Current Practices in LC-MS Untargeted Metabolomics: A Scoping Review on the Use of Pooled Quality Control Samples.

Untargeted metabolomics is an analytical approach with numerous applications serving as an effective metabolic phenotyping platform to characterize small molecules within a biological system. Data quality can be challenging to evaluate and demonstrate in metabolomics experiments. This has driven the use of pooled quality control (QC) samples for monitoring and, if necessary, correcting for analytical variance introduced during sample preparation and data acquisition stages. Described herein is a scoping literature review detailing the use of pooled QC samples in published untargeted liquid chromatography-mass spectrometry (LC-MS) based metabolomics studies. A literature query was performed, the list of papers was filtered, and suitable articles were randomly sampled. In total, 109 papers were each reviewed by at least five reviewers, answering predefined questions surrounding the use of pooled quality control samples. The results of the review indicate that use of pooled QC samples has been relatively widely adopted by the metabolomics community and that it is used at a similar frequency across biological taxa and sample types in both small- and large-scale studies. However, while many studies generated and analyzed pooled QC samples, relatively few reported the use of pooled QC samples to improve data quality. This demonstrates a clear opportunity for the field to more frequently utilize pooled QC samples for quality reporting, feature filtering, analytical drift correction, and metabolite annotation. Additionally, our survey approach enabled us to assess the ambiguity in the reporting of the methods used to describe the generation and use of pooled QC samples. This analysis indicates that many details of the QC framework are missing or unclear, limiting the reader's ability to determine which QC steps have been taken. Collectively, these results capture the current state of pooled QC sample usage and highlight existing strengths and deficiencies as they are applied in untargeted LC-MS metabolomics.

Investigation of nucleic acid damage induced by a novel ruthenium anti-cancer drug using multiple analytical techniques: Sequence specificity and damage kinetics.

Cis-diacetonitrilo-bis(bipyridine) ruthenium(II) chloride is a recently introduced cis-platin analogue that has anti-cancer properties with lower side effects. However, the sequence dependence of its DNA damaging mechanism is unclear. Here, we present a simple, sensitive, multiplexed mix-and-read assay for ascertaining the molecular mechanism of DNA damage induced by the studied ruthenium complex (Ru-complex). The damage kinetics and sequence specificity for the Ru-complex induced DNA damage are examined by studying the induced damage in various oligonucleotide sequences by EvaGreen-DNA intercalator probe. High-through-put measurements were established using a 96-well microplate platform that allows multiple sequences to be measured simultaneously. The results show that the extent of damage increases with an increasing number of guanines, with considerable amount of damage at GA, GT and GC sites, in particular. Furthermore, the interaction of Ru-complex with DNA was confirmed using thermal analysis and MALDI-TOF-MS. Results indicate that the activated Ru-complex preferentially binds via both mono- and di-adduct formation at G and GG sites, respectively. Moreover, the developed method was successfully applied for the determination of the potency of the studied Ru-complex to induce DNA damage in K-Ras and N-Ras family of genes, one of the most common oncogenic events in cancer.

Welding Fume Exposure and Health Risk Assessment in a Cohort of Apprentice Welders.

Welding fumes vary in composition depending on the materials and processes used, and while health outcomes in full-time welders have been widely studied, limited research on apprentices exists. Besides, few data are available for metals such as vanadium and antimony. This study aimed to look at individual metals present in welding fumes in the learning environment of apprentice welders. Forty-three welders and 41 controls were chosen from trade programmes at the Northern Alberta Institute of Technology. Ambient and personal air samples were collected at days 0, 1, 7, and 50 of their training and analysed for mass and metal concentrations using Inductively Coupled Plasma Mass Spectrometry. Results showed increases in particle and metal concentrations as apprentices progressed throughout their education and that concentrations at day 50 were similar to levels found in the literature for professional welders. Variable concentrations indicate that some individuals may not properly use the local exhaust ventilation system. Other possible explanation for variations are the position of the sampler on the shoulder, the time spent welding and in each welding position, and the skills of the welders. Strong relationships were observed between particle and metal concentrations, suggesting that these relationships could be used to estimate metal exposure in welders from particle exposure. Welding processes were the most important determinant of exposure in apprentice welders, with Metal Core Arc Welding producing the largest particle concentrations followed by oxyacetylene cutting, and Gas Metal Arc Welding. Health risk assessment showed that welder apprentices are at risk for overexposure to manganese, which suggests that professional welders should be monitored for manganese as they are exposed more than apprentices. Training in proper positioning of local exhaust ventilation system and proper use of respirators are recommended in training facilities.

Comparison of K-Ras and N-Ras Mutagenic Hot Spots for UVC Damage.

It has been well established that mutations in K-Ras and N-Ras proto-oncogenes can convert them into active oncogenes. Current molecular cancer research has been focused on determining the key steps by which cellular genes become oncogenes and not on the underlying and fundamental chemical damage mechanism and susceptibility to damage. In this study, we investigate the damage hot spots present in the N-Ras and K-Ras genes upon exposure to UVC radiation. Detection of damage is accomplished by a simple, sensitive, mix-and-read assay using an EvaGreen probe in a 96-well microtiter plate. Our results show that, although there is high degree of sequential similarities among K-Ras and N-Ras genes, they show different degrees of UV damage in different portions of their genomes. Our experiments demonstrate that overall, the K-Ras genome is more prone to UVC damage than the N-Ras genome. We observe that the extent of damage increases with increasing number of TTs in a sequence, consistent with previous results that show that thymine cyclobutyl photodimers are the primary DNA damage photoproducts upon UVC irradiation. This understanding of the effect of UVC radiation on various codons of K-Ras and N-Ras genes will help to increase our understanding about hot spots of DNA damage and the chemical damage mechanism.

Multiplexed, UVC-induced, sequence-dependent DNA damage detection.

The exposure of DNA to ultraviolet (UV) radiation causes sequence-dependent damage. Thus, there is a need for an analytical technique that can detect damage in large numbers of DNA sequences simultaneously. In this study, we have designed an assay for UVC-induced DNA damage in multiple oligonucleotides simultaneously by using a 96-well plate and a novel automated sample mover. The UVC-induced DNA damage is measured using smart probes, analogs of molecular beacons in which guanosine nucleotides act as the fluorescence quencher. Our results show that the oligonucleotide damage constants obtained with this method are reproducible and similar to those obtained in cuvettes. The calibration curve for poly-dT shows good linearity (R(2) = 0.96), with limits of detection (LOD) and quantification (LOQ) equal to 55 and 183 nm, respectively. The results show that the damage kinetics upon irradiation is sensitive to the different types of photoproducts formed in the different sequences used; i.e. poly-A oligonucleotides containing guanine are damaged at a faster rate than poly-A oligonucleotides containing either thymine or cytosine. Thus, detecting DNA damage in a 96-well plate and quantifying the damage with smart probes are a simple, fast and inexpensive mix-and-read technique for multiplexed, sequence-specific DNA damage detection.