A high-throughput alpha particle irradiation system for monitoring DNA damage repair, genome instability and screening in human cell and yeast model systems.

Ionizing radiation (IR) is environmentally prevalent and, depending on dose and linear energy transfer (LET), can elicit serious health effects by damaging DNA. Relative to low LET photon radiation (X-rays, gamma rays), higher LET particle radiation produces more disease causing, complex DNA damage that is substantially more challenging to resolve quickly or accurately. Despite the majority of human lifetime IR exposure involving long-term, repetitive, low doses of high LET alpha particles (e.g. radon gas inhalation), technological limitations to deliver alpha particles in the laboratory conveniently, repeatedly, over a prolonged period, in low doses and in an affordable, high-throughput manner have constrained DNA damage and repair research on this topic. To resolve this, we developed an inexpensive, high capacity, 96-well plate-compatible alpha particle irradiator capable of delivering adjustable, low mGy/s particle radiation doses in multiple model systems and on the benchtop of a standard laboratory. The system enables monitoring alpha particle effects on DNA damage repair and signalling, genome stability pathways, oxidative stress, cell cycle phase distribution, cell viability and clonogenic survival using numerous microscopy-based and physical techniques. Most importantly, this method is foundational for high-throughput genetic screening and small molecule testing in mammalian and yeast cells.

Direct identification of rituximab main isoforms and subunit analysis by online selective comprehensive two-dimensional liquid chromatography-mass spectrometry.

In this proof-of-concept study, rituximab, which is a reference therapeutic monoclonal antibody (mAb), was characterized through the implementation of online, selective comprehensive two-dimensional liquid chromatography (sLC×LC) coupled with mass spectrometry (MS), using a middle-up approach. In this setup, cation exchange chromatography (CEX) and reverse-phase liquid chromatography (RPLC) were used as the first and second separation dimensions, respectively. As illustrated in this work, the combination of these two chromatographic modes allows a direct assignment of the identities of CEX peaks, using data from the TOF/MS detector, because RPLC is directly compatible with MS detection, whereas CEX is not. In addition, the resolving power of CEX is often considered to be limited; therefore, this 2D approach provides an improvement in peak capacity and resolution when high-performance second-dimension separations are used, instead of simply using the second-dimension separation as a desalting step. This was particularly relevant when separating rituximab fragments of medium size (25 kDa), whereas most of the resolution was provided by CEX in the case of intact rituximab samples. The analysis of a commercial rituximab sample shows that online sLC×LC-TOF-MS can be used to rapidly characterize mAb samples, yielding the identification of numerous variants, based on the analysis of intact, partially digested, and digested/reduced mAb samples.

Profound DNA methylomic differences between single- and multi-fraction alpha irradiations of lung fibroblasts.

BACKGROUND: Alpha (α)-radiation is a ubiquitous environmental agent with epigenotoxic effects. Human exposure to α-radiation at potentially harmful levels can occur repetitively over the long term via inhalation of naturally occurring radon gas that accumulates in enclosed spaces, or as a result of a single exposure from a nuclear accident. Alterations in epigenetic DNA methylation (DNAm) have been implicated in normal aging and cancer pathogenesis. Nevertheless, the effects of aberrations in the methylome of human lung cells following exposure to single or multiple α-irradiation events on these processes remain unexplored. RESULTS: We performed genome-wide DNAm profiling of human embryonic lung fibroblasts from control and irradiated cells using americium-241 α-sources. Cells were α-irradiated in quadruplicates to seven doses using two exposure regimens, a single-fraction (SF) where the total dose was given at once, and a multi-fraction (MF) method, where the total dose was equally distributed over 14 consecutive days. Our results revealed that SF irradiations were prone to a decrease in DNAm levels, while MF irradiations mostly increased DNAm. The analysis also showed that the gene body (i.e., exons and introns) was the region most altered by both the SF hypomethylation and the MF hypermethylation. Additionally, the MF irradiations induced the highest number of differentially methylated regions in genes associated with DNAm biomarkers of aging, carcinogenesis, and cardiovascular disease. The DNAm profile of the oncogenes and tumor suppressor genes suggests that the fibroblasts manifested a defensive response to the MF α-irradiation. Key DNAm events of ionizing radiation exposure, including changes in methylation levels in mitochondria dysfunction-related genes, were mainly identified in the MF groups. However, these alterations were under-represented, indicating that the mitochondria undergo adaptive mechanisms, aside from DNAm, in response to radiation-induced oxidative stress. CONCLUSIONS: We identified a contrasting methylomic profile in the lung fibroblasts α-irradiated to SF compared with MF exposures. These findings demonstrate that the methylome response of the lung cells to α-radiation is highly dependent on both the total dose and the exposure regimen. They also provide novel insights into potential biomarkers of α-radiation, which may contribute to the development of innovative approaches to detect, prevent, and treat α-particle-related diseases.

Consequences of changing Canadian activity patterns since the COVID-19 pandemic include increased residential radon gas exposure for younger people.

The COVID-19 pandemic has produced widespread behaviour changes that shifted how people split their time between different environments, altering health risks. Here, we report an update of North American activity patterns before and after pandemic onset, and implications to radioactive radon gas exposure, a leading cause of lung cancer. We surveyed 4009 Canadian households home to people of varied age, gender, employment, community, and income. Whilst overall time spent indoors remained unchanged, time in primary residence increased from 66.4 to 77% of life (+ 1062 h/y) after pandemic onset, increasing annual radiation doses from residential radon by 19.2% (0.97 mSv/y). Disproportionately greater changes were experienced by younger people in newer urban or suburban properties with more occupants, and/or those employed in managerial, administrative, or professional roles excluding medicine. Microinfluencer-based public health messaging stimulated health-seeking behaviour amongst highly impacted, younger groups by > 50%. This work supports re-evaluating environmental health risks modified by still-changing activity patterns.

Chromatin and the Cellular Response to Particle Radiation-Induced Oxidative and Clustered DNA Damage.

Exposure to environmental ionizing radiation is prevalent, with greatest lifetime doses typically from high Linear Energy Transfer (high-LET) alpha particles via the radioactive decay of radon gas in indoor air. Particle radiation is highly genotoxic, inducing DNA damage including oxidative base lesions and DNA double strand breaks. Due to the ionization density of high-LET radiation, the consequent damage is highly clustered wherein ≥2 distinct DNA lesions occur within 1-2 helical turns of one another. These multiply-damaged sites are difficult for eukaryotic cells to resolve either quickly or accurately, resulting in the persistence of DNA damage and/or the accumulation of mutations at a greater rate per absorbed dose, relative to lower LET radiation types. The proximity of the same and different types of DNA lesions to one another is challenging for DNA repair processes, with diverse pathways often confounding or interplaying with one another in complex ways. In this context, understanding the state of the higher order chromatin compaction and arrangements is essential, as it influences the density of damage produced by high-LET radiation and regulates the recruitment and activity of DNA repair factors. This review will summarize the latest research exploring the processes by which clustered DNA damage sites are induced, detected, and repaired in the context of chromatin.

Expansion of rDNA and pericentromere satellite repeats in the genomes of bank voles Myodes glareolus exposed to environmental radionuclides.

Altered copy number of certain highly repetitive regions of the genome, such as satellite DNA within heterochromatin and ribosomal RNA loci (rDNA), is hypothesized to help safeguard the genome against damage derived from external stressors. We quantified copy number of the 18S rDNA and a pericentromeric satellite DNA (Msat-160) in bank voles (Myodes glareolus) inhabiting the Chernobyl Exclusion Zone (CEZ), an area that is contaminated by radionuclides and where organisms are exposed to elevated levels of ionizing radiation. We found a significant increase in 18S rDNA and Msat-160 content in the genomes of bank voles from contaminated locations within the CEZ compared with animals from uncontaminated locations. Moreover, 18S rDNA and Msat-160 copy number were positively correlated in the genomes of bank voles from uncontaminated, but not in the genomes of animals inhabiting contaminated, areas. These results show the capacity for local-scale geographic variation in genome architecture and are consistent with the genomic safeguard hypothesis. Disruption of cellular processes related to genomic stability appears to be a hallmark effect in bank voles inhabiting areas contaminated by radionuclides.

Characterization of therapeutic antibodies and related products by two-dimensional liquid chromatography coupled with UV absorbance and mass spectrometric detection.

The development of analytical tools for the characterization of large biomolecules is an emerging and rapidly evolving area. This development activity is motivated largely by the current trend involving the increase in development and use of large biomolecules for therapeutic uses. Given the inherent complexity of these biomolecules, which arises from their sheer size and possibilities for chemical modification as well as changes over time (e.g., through modification in solution, aggregation), two-dimensional liquid chromatography (2D-LC) has attracted considerable interest as an analytical tool to address the challenges faced in characterizing these materials. The immediate potential benefits of 2D-LC over conventional one-dimensional liquid chromatography in this context include: (1) higher overall resolving power; (2) complementary information gained from two dimensions of separation in a single analysis; and (3) enabling indirect coupling of separation modes that are inherently incompatible with mass spectrometric (MS) detection (e.g., ion-exchange, because of high-salt eluents) to MS through a more compatible second dimension separation such as reversed-phase LC. In this review we summarize the work in this area, most of which has occurred in the past five years. Although the future is bright for further development in this area, some challenges have already been addressed through new 2D-LC methods. These include: (1) deep characterization of monoclonal antibodies to understand charge heterogeneity, glycosylation patterns, and other modifications; (2) characterization of antibody-drug conjugates to understand the extent and localization of small molecule conjugation; (3) detailed study of excipients in protein drug formulations; and (4) detection of host-cell proteins on biotherapeutic molecule preparations. We fully expect that in the near future we will see this list expanded, and that continued development will lead to methods with further improved performance metrics.