dr. David Rubinsztein (Cambridge Institute for Medical Research) and Dr. Kay F. Macleod (University of Chicago). Key discussions included the context-dependent roles and mechanisms of dysregulation of autophagy in diseases and the corresponding need to consider context-dependent autophagy modulation strategies. Additional highlights included the differential roles of bulk autophagy versus selective autophagy, novel autophagy regulators, and emerging chemical tools to study autophagy inhibition. Interdisciplinary discussions focused on addressing questions such as which stage of disease to target, which type of autophagy to target and which component to target for autophagy modulation. : AD: Alzheimer disease; AMFR/Gp78: autocrine motility factor receptor; CCCP: carbonyl cyanide -chlorophenylhydrazone; CML: chronic myeloid leukemia; CVB3: coxsackievirus B3; DRPLA: dentatorubral-pallidoluysian atrophy; ER: endoplasmic reticulum; ERAD: ER-associated degradation; FA: focal adhesion; HCQ: hydroxychloroquine; HD: Huntingtin disease; HIF1A/Hif1α: hypoxia inducible factor 1 subunit alpha; HTT: huntingtin; IM: imatinib mesylate; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; NBR1: neighbour of BRCA1; OGA: O-GlcNAcase; PDAC: pancreatic ductal adenocarcinoma; PLEKHM1: pleckstrin homology and RUN domain containing M1; polyQ: poly-glutamine; ROS: reactive oxygen species; RP: retinitis pigmentosa; SNAP29: synaptosome associated protein 29; SPCA3: spinocerebellar ataxia type 3; TNBC: triple-negative breast cancer.
Methods in molecular biology (Clifton, N.J.), 2019
The broad utility of mass spectrometry (MS) for investigating the proteomes of a diverse array of sample types has significantly expanded the use of this technology in biological studies. This widespread use has resulted in a substantial collection of protocols and acquisition approaches designed to obtain the highest-quality data for each experiment. As a result, distilling this information to develop a standard operating protocol for essential workflows, such as bottom-up quantitative shotgun whole proteome analysis, can be complex for users new to MS technology. Further complicating this matter, in-depth description of the methodological choices is seldom given in the literature. In this work, we describe a workflow for quantitative whole proteome analysis that is suitable for biomarker discovery, giving detailed consideration to important stages, including (1) cell lysis and protein cleanup using SP3 paramagnetic beads, (2) quantitative labeling, (3) offline peptide fractionation, (4) MS analysis, and (5) data analysis and interpretation. Special attention is paid to providing comprehensive details for all stages of this proteomics workflow to enhance transferability to external labs. The standardized protocol described here will provide a simplified resource to the proteomics community toward efficient adaptation of MS technology in proteomics studies.
PloS one, 2019
Inflammation contributes to breast cancer development through its effects on cell damage. This damage is usually dealt with by key genes involved in apoptosis and autophagy pathways.
Nature protocols, 2019
A critical step in proteomics analysis is the optimal extraction and processing of protein material to ensure the highest sensitivity in downstream detection. Achieving this requires a sample-handling technology that exhibits unbiased protein manipulation, flexibility in reagent use, and virtually lossless processing. Addressing these needs, the single-pot, solid-phase-enhanced sample-preparation (SP3) technology is a paramagnetic bead-based approach for rapid, robust, and efficient processing of protein samples for proteomic analysis. SP3 uses a hydrophilic interaction mechanism for exchange or removal of components that are commonly used to facilitate cell or tissue lysis, protein solubilization, and enzymatic digestion (e.g., detergents, chaotropes, salts, buffers, acids, and solvents) before downstream proteomic analysis. The SP3 protocol consists of nonselective protein binding and rinsing steps that are enabled through the use of ethanol-driven solvation capture on the surface of hydrophilic beads, and elution of purified material in aqueous conditions. In contrast to alternative approaches, SP3 combines compatibility with a substantial collection of solution additives with virtually lossless and unbiased recovery of proteins independent of input quantity, all in a simplified single-tube protocol. The SP3 protocol is simple and efficient, and can be easily completed by a standard user in ~30 min, including reagent preparation. As a result of these properties, SP3 has successfully been used to facilitate examination of a broad range of sample types spanning simple and complex protein mixtures in large and very small amounts, across numerous organisms. This work describes the steps and extensive considerations involved in performing SP3 in bottom-up proteomics, using a simplified protein cleanup scenario for illustration.
Nucleic acids research, 2019
Tissues used in pathology laboratories are typically stored in the form of formalin-fixed, paraffin-embedded (FFPE) samples. One important consideration in repurposing FFPE material for next generation sequencing (NGS) analysis is the sequencing artifacts that can arise from the significant damage to nucleic acids due to treatment with formalin, storage at room temperature and extraction. One such class of artifacts consists of chimeric reads that appear to be derived from non-contiguous portions of the genome. Here, we show that a major proportion of such chimeric reads align to both the 'Watson' and 'Crick' strands of the reference genome. We refer to these as strand-split artifact reads (SSARs). This study provides a conceptual framework for the mechanistic basis of the genesis of SSARs and other chimeric artifacts along with supporting experimental evidence, which have led to approaches to reduce the levels of such artifacts. We demonstrate that one of these approaches, involving S1 nuclease-mediated removal of single-stranded fragments and overhangs, also reduces sequence bias, base error rates, and false positive detection of copy number and single nucleotide variants. Finally, we describe an analytical approach for quantifying SSARs from NGS data.
Journal of clinical oncology : official journal of the American Society of Clinical Oncology, 2019
High-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements (HGBL-DH/TH) has a poor outcome after standard chemoimmunotherapy. We sought to understand the biologic underpinnings of HGBL-DH/TH with BCL2 rearrangements (HGBL-DH/TH- BCL2) and diffuse large B-cell lymphoma (DLBCL) morphology through examination of gene expression.