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DNA damage is a common cellular feature seen in cancer and neurodegenerative disease, but fast and accurate methods for quantifying DNA damage are lacking. Comet assays are a biochemical tool to measure DNA damage based on the migration of broken DNA strands towards a positive electrode, which creates a quantifiable 'tail' behind the cell. However, a major limitation of this approach is the time needed for analysis of comets in the images with available open-source algorithms. The requirement for manual curation and the laborious pre- and post-processing steps can take hours to days. To overcome these limitations, we developed AutoComet, a new open-source algorithm for comet analysis that utilizes automated comet segmentation and quantification of tail parameters. AutoComet first segments and filters comets based on size and intensity and then filters out comets without a well-connected head and tail, which significantly increases segmentation accuracy. Because AutoComet is fully automated, it minimizes curator bias and is scalable, decreasing analysis time over ten-fold, to less than 3 s per comet. AutoComet successfully detected statistically significant differences in tail parameters between cells with and without induced DNA damage, and was more comparable to the results of manual curation than other open-source software analysis programs. We conclude that the AutoComet algorithm provides a fast, unbiased and accurate method to quantify DNA damage that avoids the inherent limitations of manual curation and will significantly improve the ability to detect DNA damage.

Consumption of red meat contributes to the intake of many essential nutrients in the human diet including protein, essential fatty acids, and several vitamins and trace minerals, with high iron content, particularly in meats with high myoglobin content. Demand for red meat continues to increase worldwide, particularly in developing countries where food nutrient density is a concern. Dietary and genetic manipulation of livestock can influence the nutritional value of meat products, providing opportunities to enhance the nutritional value of meat. Studies have demonstrated that changes in livestock nutrition and breeding strategies can alter the nutritional value of red meat. Traditional breeding strategies, such as genetic selection, have influenced multiple carcass and meat quality attributes relevant to the nutritional value of meat including muscle and fat deposition. However, limited studies have combined both genetic and nutritional approaches. Future studies aiming to manipulate the composition of fresh meat should aim to balance potential impacts on product quality and consumer perception. Furthermore, the rapidly emerging fields of phenomics, nutrigenomics, and integrative approaches, such as livestock precision farming and systems biology, may help better understand the opportunities to improve the nutritional value of meat under both experimental and commercial conditions.

Evaluating RNA quality and transcriptomic profile of beef muscle over time post-mortem may provide insight into RNA degradation and underlying biological and functional mechanisms that accompany biochemical changes occurring post-mortem during transformation of muscle to meat. RNA was extracted from longissimus thoracis (LT) sampled from British Continental crossbred heifer carcasses (n = 7) stored at 4°C in an abattoir drip cooler at 5 time points post-mortem, i.e., 45 min (0 h), 6 h, 24 h, 48 h, and 72 h. Following RNA-Sequencing, processed reads were aligned to the ARS-UCD1.2 bovine genome assembly. Subsequent differential expression (DE) analysis identified from 51 to 1434 upregulated and 27 to 2256 downregulated DE genes at individual time points compared to time 0 h, showing a trend for increasing counts of both upregulated and downregulated genes over time. Gene ontology and biological pathway term enrichment analyses on sets of DE genes revealed several processes and their timelines of activation/deactivation that accompanied or were involved with muscle transformation to meat. Although the quality of RNA in refrigerated LT remained high for several days post-mortem, the expression levels of several known biomarker genes for meat quality began to change from 24 h onwards. Therefore, to ensure accuracy of predictions on meat quality traits based on the expression levels of those biomarker genes in refrigerated beef muscle tissue, it is crucial that those expression measurements be made on RNA sampled within 24 h post-mortem. The present study also highlighted the need for more research on the roles of mitochondrial genes and non-coding genes in orchestrating muscle tissue processes after death, and how pre-mortem immune status might influence post-mortem meat quality.

Chronic limb oedema can have a wide variety of causes. An accurate diagnosis can guide effective management. In addition to the treatment of any underlying cause, the principles of lymphoedema management can be applied to improve symptoms, reduce complications and improve quality of life for patients, regardless of the cause of chronic oedema. [ABSTRACT FROM AUTHOR]

Background: Detection of del(17p) in myeloma is generally performed by fluorescence in situ hybridization (FISH) on a slide with analysis of up to 200 nuclei. The small cell sample analyzed makes this a low precision test. We report the utility of an automated FISH method, called "immuno‐flowFISH", to detect plasma cells with adverse prognostic risk del(17p) in bone marrow and blood samples of patients with myeloma. Methods: Bone marrow (n = 31) and blood (n = 19) samples from 35 patients with myeloma were analyzed using immuno‐flowFISH. Plasma cells were identified by CD38/CD138‐immunophenotypic gating and assessed for the 17p locus and centromere of chromosome 17. Cells were acquired on an AMNIS ImageStreamX MkII imaging flow cytometer using INSPIRE software. Results: Chromosome 17 abnormalities were identified in CD38/CD138‐positive cells in bone marrow (6/31) and blood (4/19) samples when the percent plasma cell burden ranged from 0.03% to 100% of cells. Abnormalities could be identified in 14.5%–100% of plasma cells. Conclusions: The "immuno‐flowFISH" imaging flow cytometric method could detect del(17p) in plasma cells in both bone marrow and blood samples of myeloma patients. This method was also able to detect gains and losses of chromosome 17, which are also of prognostic significance. The lowest levels of 0.009% (bone marrow) and 0.001% (blood) for chromosome 17 abnormalities was below the detection limit of current FISH method. This method offers potential as a new means of identifying these prognostically important chromosomal defects, even when only rare cells are present and for serial disease monitoring. [ABSTRACT FROM AUTHOR]

Electron microscopy (EM) is the premiere technique for high-resolution imaging of cellular ultrastructure. Unambiguous identification of specific proteins or cellular compartments in electron micrographs, however, remains challenging because of difficulties in delivering electron-dense contrast agents to specific subcellular targets within intact cells. We recently reported enhanced ascorbate peroxidase 2 (APEX2) as a broadly applicable genetic tag that generates EM contrast on a specific protein or subcellular compartment of interest. This protocol provides guidelines for designing and validating APEX2 fusion constructs, along with detailed instructions for cell culture, transfection, fixation, heavy-metal staining, embedding in resin, and EM imaging. Although this protocol focuses on EM in cultured mammalian cells, APEX2 is applicable to many cell types and contexts, including intact tissues and organisms, and is useful for numerous applications beyond EM, including live-cell proteomic mapping. This protocol, which describes procedures for sample preparation from cell monolayers and cell pellets, can be completed in 10 d, including time for APEX2 fusion construct validation, cell growth, and solidification of embedding resins. Notably, the only additional steps required relative to a standard EM sample preparation are cell transfection and a 2- to 45-min staining period with 3,3-diaminobenzidine (DAB) and hydrogen peroxide (H2O2).

APEX is an engineered peroxidase that functions as an electron microscopy tag and a promiscuous labeling enzyme for live-cell proteomics. Because limited sensitivity precludes applications requiring low APEX expression, we used yeast-display evolution to improve its catalytic efficiency. APEX2 is far more active in cells, enabling the use of electron microscopy to resolve the submitochondrial localization of calcium uptake regulatory protein MICU1. APEX2 also permits superior enrichment of endogenous mitochondrial and endoplasmic reticulum membrane proteins.

Gastrointestinal nematodes (GINs) can be a major constraint and global challenge to the sheep industry. These nematodes infect the small intestine and abomasum of grazing sheep, causing symptoms such as weight loss, diarrhea, hypoproteinemia, and anemia, which can lead to death. The use of anthelmintics to treat infected animals has led to GIN resistance, and excessive use of these drugs has resulted in residue traced in food and the environment. Resistance to GINs can be measured using multiple traits, including fecal egg count (FEC), Faffa Malan Chart scores, hematocrit, packed cell volume, eosinophilia, immunoglobulin (Ig), and dagginess scores. Genetic variation among animals exists, and understanding these differences can help identify genomic regions associated with resistance to GINs in sheep. Genes playing important roles in the immune system were identified in several studies in this review, such as the CFI and MUC15 genes. Results from several studies showed overlapping quantitative trait loci (QTLs) associated with multiple traits measuring resistance to GINs, mainly FEC. The discovery of genomic regions, positional candidate genes, and QTLs associated with resistance to GINs can help increase and accelerate genetic gains in sheep breeding programs and reveal the genetic basis and biological mechanisms underlying this trait. [ABSTRACT FROM AUTHOR]

In this viewpoint paper, we argue that the predominance of single-generation research approaches in the growing literature on youth climate activism risks obscuring the intergenerational nature of youth(-led) activism that often encompasses people of many ages enmeshed in cross-generational support structures working collaboratively for change. The partitioning of generations in research on climate activism is rooted in adultist tendencies, which are in turn rooted in colonial logics that segment the human lifespan and subordinate young people’s perspectives and experiences. Towards resisting often-unquestioned colonial taxonomies, while more fully accounting for the intergenerational complexities of youth climate activism, we draw on our own and others’ recent research to advocate for selected critical methodologies and related decolonial research practices—emphasizing arts-based and participatory methods as well as long-term research collaborations—that better enable scholar-activists to aid and accompany the movement through research and action that recognizes and facilitates intergenerational solidarities. Doing so, we argue, will not only more accurately reflect the already-intergenerational nature of the climate movement, but also—through decolonial research practices—foster powerful connections across intergenerational lines that can enable people young and old to imagine and enact climate-just futures. [ABSTRACT FROM AUTHOR]

Intradialytic hypotension (IDH) is a common adverse event resulting in premature interruption of hemodialysis, and consequently, inadequate fluid and solute removal. IDH occurs in response to the reduction in blood volume during ultrafiltration and subsequent poor compensatory mechanisms due to abnormal cardiac function or autonomic or baroreceptor failure. Pediatric patients are inherently at risk for IDH due to the added difficulty of determining and attaining an accurate dry weight. While frequent blood pressure monitoring, dialysate sodium profiling, ultrafiltration-guided blood volume monitoring, dialysate cooling, hemodiafiltration, and intradialytic mannitol and midodrine have been used to prevent IDH, they have not been extensively studied in pediatric population. Lack of large-scale studies on IDH in children makes it difficult to develop evidence-based management guidelines. Here, we aim to review IDH preventative strategies in the pediatric population and outlay recommendations from the Pediatric Continuous Renal Replacement Therapy (PCRRT) Workgroup. Without strong evidence in the literature, our recommendations from the expert panel reflect expert opinion and serve as a valuable guide., Author(s): Rupesh Raina [sup.1] [sup.2] , Stephanie Lam [sup.3] , Hershita Raheja [sup.4] , Vinod Krishnappa [sup.2] [sup.5] , Daljit Hothi [sup.6] , Andrew Davenport [sup.7] , Deepa Chand [sup.8] [...]

Growing recognition of the multi-faceted injustices of climate change has resulted in shifting public and policy discourse around how to understand and address climate change, yet justice considerations are rarely present in climate change education (CCE). With aims of bringing visibility to existing efforts and building a foundation for more widespread justice-driven CCE, this systematic review examined how justice frameworks are being employed in a range of CCE contexts, within and beyond the classroom. Analyses of 55 peer-reviewed articles with data from 57 countries published between 2007 and 2020 demonstrate that the empirical literature on justice in CCE has grown significantly in recent years. Notably, justice-driven CCE in this review took place within and beyond STEM education settings, was fueled more by people-focused aims (e.g. advancing equity) compared to planet-focused aims (e.g. protecting the environment), and encompassed a remarkable diversity of educational processes, goals, and outcomes—with learners of all ages—across formal, community-based, and activist learning contexts. Findings have implications for scholars, educators, and practitioners across disciplines committed to educating for justice in the face of climate change. [ABSTRACT FROM AUTHOR]

Deconditioning describes functional decline, progressive weakness and physiological changes that result from prolonged bed rest and inactivity. Frailty is a syndrome in which age-related physiological changes are accelerated or exacerbated in multiple systems. Deconditioning is more likely in patients who experience frailty. Due to the complexities of these conditions, optimal management involves a multidisciplinary team comprised of GPs, specialists and allied health professionals. [ABSTRACT FROM AUTHOR]